Luisa De Risio Simon Platt October 2013

Several decades have been devoted to the study of the pathophysiology of epilepsy. Increasing knowledge in the field has only contributed to a partial understanding of the underlying mechanisms. Nevertheless, insight into the pathophysiology of epilepsy and its underlying histological and neurochemical alterations has contributed to rational development strategies of new antiepileptic medications (AEMs). Although various epileptic syndromes in people have been shown to differ pathophysiologically, they apparently share common ictogenesisrelated characteristics such as increased neuronal excitability and synchronicity. Emerging insights point to alterations of synaptic functions and intrinsic properties of neurons as common mechanisms underlying hyperexcitability. Progress in the field of molecular genetics has revealed arguments in favour of this hypothesis as mutations of genes encoding ion channels were recently discovered in some forms of human epilepsy.

Epileptic seizures arise from an excessively synchronous and sustained discharge of a group of neurons. The single feature of all epileptic syndromes is a persistent increase of neuronal excitability. Abnormal cellular discharges may be associated with a variety of causative factors such as trauma, oxygen deprivation, tumours, infection and metabolic derangements. However, no specific causative factors are found in many dogs and cats suffering from epilepsy.

Underlying causes and pathophysiological mechanisms are (partially) understood for some forms of epilepsy, at least in people, e.g. epilepsies caused by disorders of neuronal migration and monogenic epilepsies. For several other types of epilepsy, current knowledge is only fragmentary. This chapter will review several areas that are understood to contribute to the evolution and maintenance of epilepsy. The genetics of epilepsy are discussed in Chapter 6.

The Electrical Basis of Nerve Cell Function

At the most fundamental level, the nervous system is a function of its ionic milieu, the chemical and electrical gradients that create the setting for electrical activity. Therefore, some of the most easily appreciated controls on excitability are the ways the nervous system maintains the ionic environment. An example is the electrical basis of resting membrane potential. Resting potential is set normally so that neurons are not constantly firing but are close enough to threshold so that it is still possible that they can discharge, given that action potential generation is essential to CNS function. The control of resting potential

(L. De Risio and S. Platt)

becomes critical to prevent excessive discharge that is typically associated with seizures.

Normally a high concentration of potassium exists inside a neuron and there is a high extracellular sodium concentration, as well as additional ions, leading to a net transmembrane potential of −60 mV (Scharfman, 2007). If the balance is perturbed (e.g. if potassium is elevated in the extracellular space), this can lead to depolarization that promotes abnormal activity in many ways (Somjen, 2002): terminals may depolarize, leading to transmitter release, and neurons may depolarize, leading to action potential discharge. Pumps are present in the plasma membrane to maintain the chemical and electrical gradients, such as the sodium–potassium ATPase, raising the possibility that an abnormality in these pumps could facilitate seizures. Indeed, blockade of the sodium–potassium ATPase can lead to seizure activity in experimental preparations (Vaillend et al., 2002), suggesting a role in epilepsy (Grisar et al., 1992). The sodium–potassium pump is very interesting because it does not develop in the rodent until several days after birth, and this may contribute to the greater risk of seizures in early life (Haglund et al., 1985; Fukuda and Prince, 1992). In addition to pumps, glia also provide important controls on extracellular ion concentration, which has led many to believe that glia are just as important as neurons in the regulation of seizure activity (Duffy and MacVicar, 1999; Fellin and Haydon, 2005). Thus, the control of the ionic environment provides many potential targets for novel anticonvulsants. It is important to bear in mind that seizures, by themselves, can lead to the changes in the transmembrane gradients. For example, seizures are followed by a rise in extracellular potassium, a result of excess discharge. This can lead to a transient elevation in extracellular potassium that can further depolarize neurons. Thus, the transmembrane potential is a control point that, if perturbed, could elicit seizures and begin a ‘vicious’ cycle, presumably controlled by many factors that maintain homeostasis, such as pumps and glia.

The ionic basis of the action potential is another example of a fundamental aspect of neurobiology that can suggest potential mechanisms of seizures. Neurons are designed to discharge because of an elegant orchestration of sodium and potassium channels that rely on chemical and ionic gradients across the cell membrane. Abnormalities in the sodium channel might lead to a decrease in the threshold for an action potential to occur if the method by which sodium channel activation is controlled alters in any way (i.e. sodium channels are activated at more negative resting potentials or sodium channel inactivation is impaired). Indeed, it has been shown that mutations in the subunits of the voltagedependent sodium channels can lead to epilepsy. A specific syndrome, generalized epilepsy with febrile seizures, is caused by mutations in selected genes responsible for subunits of the voltagedependent sodium channel (Meisler et al., 2001). The mutation does not block sodium channels, presumably because such a mutation would be lethal, but they modulate sodium channel function. This concept, that modulation, rather than essential function, is responsible for genetic epilepsies, has led to a greater interest in directing the development of new anticonvulsants at targets that are not essential to, but simply influence, CNS function.

Synaptic Transmission

Research into seizures has gravitated to mechanisms associated with synaptic transmission, because of its critical role in maintaining the balance between excitation and inhibition. As more research has identified the molecular mechanisms of synaptic transmission, it has become appreciated that defects in almost every step can lead to seizures. Glutamatergic and gaminobutyric acid (GABA)ergic transmission, as the major excitatory and inhibitory transmitters of the nervous system, respectively, have been examined in great detail. It is important to point out, however, that both glutamate and GABA may not have a simple, direct relationship to seizures. One reason is that desensitization of glutamate and GABA receptors can reduce effects, depending on the timecourse of exposure. In addition, there are other reasons. GABAergic transmission can lead to depolarization rather than hyperpolarization if the gradients responsible for ion flow through GABA receptors are altered. For example, chloride is the major ion that carries current through GABAA receptors, and it usually hyperpolarizes neurons because chloride flows into the cell from the extracellular space. However, the K⁺Cl⁻ cotransporters (KCCs) that are pivotal to the chloride gradient are not constant. In development, transporter expression changes, and this has led to evidence that one of the transporters, NKCC1, may explain seizure susceptibility early in life (Dzhala et al., 2005). The relationship of glutamate to excitation may not always be simple either. One reason is that glutamatergic synapses innervate both glutamatergic neurons and GABAergic neurons in many neuronal systems. Exposure to glutamate could have little net effect as a result, or glutamate may paradoxically increase inhibition of principal cells because the GABAergic neurons typically require less depolarization by glutamate to reach threshold. It is surprisingly difficult to predict how glutamatergic or GABAergic modulation will influence seizure generation in vivo, given these basic characteristics of glutamatergic and GABAergic transmission.

Synchronization

Excessive discharge alone does not necessarily cause a seizure. Synchronization of a network of neurons is involved. Therefore, how synchronization occurs becomes important to consider. There are many ways neurons can synchronize. In 1964, Matsumoto and AjmoneMarsan found that the electrographic events recorded at the cortical surface during seizures corresponded to paroxysmal depolarization shifts (PDS) of cortical pyramidal cells occurring synchronously (Matsumoto and Marsan, 1964). These studies led to efforts to understand how neurons begin to fire in concert when normally they do not. Glutamatergic interconnections are one example of a mechanism that can lead to synchronization. Indeed, studies of the PDS suggested that the underlying mechanism was a ‘giant’ excitatory postsynaptic potential, although it was debated widely at that time if this was the only cause (Johnston and Brown, 1984). Thus, pyramidal cells of cortex are richly interconnected to one another by glutamatergic synapses. Gap junctions on cortical neurons are another mechanism for synchronization. Gap junctions allow a lowresistance pathway of current flow from one cell to another, so that coupled neurons are rapidly and effectively synchronized. It was thought that gap junctions were rare, so it was unlikely that they could play a major role, but further study led to the appreciation that even a few gap junctions may have a large impact on network function (Traub et al., 2004). Another mechanism of synchronization involves, paradoxically, inhibition.

Many GABAergic neurons that innervate cortical pyramidal cells, such as the cell type that controls somatic inhibition (the basket cell), make numerous connections to pyramidal cells in a local area. Therefore, discharge of a single interneuron can synchronously hyperpolarize a population of pyramidal cells. As GABAergic inhibition wanes, voltagedependent currents of pyramidal cells become activated. These currents, such as Ttype calcium channels and others, are relatively inactive at resting potential, but hyperpolarization relieves this inhibition. The result is a depolarization that is synchronous in a group of pyramidal cells (Scharfman, 2007).

Some of the changes that develop within the brain of individuals with epilepsy also promote synchronization. Such changes are of interest in themselves because they may be one of the reasons why the seizures are recurrent. These changes include growth of axon collaterals of excitatory neurons, typically those that use glutamate as a neurotransmitter and are principal cells. An example is the dentate gyrus granule cell of hippocampus. In animal models of epilepsy and in patients with intractable temporal lobe epilepsy (TLE), the axons of the granule cells develop new collaterals and the new collaterals extend for some distance. They do not necessarily terminate in the normal location but in a novel lamina, one that contains numerous granule cell dendrites. Electron microscopy has shown that the new collaterals innervate granule cell dendrites, potentially increasing recurrent excitatory circuits. Some argue that recurrent inhibition increases as well as recurrent excitation, but the

fact remains that new synaptic excitatory circuits develop that are sparse or absent in the normal brain (Nadler, 2003; Sloviter et al., 2006). The resultant ‘synaptic reorganization’ not only can support synchronization, potentially, but it also illustrates how the plasticity of the nervous system may contribute to epileptogenesis.

Kindling and Epileptogenesis

Goddard (1967) was the first to describe that periodic stimulation of neural pathways progressively leads to recurrent behavioural and electrographic seizures. Kindling procedures have provided a substrate for the study of the role of enhanced synaptic efficacy in seizure disorders. It is now considered to be a first choice experimental procedure in the study of the potential mechanisms of epileptogenesis. The phenomenon can be evoked in various brain regions, but amygdala kindling is most frequently used in epilepsy research as a model for complex focal (partial) seizures (Fisher, 1989). Although kindling has been shown to be phenomenologically different from other types of plastic changes in the central nervous system, there are many points of similarity between kindling and the process of longterm potentiation (Sutula et al., 1989).

Kindling has been shown to depend upon functional as well as structural changes in glutamatergic synapses. The anticonvulsant effects of glutamate receptor blocking agents like NmethylDaspartate (NMDA) antagonists seem to be at least partly due to their inhibitory effects on in vitro kindling.

ictogenesis

Excitability is a key feature of ictogenesis that may originate from individual neurons, neuronal environment or a population of neurons. Excitability arising from single neurons may be caused by alterations in membrane or metabolic properties of individual neurons (Traub et al., 1996). When regulation of environmental, extracellular concentrations of ions or neurotransmitters is suboptimal, the resulting imbalance might enhance neuronal excitation. Collective anatomic or physiologic neuronal alterations may convert neurons into a hyperexcitable neuronal population. In reality, these three theoretical mechanisms are thought to interact during specific ictal episodes. Each epileptic focus is unique as the differential contribution of these three concepts leading to ictal events is thought to differ from focus to focus.

Excitability arising from individual neurons

Functional and perhaps structural changes occur in the postsynaptic membrane, thus altering the character of receptor proteinconductance channels, thereby favouring development of paroxysmal depolarizing shifts (PDS) and enhanced excitability. Epileptic neurons appear to have increased Ca²⁺ conductance. It may be that latent Ca²⁺ channels are used, that the efficacy of Ca²⁺ channels is increased or that the number of Ca²⁺ channels is chronically elevated. However, development of burst activity depends on the net inward current and not on the absolute magnitude of the inward current. When extracellular K⁺ concentrations are increased (as during seizure activity), the K⁺ equilibrium across the neuronal membrane is reduced, resulting in reduced outward K⁺ currents. The net current will become inward, depolarizing the neuron to the extent that Ca²⁺ currents will be triggered. This results in a PDS and a burst of spikes (Dichter and Ayala, 1987).

Excitability arising from neuronal microenvironment

Both functional and structural alterations occur in epileptic foci. The functional changes involve concentrations of cations and anions, metabolic alterations, and changes in neurotransmitter levels. The structural changes involve both neurons and glia. Excessive extracellular K⁺ depolarizes neurons and leads to spike discharge. During seizures, changes in extracellular Ca²⁺ (a decrease of 85%) precede those of K⁺ by milliseconds and Ca²⁺ levels return to normal more quickly than K⁺. Glia are able to clear neurotransmitters from the extracellular space and to buffer K⁺, thus correcting the increased extracellular K⁺ concentrations that occur during seizures. Epileptic foci may show proliferation of glia that differ however in morphological and physiological properties (Bordey and Sontheimer, 1998). Gliosis will affect glial K⁺ buffering capacity and hence may contribute to seizure generation.

The epileptic cell population

Collective anatomic or physiologic neuronal alterations might produce progressive, networkdependent facilitation of excitability, perhaps coupled with a decrease of inhibitory influences, e.g. due to selective loss of inhibitory neurons. Mossy fibre sprouting (MFS) is an example of neuronal alterations leading to increased excitability (Cavazos et al., 1991). MFS was demonstrated in patients with refractory temporal lobe epilepsy with hippocampal sclerosis on neuroimaging as well as in numerous animal models of temporal lobe epilepsy (Sutula et al., 1988, 1989). In normal conditions, the dentate granule cells limit seizure propagation through the hippocampal network. However, the formation of recurrent excitatory synapses between dentate granule cells, as is thought to occur after MFS, may transform the dentate granule cells into an epileptogenic population of neurons (McNamara, 1999). Possibly, a vicious cycle develops: seizures cause neuronal death, which results in MFS, which in turn increases seizure frequency.

mechanisms of interictal–ictal Transition

Mechanisms producing signal amplification, synchronicity and spread of activity are likely to be involved in interictal–ictal transitions. In vivo, interictal–ictal transition can seldom be attributed to one theoretical mechanism, but often results from the interaction of different mechanisms.

Nonsynaptic mechanisms

Alterations in ionic microenvironment

Repetitive ictal and interictal activity causes increases in extracellular K⁺ leading to increased neuronal excitability (Moody et al., 1974). Some neurons are very sensitive to changes in membrane K⁺ currents, e.g. pyramidal cells in the CA1 region of the hippocampus (Rutecki et al., 1985).

Active ion transport

Activation of the Na⁺–K⁺ pump is important for regulation of neuronal excitability during excessive neuronal discharges. Substances like ouabain that block the Na⁺–K⁺ pump can induce epileptogenesis in animal models. Hypoxia or ischaemia can result in Na⁺–K⁺ pump failure thus promoting interictal–ictal transition. A Cl⁻–K⁺ cotransport mechanism controls the intracellular Cl⁻ concentration and the Cl⁻ gradient across the cell membrane, which regulates effectiveness of GABAactivated inhibitory Cl⁻ currents. Interference with this process could cause a progressive decrease in the effectiveness of GABAergic inhibition leading to increased excitability (Engelborghs et al., 2000).

Presynaptic terminal bursting

The amount of transmitter released is related to depolarization of presynaptic terminals. Changes in axon terminal excitability will have effects on synaptic excitation (Engelborghs et al., 2000). Abnormal bursts of action potentials occur in the axonal arborizations of thalamocortical relay cells during epileptogenesis. Since one thalamocortical relay cell ends on a large number of cortical neurons, synchronization can occur, which might play an important role in interictal–ictal transition (Engelborghs et al., 1998a).

Ephaptic interaction

Ephaptic interactions are produced when currents from activated neurons excite adjacent neurons in the absence of synaptic connections. Ephaptic effects are strongly dependent

on the size of the extracellular space. When extracellular space is small, ephaptic interactions are promoted (Traub et al., 1985).

Synaptic mechanisms

Two theoretical mechanisms can occur: decreased effectiveness of inhibitory synaptic mechanisms or facilitation of excitatory synaptic events. Both mechanisms will be discussed below.

Neurochemical mechanisms Underlying Epilepsy

GABA

The GABA hypothesis of epilepsy implies that a reduction of GABAergic inhibition results in epilepsy whereas an enhancement of GABAergic inhibition results in an antiepileptic effect (Wong and Watkins, 1982; De Deyn and Macdonald, 1990; De Deyn et al., 1990). Inhibitory postsynaptic potentials (IPSPs) gradually decrease in amplitude during repetitive activation of cortical circuits. This phenomenon might be caused by decreases in GABA release from terminals, desensitization of GABA receptors that are coupled to increases in Cl⁻ conductance or alterations in the ionic gradient because of intracellular accumulation of Cl⁻ (Wong and Watkins, 1982). In case of intracellular accumulation of Cl⁻, passive redistribution is ineffective. Moreover, Cl⁻–K⁺ cotransport becomes less effective during seizures as it depends on the K⁺ gradient. As Cl⁻–K⁺ cotransport depends on metabolic processes, its effectiveness may be affected by hypoxia or ischaemia as well. These mechanisms may play a critical role in ictogenesis and interictal–ictal transition. Several studies have shown that GABA is involved in pathophysiology of epilepsy in both animal models and patients suffering from epilepsy. GABA levels and glutamic acid decarboxylase (GAD) activity were shown to be reduced in epileptic foci surgically excised from patients with intractable epilepsy and in CSF of patients with certain types of epilepsy (De Deyn et al., 1990).

A reduction of 3H–GABA binding has been reported in human brain tissue from epileptic patients whereas PET studies demonstrated reduced benzodiazepine receptor binding in human epileptic foci (Savic et al., 1996). The degree of benzodiazepine receptor reduction showed a positive correlation with seizure frequency. The GABA receptor complex is involved in various animal models of epilepsy as well. Low CSF levels of GABA were revealed in dogs with epilepsy (Loscher and SchwartzPorsche, 1986). Reduced GAD levels were revealed in the substantia nigra of amygdalakindled rats (Loscher and Schwark, 1985). Significant alterations in GABA and benzodiazepine binding have been shown in the substantia nigra of genetically seizureprone gerbils (Olsen et al., 1985). Mice with a genetic susceptibility to audiogenic seizures have a lower number of GABA receptors than animals of the same strain that are not seizure prone (Horton et al., 1982). Several endogenous (guanidino compounds) and exogenous

(e.g. bicuculline, picrotoxin, penicillin, pilocarpine, pentylenetetrazol) convulsants inhibit GABAergic transmission through inhibition of GABA synthesis or through interaction with distinct sites at the postsynaptic GABA_A receptor (De Deyn and Macdonald, 1990; D’Hooge et al., 1996). Convulsant agents that block synaptic GABAmediated inhibition, amplify the dendritic spikegenerating mechanism that involves Ca²⁺ (Dichter and Ayala, 1987; Fisher, 1989). Synaptic inputs are thought to trigger and synchronize this process throughout a population of cells, which then might result in an epileptic seizure. Several AEMs are GABA analogues, block GABA metabolism or facilitate postsynaptic effects of GABA. However, a study evaluating dosedependent behavioural effects of single doses of vigabatrin in audiogenic sensitive rats, suggests that the antiepileptic properties of vigabatrin not only depend on GABAergic neurotransmission but might also be explained by decreased central nervous system levels of excitatory amino acids or increased glycine concentrations (Engelborghs et al., 1998b).

Glutamate

In rodent models, altering glutamate receptor or glutamate transporter expression by knockout

or knockdown procedures can induce or suppress epileptic seizures (Chapman et al., 1996; Kabova et al., 1999; Chapman, 2000). Regardless of the primary cause, synaptically released glutamate acting on ionotropic and metabotropic receptors appears to play a major role in the initiation and spread of seizure activity (Meldrum, 1994; Chapman et al., 1996; Chapman, 2000). Glutamatergic synapses play a critical role in all epileptic phenomena. Activation of both ionotropic and metabotropic postsynaptic glutamate receptors is proconvulsant. Antagonists of NmethylDaspartate (NMDA) receptors are powerful anticonvulsants in many animal models of epilepsy. Several genetic alterations have been shown to be epileptogenic in animal models.

Glutamate receptors

Studies of epileptiform discharges in hippocampal slices show that the characteristic burst discharge, associated with a ‘paroxysmal depolarizing shift’, is dependent on activation of AMPA receptors for its initial components and NMDA receptors for the later elements (Bengzon et al., 1999; Mazarati and Wasterlain, 1999; Meldrum et al., 1999).

AMPA

AMPA receptor antagonists, either competitive or noncompetitive, are anticonvulsant in rodent models (Rogawski and Donevan, 1999). Thus, altered function of AMPA receptors could contribute to proconvulsant or anticonvulsant effects (Meldrum et al., 1999). Evidence has accumulated that Ca²⁺permeable AMPA receptors may play a role in epileptogenesis and the brain damage occurring during the prolonged seizures (Rogawski and Donevan, 1999). Because Ca²⁺permeable AMPA receptors are predominantly expressed in GABAergic interneurons, it is hypothesized that some forms of epilepsy might be caused by reduced GABA inhibition resulting from Ca²⁺permeable AMPA receptormediated excitotoxic death of interneurons (Rogawski and Donevan, 1999).

NMDA

NMDA receptor antagonists are potent anticonvulsants in many animal models, suggesting a role for these receptors in epileptogenesis (Patrylo et al., 1999). It is known that enhancing NMDA receptormediated excitatory actions (e.g. by lowering extracellular Mg) produces epileptiform activity in experimental models of ‘kindled’ epilepsy (Chapman, 1998, 2000). It has been postulated that NMDA receptors may change after neuronal damage (Rice and DeLorenzo, 1998). New receptors are formed that have either less sensitivity to ambient Mg or more sensitivity to ambient glycine; increased excitability could occur within local circuits where the circuitry itself is not altered (or may occur in addition to circuit alterations) (Meldrum et al., 1999). As it is known that the NMDA receptor is subject to modulation by a variety of endogenous agents, including glycine (as a coagonist with glutamate), polyamines, steroids, neuropeptides (Vezzani et al., 2000b), pH, the redox state of the receptor, and NO, there are many chronic alterations in NMDA receptors that could underlie longterm changes in excitability and, thereby, epilepsy. Presently, there are no data to support changes in any of these regulatory factors in chronic epilepsy, but it is distinctly possible that alterations in one or more of these will be shown to be responsible for one or another form of inherited epilepsy.

Kindling is the most extensively studied animal model of epileptogenesis, and this has demonstrated the unique importance of NMDA receptors in the creation of seizure activity (Bengzon et al., 1999; Meldrum et al., 1999). In kindling, repeated electrical stimuli in the limbic system lead to a progressive increase of seizure susceptibility. When the animal responds to stimuli with generalized convulsions, it has developed a permanent epileptic condition. Activation of NMDA receptors and levels of NMDA receptor function are critical in kindling epilepsy (Bengzon et al., 1999). Selective NMDAreceptor antagonists retard kindling development and can also, at higher doses, have an anticonvulsant effect (Bengzon et al., 1999; Trist, 2000).

Metabotropic receptors

On account of these receptors’ responsibility for regulating glutamatergic and GABAergic neurotransmission, it is not surprising that mGluRs strongly influence the induction, propagation and termination of epileptic activity in the central nervous system (CNS) (Doherty and Dingledine, 2002). Pharmacological studies with mGluR group specific agonists and antagonists provide a relatively clear picture for Group I, with agonists being convulsant and antagonists being anticonvulsant (Meldrum et al., 1999; Doherty and Dingledine, 2002; Sayin and Rutecki, 2003). The picture is more complicated for the Group II and III receptors but anticonvulsant effects have been described for agonists of both these groups (Meldrum et al., 1999).

Glutamate transporters

In addition to receptor abnormalities, glutamate transporters, responsible for the removal of glutamate from the extracellular fluid, have been implicated in epilepsy (Meldrum et al., 1999). In situ hybridization studies have shown that the mRNA responsible for the rat glial glutamate transporter (GLT) is reduced in several brain regions in epilepsyprone rats (Meldrum et al., 1999). GLT ‘knockout’ mice have been bred to provide homozygous mice, in which the GLT protein is not detected. In such mutant mice, glutamate uptake in cortical synaptosomes is 5.8% compared with the wildtype (Meldrum et al., 1999). The mutant mice show spontaneous seizures, with wild running and tonic extension, which is frequently fatal. In chronic seizure models (kindled seizures, spontaneous seizures and genetically epilepsyprone rats), there are numerous reports of increases in extracellular glutamate during seizures (Meldrum et al., 1999). This strongly suggests that in these chronic models there are sustained functional alterations in mechanisms relating to the synaptic release of glutamate or its transport. GLT1 astrocytic expression was reduced in four Shetland sheepdogs with IE (Morita et al., 2005). In these dogs it was suggested that decreased expression of the transporter might be related to development of status epilepticus.

There is not as yet any genetically determined epilepsy syndrome occurring spontaneously in man or mouse that can be ascribed to a primary gene defect involving a glutamate receptor or transporter.

Targets for treatment

In animal models of epilepsy, antagonists acting at NMDA receptors, AMPA receptors or at Group I metabotropic receptors have potent anticonvulsant actions (Meldrum and Chapman, 1999; Rogawski and Donevan, 1999; Chapman, 2000; Moldrich et al., 2003).

NMDA receptor antagonists have been successful in stopping the maintenance phase of selfsustaining status epilepticus (SE) in rats, which suggests that these compounds may have a promising role in the treatment of unrelenting seizure activity such as SE (Mazarati and Wasterlain, 1999). Studies with selective AMPA receptor antagonists have indicated that AMPA receptors are potentially promising anticonvulsant drug targets, but at present this is uncertain (Rogawski and Donevan, 1999).

In genetic mouse models, mGlu1/5 antagonists and mGlu2/3 agonists are effective against absence seizures. Thus, antagonists at Group I mGlu receptors and agonists at Groups II and III mGlu receptors are potential antiepileptic agents, but their clinical usefulness will depend on their acute and chronic sideeffects (Moldrich et al., 2003). Potential also exists for combining mGlu receptor ligands with other glutamatergic and nonglutamatergic agents to produce an enhanced anticonvulsant effect (Moldrich et al., 2003).

The Veterinary Perspective

Idiopathic epilepsy (see Chapter 6) is the most common cause of seizures in dogs (Podell and Hadjiconstantinou, 1997). Low levels of GABA and high levels of glutamate have been detected in the cerebrospinal fluid of epileptic dogs independent of time relation to recent seizure activity (Podell and Hadjiconstantinou, 1997). The glutamate elevations are not related whether the seizures were focal or generalized in character (Podell and Hadjiconstantinou, 1997). These findings may indicate the brains of epileptic dogs are under a state of chronic overexcitation. Although a separate study found that lower CSF GABA concentration was associated with a reduced response to phenobarbital therapy in dogs, there was no association between CSF glutamate and response to this therapy (Podell and Hadjiconstantinou, 1999). However, a negative association was found between CSF glutamate:GABA ratio and response to phenobarbital therapy (Podell and Hadjiconstantinou, 1999). Therefore glutamatemediated mechanisms may be useful targets for anticonvulsant therapy in dogs. Intracerebral microdialysis was used to demonstrate elevation of extracellular levels of glutamate in four Shetland sheepdogs with IE, suggesting an important role in the occurrence of seizure activity (Morita et al., 2005).

Gabapentin (see Chapter 17), a relatively new human anticonvulsant, has been evaluated in dogs refractory to phenobarbitone and potassium bromide with an approximate 50% success rate.

Gabapentin has been shown to modestly decrease glutamate levels in the brain (Errante and Petroff, 2003). Another new anticonvulsant, topiramate (see Chapter 19), produces its antiepileptic effect by several mechanisms, one of which is inhibition of kainitemediated glutamate receptors (Angehagen et al., 2003a). This drug has also been demonstrated to protect neurons from excitotoxic levels of glutamate, potentially preventing brain damage during seizure activity (Angehagen et al., 2003b).

Catecholamines

Abnormalities of CNS catecholamines have been reported in several genetic models of epilepsy. In the spontaneous epileptic rat, dopamine was decreased in the caudate nucleus whereas noradrenaline was increased in the midbrain and brainstem (Hara et al., 1993). Decreased levels of dopamine have been found in epileptic foci of epilepsy patients (Mori et al., 1987). In animal models of absence epilepsy, seizures are exacerbated by dopamine antagonists while alleviated by dopamine agonists (Snead, 1995). These results suggest that decreased dopamine facilitates appearance of seizures by lowering the threshold triggering such seizures. Tottering mice have an absencelike syndrome that is characterized by episodes of behavioural arrest associated with 6 to 7 Hz cortical SW EEG discharges. Selective destruction of the ascending noradrenergic system at birth prevents the onset of the syndrome. Therefore, it has been suggested that the syndrome is caused by a noradrenergic hyperinnervation of the forebrain (Engelborghs et al., 2000). Recent data indicate that the serotonergic system regulates epileptiform activity in a genetic rat model of absence epilepsy as intraperitoneal or intracerebroventricular administration of 8 OHDPAT caused marked and dosedependent increases in number and duration of discharges (Gerber et al., 1998).

Opioid peptides

In experimental studies, opioids and opioid peptides had both convulsant and anticonvulsant properties (Engelborghs et al., 2000). Kappa agonists suppress electrical discharges in an animal model of absence epilepsy (Przewlocka et al., 1995). Peptides with a magonist action induce hippocampal or limbic seizures when administered intraventricularly possibly due to inhibition of inhibiting interneurons. In patients with complex partial seizures, PET studies pointed out that mreceptor density is increased in the temporal cortex (Mayberg et al., 1991).

inflammatory mechanisms Underlying Epilepsy

Over the past 10 years an increasing body of clinical and experimental evidence has provided strong support to the hypothesis that inflammatory processes within the brain might constitute a common and crucial mechanism in the pathophysiology of seizures and epilepsy (Vezzani et al., 2011). The first insights into the potential role of inflammation in human epilepsy were derived from clinical evidence indicating that steroids and other antiinflammatory treatments displayed anticonvulsant activity in some drugresistant epilepsies (Wirrell et al., 2005; Wheless et al., 2007). Additional evidence came from febrile seizures in people, which always coincide with, and are often caused by, a rise in the levels of proinflammatory agents (Dube et al., 2007). Evidence of immune system activation in some patients with seizure disorders, the high incidence of seizures in autoimmune diseases, and the discovery of limbic encephalitis as a cause of epilepsy led to the suggestion that immune and inflammatory mechanisms have roles in some forms of epilepsy (Aarli, 2000; Bien et al., 2007; Vincent and Bien, 2008; Vezzani et al., 2011).

Evidence is emerging that inflammation might be a consequence as well as a cause of epilepsy. Several inflammatory mediators have been detected in surgically resected brain tissue from human patients with refractory epilepsies, including temporal lobe epilepsy (TLE) and cortical dysplasiarelated epilepsy (Choi et al., 2009; Vezzani et al., 2011). The finding that brain inflammation occurred in epilepsies that were not classically linked to immunological dysfunction highlighted the possibility that chronic inflammation might be intrinsic to some epilepsies, irrespective of the initial insult or cause, rather than being only a consequence of a specific underlying inflammatory or autoimmune aetiology. The mounting evidence for a role for inflammatory processes in human epilepsy has led to the use of experimental rodent models to identify putative triggers of brain inflammation in epilepsy, and to provide mechanistic insights into the reciprocal causal links between inflammation and seizures (Vezzani et al., 2011). Experimental studies have shown that seizure activity per se can induce brain inflammation, and that recurrent seizures perpetuate chronic inflammation. Seizureassociated cell loss can contribute to inflammation but is not a prerequisite for inflammation to occur. In addition, models of systemic or CNS infections suggested that preexisting brain inflammation increases the predisposition to seizures, associated with alterations in neuronal excitability and enhanced seizureinduced neuropathology. Additional mechanistic insights into the role of inflammation in seizures and the development of epilepsy have been gained through use of pharmacological approaches that interfere with specific inflammatory mediators and from changes in seizure susceptibility in genetically modified mice with perturbed inflammatory pathways (Campbell et al., 1993; Kelley et al., 1999; Vezzani et al., 2000a; Balosso et al., 2005).

Inflammation consists of the production of a cascade of inflammatory mediators (a dynamic process), as well as antiinflammatory molecules and other molecules induced to resolve inflammation, as a response to noxious stimuli (such as infection or injury), or immune stimulation, and is designed to defend the host against pathogenic threats. Inflammation is characterized by the production of an array of inflammatory mediators from tissueresident or bloodcirculating immunocompetent cells, and involves activation of innate and adaptive immunity. Both innate and adaptive immunity have been implicated in epilepsy, and microglia, astrocytes and neurons are believed to contribute to the innate immunitytype processes that cause inflammation of the brain. The brain has traditionally been considered an immunoprivileged site because of the presence of the blood–brain barrier (BBB), the lack of a conventional lymphatic system, and the limited trafficking of peripheral immune cells. Nevertheless, both the innate and adaptive immune responses are readily evoked within the CNS in response to pathogens, selfantigens, or tissue injury of several aetiologies. Microglia, astrocytes, neurons, BBB endothelial cells, and peripheral immune cells extravasating into brain parenchyma can all produce proinflammatory and anti inflammatory molecules (Ransohoff et al., 2003; Banks and Erickson, 2010).

The contribution of each cell population to brain inflammation depends on the origin (for example, CNS versus systemic) and the type (for example, infectious versus sterile) of the initial precipitating event (Glass et al., 2010). The BBB represents a key regulatory element of the communication between intrinsic brain cells and peripheral immunocompetent cells. As noted above, an inflammatory response in the CNS can be induced in the absence of infection. Brain inflammation has been reported following ischaemic stroke or traumatic brain injury (TBI), and during chronic neurodegenerative diseases. In all these conditions, pronounced activation of microglia and astrocytes takes place in brain regions affected by the specific disease, and these cells act as major sources of inflammatory mediators. Recruitment of peripheral immune cells might also occur (Nguyen et al., 2002; Glass et al., 2010). The activation of innate immunity and the transition to adaptive immunity are mediated by a large variety of inflammatory mediators, among which cytokines, polypeptides that act as soluble mediators of inflammation, have a pivotal role (Akira et al., 2001; Nguyen et al., 2002).

These molecules include interleukins (ILs), interferons (IFNs), tumour necrosis factors (TNFs) and growth factors (for example, transforming growth factor (TGF)b). Cytokines are released by immunocompetent and endothelial cells, as well as by glia and neurons in the CNS, thereby enabling communication between effector and target cells during an immune challenge or tissue injury. Following their release, cytokines interact with one or more cognate receptors. The most extensively studied prototypical inflammatory cytokines in the CNS are IL1b, TNF and IL6 (Allan and Rothwell, 2001; Bartfai et al., 2007). Cytokine activity can be regulated at multiple levels, including gene transcription, cleavage of cytokine precursors (for example, proIL1b, proTNF) by specific proteolytic enzymes, and cellular release, as well as through receptor signalling (discussed below). All cell types in the brain seem capable of expressing cytokines and their receptors, with low basal expression of these molecules being rapidly upregulated following CNS insults. Chemokines comprise a specific class of cytokines that act as chemoattractants to guide the migration of leukocytes from blood through the endothelial barrier into sites of infection or injury (Wilson et al., 2010). These cytokines also regulate microglial motility and neural stem cell migration, provide axon guidance during brain development, and promote angiogenesis, neurogenesis and synaptogenesis (Szekanecz and Koch, 2001; Semple et al., 2010). The release of chemokines is often stimulated by proinflammatory cytokines such as IL1b.

Several mechanisms have been identified that attenuate the inflammatory response, indicating the importance of such strict control for homeostasis and prevention of injury. Regulatory mechanisms include production of proteins that compete with cytokines to bind their receptors, such as IL1 receptor antagonist protein (IL1ra), and decoy receptors that bind cytokines and chemokines but are incapable of signalling, thereby acting as molecular traps to prevent such ligands from interacting with biologically active receptors (Mantovani et al., 2001; Dinarello, 2009). Proteins that inhibit cytokineinduced signal transduction (for example, suppressor of cytokine signalling proteins) or transcription (for example, Nurr1CoREST or activity transcription factor 3), as well as an array of soluble mediators with antiinflammatory activities (such as IL10 and TGFb), are produced concomitantly with proinflammatory molecules to resolve inflammation (Blobe et al., 2000; Khuu et al., 2007; Baker et al., 2009). For example, glucocorticoids, via activation of glucocorticoid receptors and, consequently, downregulation of nuclear factorkB (NFkB) and activator protein 1 activity, inhibit innate immune responses and, hence, act as an endogenous antiinflammatory feedback system. Proinflammatory cytokines are powerful enhancers of glucocorticoid levels in adrenal glands via corticotropinreleasing hormone and adrenocorticotropic hormone (ACTH). Glucocorticoids also elicit immunosuppressive effects through inhibition of leukocyte extravasation from the vasculature, and through regulation of T helper cell differentiation (Sapolsky et al., 1987; Elenkov et al., 1999). The CNS can also negatively regulate the inflammatory response in a reflexive manner, using the efferent activity of the vagus nerve to inhibit release of proinflammatory molecules from tissue macrophages (Vezzani et al., 2000a, 2011; Tracey, 2002).

Do seizures cause inflammation?

In adult rats and mice, induction of recurrent short seizures or single prolonged seizures (status epilepticus; defined as a seizure lasting >30 min) by chemoconvulsants or electrical stimulation triggers rapid induction of inflammatory mediators in brain regions of seizure activity onset and propagation (Vezzani et al., 2000a, 2011; Crespel et al., 2002). Immunohistochemical studies on rodent brains after induction of status epilepticus demonstrated subsequent waves of inflammation during the epileptogenic process (that is, the process underlying the onset and chronic recurrence of spontaneous seizures after an initial precipitating event), involving various cell populations. Findings from these and other studies show that proinflammatory cytokines (IL1b, TNF and IL6) are first expressed in activated microglia and astrocytes, and cytokine receptor expression is upregulated in microglia, astrocytes and neurons (Vezzani and Granata, 2005). These initial events are followed by the induction of cyclooxygenase2 (COX2) and, hence, prostaglandins, and upregulation of components of the complement system in microglia, astrocytes and neurons (Yoshikawa et al., 2006; Aronica et al., 2007; Kulkarni and Dhir, 2009; Xu et al., 2009).

In addition to the molecules mentioned above, chemokines and their receptors are produced – predominantly in neurons and in activated astrocytes – days to weeks after status epilepticus (Wu et al., 2008; Xu et al., 2009; Fabene et al., 2010). An ensuing wave of inflammation is induced in brain endothelial cells by seizures, and includes upregulation of IL1b and its receptor IL1R1, the complement system, and adhesion molecules (Pselectin, Eselectin, intercellular adhesion molecule 1 (ICAM) and vascular cell adhesion molecule 1) (Vezzani and Granata, 2005; Aronica et al., 2007; Fabene et al., 2008; Vezzani et al., 2011). The presumed cascade of events leading to this vascular inflammation involves seizureinduced activation of perivascular glia, which produce and release cytokines and prostaglandins. Importantly, no peripheral immune cells or bloodderived inflammatory molecules are required for vascular inflammation, as such events have been replicated in vitro in isolated guinea pig brain undergoing seizure activity (Vezzani and Granata, 2005; Vezzani et al., 2011).

The presence of inflammation originating from the brain might promote the recruitment of peripheral inflammatory cells. Indeed, chemokines expressed by neurons and glia and in the cerebrovasculature following seizures might direct blood leukocytes into the brain, which would be consistent with the reported emergence of granulocytes during epileptogenesis, and sparse T lymphocytes in chronic epileptic tissue from TLE models and humans (Ravizza et al., 2008). As in human epileptic brain specimens, brain tissue from rodents with experimental chronic TLE contains both activated astrocytes and microglia expressing inflammatory mediators (Crespel et al., 2002; Dube et al., 2007; Ravizza et al., 2008). Evidence for brain vessel inflammation associated with BBB breakdown is also prevalent (Fabene et al., 2008). A recent veterinary study evaluated the relationship of microglial activation to seizureinduced neuronal death in the cerebral cortex of Shetland sheepdogs with familial epilepsy (Sakurai et al., 2013). Cadavers of ten Shetland sheepdogs from the same family (six dogs with seizures and four dogs without seizures) and four agematched unrelated Shetland sheepdogs were evaluated. Samples of brain tissues were collected after euthanasia and sectioned for H&E staining and immunohistochemical analysis. Evidence of seizureinduced neuronal death was detected exclusively in samples of cerebral cortical tissue from the dogs with familial epilepsy in which seizures had been observed. The seizureinduced neuronal death was restricted to tissues from the cingulate cortex and sulci surrounding the cerebral cortex. In almost the same locations as where seizureinduced neuronal death was identified, microvessels appeared longer and more tortuous and the number of microvessels was greater than in the dogs without seizures and control dogs. Immunohistochemical results for neurons and glial cells (astrocytes and microglia) were positive for vascular endothelial growth factor, and microglia positive for ionized calciumbinding adapter molecule 1 were activated

(i.e. had swollen cell bodies and long processes) in almost all the same locations as where seizureinduced neuronal death was detected. Doublelabel immunofluorescence techniques revealed that the activated microglia had positive results for TNFa, IL6 and vascular endothelial growth factor receptor 1. These findings were not observed in the cerebrum of dogs without seizures, whether the dogs were from the same family as those with epilepsy or were unrelated to them. The suggested conclusion of this study was that microglial activation induced by vascular endothelial growth factor and associated proinflammatory cytokine production may accelerate seizureinduced neuronal death in dogs with epilepsy (Sakurai et al., 2013).

The findings discussed above show that brain inflammation induced by status epilepticus develops further during epileptogenesis and demonstrate that this phenomenon persists in chronic epileptic tissue, thereby supporting the idea that inflammation might be intrinsic to, and perhaps a biomarker of, the epileptogenic process (Dube et al., 2007).

Does inflammation cause seizures?

Although the functions of many inflammatory mediators remain unresolved, clear evidence exists for an active role for IL1b, TNF, IL6, prostaglandin E2 (PGE2) and the complement cascade in seizure generation and exacerbation (Xiong et al., 2003). Seizure activity leads to the production of inflammatory molecules that, in turn, affect seizure severity and recurrence, and this action takes place through mechanisms distinct from the transcriptional events traditionally activated during systemic inflammation. Cerebrospinal fluid studies in children and animal models have implicated the release of endogenous cytokines, especially IL1b, in the generation of febrile seizures and, possibly, in the development of epilepsy after febrile seizures (Haspolat et al., 2002; Virta et al., 2002; Dube et al., 2005; Heida and Pittman, 2005; Vezzani et al., 2013).

A positive feedback pathway has been identified in rat models between seizure activity and the presence of inflammation (Vezzani et al., 2011). However, the role of inflammation in epilepsy in veterinary medicine has really only been described clinically in cats with hippocampal necrosis (Fatzer et al., 2000). Hippocampal lesions of 38 cats with seizures have been described and seemed to reflect different stages of disease consisting of acute neuronal degeneration to complete malacia, affecting mainly the layer of the large pyramidal cells but sometimes also the neurons of the dentate gyrus and the piriform lobe. The clinical, neuropathologic and epidemiologic findings suggest that the seizures in these cats were triggered by primary structural brain damage, perhaps resulting from excitotoxicity, but secondary inflammation cannot be ruled out in these cases.

Does inflammation cause cell loss?

Available studies suggest that seizurerelated or injuryrelated inflammation might contribute to cell loss and synaptic reorganization, which are important mediators of the development of hyperexcitable circuits that lead to epilepsy after insults such as status epilepticus or TBI in the adult rodent brain (Bartfai and Schultzberg, 1993; Buckmaster and Dudek, 1997; Pitkanen and Sutula, 2002). Inflammation is induced rapidly following such insults, preceding neurodegeneration in lesional models of seizures (Rizzi et al., 2003; Ravizza and Vezzani, 2006). This finding is consistent with the idea that inflammation augments cell death, which is further supported by data from studies involving injection of inflammatory mediators together with excitotoxic stimuli (Allan et al., 2005). Activation of microglia and astrocytes and production of cytokines and PGE2 can occur in seizure models where cell loss is not detected in immature or adult rodents (Vezzani et al., 1999, 2000a; Rizzi et al., 2003; Kovacs et al., 2006; Dube et al., 2010). Such observations suggest that rather than being a consequence of cell loss, seizureinduced brain inflammation can contribute to cell death (Vezzani and Baram, 2007). Additional interactions between inflammation and cell death in the context of epilepsy have been observed. Brain injury, such as TBI, causes tissue inflammation that seems to contribute to both cell death and longterm hyperexcitability (Clausen et al., 2009; Longhi et al., 2009). In the context of CNS injury (for example, in chronic neurodegenerative diseases or acute stroke), inflammation can have a neuroprotective role (Liesz et al., 2009; Schwartz and Shechter, 2010). Indeed, whether microglia, macrophages and/or T cells are destructive or neuroprotective seems to depend on their activation status, which is orchestrated by the specific inflammatory environment (Rothwell, 1989; Schwartz and Shechter, 2010). This balance, together with the specific brain regions in which inflammation develops, might account for the relatively low incidence of seizures in other neurological disorders associated with brain inflammation (Vezzani et al., 2013).

mechanistic insights

Several established and novel mechanisms could mediate the effects of inflammatory mediators on neuronal excitability and epilepsy. Some of these mechanisms could be involved in the precipitation and recurrence of seizures, while others are implicated in the development of epileptogenesis (Vezzani and Baram, 2007). These mechanisms constitute potential molecular targets for drug design, and are briefly summarized here. As discussed above, IL1b and HMGB1 activate convergent signalling cascade through binding to IL1R1 and TLR4, respectively (Akira et al., 2001; Perkins, 2007; Hoebe and Beutler, 2008). The downstream pathways activated by these ligands converge with the TNF pathways at the transcription factor NFkB, which regulates the synthesis of chemokines, cytokines, enzymes (for example, COX2) and receptors (for example, TLRs, IL1R1, and TNF p55 and p75 receptors) (Gilmore, 2006). This transcriptional pathway modulates the expression of genes involved in neurogenesis, cell death and survival, and in synaptic molecular reorganization and plasticity (processes that occur concomitantly with epileptogenesis in experimental models) (Buckmaster and Dudek, 1997; Pitkanen and Lukasiuk, 2009).

immune and anti-inflammatory therapies

If immune mechanisms and inflammation do indeed have a role in the generation of seizures, immunemodulating and antiinflammatory therapies might be effective treatments for some or all forms of epilepsy. Therapies such as ACTH, corticosteroids, plasmapheresis and intravenous immunoglobulin (IVIg) have been employed to treat seizures and/or epilepsy, with varying success. These therapies have all been employed in human patients with presumed autoimmune limbic encephalitis, where early and aggressive treatment often seems to be useful (Vincent et al., 2010).

The presumed mechanism of action of the therapeutic agents listed above is suppression of inflammation; however, other modes of action might also be involved, including direct effects on brain excitability, and suppression of endogenous proconvulsant brain agents (Baram and Hatalski, 1998; Joels and Baram, 2009).

The use of steroids in various forms is common for more severe, treatmentresistant forms of childhood epilepsy. ACTH, steroids and IVIg have all been employed to treat AEMunresponsive paediatric epilepsies, difficult focal (partial) epilepsies and myoclonic epilepsies (You et al., 2008). Unfortunately, determination of whether patients received benefit from these treatments is problematic, since most of these epilepsies are extremely heterogeneous in aetiology and severity, and exhibit notoriously variable courses. In addition, most of the clinical studies are retrospective case series, with occasional prospective case series that lack controls (Mikati et al., 2002; Verhelst et al., 2005).

Followup duration in these case series was also often variable. A recent review of investigations of IVIg in intractable childhood epilepsy found no randomized or controlled studies and, in fact, only two case series employed statistics in assessing outcome (Mikati et al., 2010). One series showed a statistically significant reduction in seizures with IVIg treatments, while the other revealed an insignificant trend with such therapy (Mikati et al., 2010). However, a Cochrane Collaboration review on the use of ACTH for other childhood epilepsies, published in 2007, found only a single randomized controlled trial, which only included five patients (Gayatri et al., 2007). The authors of this review concluded that, at present, no evidence exists to support either the safety or the efficacy of ACTH for general paediatric epilepsies (Gayatri et al., 2007).

Disorders of Neuronal migration and Seizures

The major developmental disorders noted in humans giving rise to epilepsy are disorders of neuronal migration that may have genetic or intrauterine causes (Engelborghs et al., 2000). Abnormal patterns of neuronal migration lead to various forms of agyria or pachygyria whereas lesser degrees of failure of neuronal migration induce neuronal heterotopia in the subcortical white matter. Experimental data suggest that cortical malformations can both form epileptogenic foci and alter brain development such that diffuse hyperexcitability of the cortical network occurs (ChevassusauLouis et al., 1999). Other studies revealed increases in postsynaptic glutamate receptors and decreases in GABA_A receptors in microgyric cortex, which could promote epileptogenesis (Jacobs et al., 1999).

Periventricular heterotopia is a human Xlinked dominant disorder of cerebral cortical development. Mutations in the filamin 1 gene prevent migration of cerebral cortical neurons causing periventricular heterotopia (Fox et al., 1998). Affected females present with epilepsy whereas affected males die embryonically.

Lissencephaly is a brain malformation characterized by a paucity of gyral formation and a thickening of the cerebral cortex. It is presumed to occur secondary to incomplete migration of immature neurons to the cortical plate during fetal development (Saito et al., 2002). Lissencephaly is considered to be the most severe type of neuronal migration disorder compatible with survival. In humans, it is presumed to result from an arrest of neuronal migration at approximately 3 to 4 months (Dobyns et al., 1993). Once they exit the cell cycle in the periventricular proliferative zone, immature neurons must migrate to the cortical plate along radial glial fibres (Rakic, 1988). The six layers of the cerebral cortex are formed in an ‘inside out’ pattern, with early migrating neurons forming the deep layer and later migrating neurons passing their migratory predecessors to form the superficial layers. Interruption at any stage of the process of neuronal migration may result in the arrest of neurons in an intermediate position between the periventricular zone and the cortex (Saito et al., 2002). Such an interruption may be due to a genetic lack of appropriate molecular cues, or secondary to nongenetic influences such as in utero infection or ischaemia. Secondary influences are a more common mechanism for the related cortical malformation, polymicrogyria.

In humans, mutations of two genes, LIS1 (located on 17p13.3) and DCX (located on Xq22.3), have been found to account for the majority of cases (Pilz et al., 1998). Both of these genes have been shown to have roles in neuronal migration by their interactions with the neuron microtubule network (Gleeson et al., 1999a; Sapir et al., 1999). Xlinked lissencephaly and double cortex syndrome is a disorder of neuronal migration documented in humans. Double cortex or subcortical band heterotopias often occur in females whereas more severe lissencephaly is found in affected males. A causal mutation in a gene called doublecortin has been identified (Gleeson et al., 1998). It was suggested that doublecortin acts as an intracellular signalling molecule critical for the migration of developing neurons (Allen and Walsh, 1999; Gleeson et al., 1999b). Lissencephaly has been documented in Lhasa apsos with histopathology indicating the condition to be very similar to that seen in people (Greene et al., 1976; Saito et al., 2002). This condition has also been documented in a mixed breed dog and together with either cerebellar hypoplasia in two wirehaired fox terriers and three Irish setters, with cyclopia in one German shepherdmixed breed dog, or with microencephaly in the Korat breed of cat (Saito et al., 2002; Lee et al., 2011).

Although these disorders are relatively rare, studying the underlying pathophysiological mechanisms may shed light on the pathophysiology of more common epileptic syndromes.

How Do Seizures Stop?

Most seizures are selflimited, lasting no more than a few minutes. The persistence of a seizure lasting longer than several minutes is usually a cause for alarm as physiological mechanisms terminating the seizure may have failed. Why seizures typically do not continue indefinitely, and how intrinsic anticonvulsant mechanisms in the brain lead to seizure termination, are questions that potentially offer new avenues for developing novel treatments for epilepsy, as well as offering insights into brain autoregulatory mechanisms.

mechanisms acting at the level of single neurons

Within a single neuron, prolonged depolarizations with sustained actionpotential firing may be triggered by a brief depolarizing pulse, as in the paroxysmal depolarizing shift, or may be the result of sustained excitatory synaptic input from neighbouring neurons engaged in seizure activity (Ayala, 1983). Intrinsic mechanisms of seizure termination active in a single neuron, discussed below, include: the potassium currents activated by calcium and sodium entry; the loss of ionic gradients, particularly of potassium, leading first to depolarization with increased firing, followed by depolarization blockade of membrane firing and cessation of firing; and possibly the depletion of energy substrates locally, with the decline in adenosine triphosphate (ATP), resulting in cessation of neuronal firing.

Intracellular ion-activated potassium currents

The membrane after hyperpolarization that follows bursts of action potential discharge is the result, at least in part, of potassium currents activated by the entry of calcium and sodium. Increased calcium entry during the paroxysmal depolarizing shift, or as a result of the action of glutamate at the postsynaptic membrane, activates a calciumdependent membrane potassium conductance that allows potassium efflux, membrane hyperpolarization and cessation of firing (Alger and Nicoll, 1980; Timofeev et al., 2004). Like calcium, sodium entry may also activate a sodiumdependent potassium current that reduces neuronal excitability by hyperpolarizing the membrane and increasing shunt conductance (Schwindt et al., 1989).

Transmembrane ion gradients

The effect of extracellular potassium is multifaceted. Sustained potassium efflux increases extracellular potassium concentration, depolarizing the membrane and moving the intracellular voltage toward the threshold for sodium action potential firing. As extracellular potassium continues to accumulate, there is membrane depolarization and action potential firing increases. With further accumulation, the membrane potential becomes more depolarized than the firing threshold for sodiumaction potentials, sodium channels inactivate, and neuronal firing ceases. In vitro experiments by Bikson et al. (2003) illustrate these effects of extracellular potassium accumulation. Electrographic seizurelike activity triggered in hippocampal slices by exposure to lowcalcium artificial cerebrospinal fluid (aCSF) manifested as recurrent periods of population firing followed by periods of electrographic silence lasting 12–18 s. The termination of each electrographic discharge by a period of electrographic silence resulted from transient increases in extracellular potassium to plateaus of approximately 12 mM. The depolarized state was maintained by the elevation of extracellular potassium and by the presence of persistent sodium channels that did not inactivate. Depolarization blockadeterminating seizurelike discharges have also been observed in neocortical slices in which GABAergic inhibition is partially blocked by picrotoxin (Pinto et al., 2005). Focal or localized increases in potassium may also trigger additional potassium release beyond the initial region of potassium accumulation. Shifts in extracellular potential, and oscillations seen at the end of hippocampal afterdischarges, have been attributed to a rapid rise in extracellular potassium that triggers waves of astrocyte depolarization and a propagating rise in potassium that terminates neuronal firing (Bragin et al., 1997). In addition to its direct depolarizing effects, increased extracellular potassium may also indirectly result in membrane depolarization through the action of the potassium–chloride cotransporter KCC2. The rise in extracellular potassium can increase intracellular chloride, shifting the chloride reversal potential toward membrane depolarization. In the setting of increased intracellular chloride, the action of GABA to open chloride channels could enhance membrane depolarization to the point of becoming refractory to further firing of action potentials (Jin et al., 2005; Galanopoulou, 2007).

Extracellular calcium levels also change markedly during paroxysmal neuronal firing and may affect the efficiency of neurontoneuron spread of activity. Focal seizure activity results in a decline in extracellular calcium activity of approximately 50% (Heinemann et al., 1977). This decline may inhibit synaptic transmission because synaptic vesicle fusion and neurotransmitter release are dependent on entry of extracellular calcium (King et al., 2001; Cohen and Fields, 2004). Decline in extracellular calcium also potentially affects gap junction function as hemichannel opening increases in low calcium (Thimm et al., 2005).

Energy failure

Sustained neuronal activation also markedly increases energy, namely ATP, utilization to restore ion gradients across the membrane. In some neurons, the presence of an ATPgated potassium channel (KATP) reduces neuronal activity when ATP levels decline intracellularly (Yamada et al., 2001). When the ATP level falls because energy utilization outpaces energy production, potassium channels open and produce membrane hyperpolarization. Indeed, knockout mice lacking functioning KATP channels experience a myoclonic seizure on average 8.9 ± 1.1 s following onset of hypoxia, followed by generalized convulsions and death. A similar hypoxic challenge, however, does not trigger seizures in wildtype mice, indicating that KATP channels in vivo resist membrane depolarization during energy failure. Reduced levels of energy metabolites, such as glucose, may also affect seizure duration. In vitro recordings show that decreasing extracellular glucose terminates electrographic seizurelike activity in the low magnesium hippocampal slice (Kirchner et al., 2006). The effect of hypoglycaemia on seizurelike discharges in vitro was statistically significant, but not immediate. Fifty per cent fewer seizurelike discharges occurred in the 24min period following application of low glucose artificial cerebrospinal fluid compared to the frequency of discharges in the 30 min prior to application. Low glucose also reduced the amplitude of the seizurelike discharge by 25%. These effects on the frequency and amplitude of seizurelike discharges were reversed by restoration of normal glucose levels.

mechanisms acting on a local network of neurons

While seizure initiation is driven at least in part by the burstfiring properties of the individual neurons, the evolution and spread of the seizures also requires amplification and synchronization among neurons within susceptible networks. Seizure amplification occurs through the action of recurrent excitatory collaterals that form feedback loops, returning excitatory synaptic activity to the neurons within the seizure onset zone (Rutecki et al., 1989; Coulter and DeLorenzo, 1999). Seizure spread depends on the propagation and synchronization of the seizure discharge across synapses that separate neurons in the seizure onset zone from ‘normal’ neurons synaptically connected to the seizure onset zone (MacVicar and Dudek, 1980; Miles and Wong, 1983).

Glutamate depletion

Decrease in synaptic efficacy results in milder postsynaptic excitation, and consequently diminished amplification and spread of the seizure discharge. One mechanism limiting synaptic transmission during a sustained seizure discharge is the depletion of synaptic vesicles containing neurotransmitter. Staley et al. (1998) investigated the effects of synaptic depletion in vitro using a model of CA3 electrographic seizure discharges produced by hyperkalaemia. CA3 discharges consist of recurrent neuronal depolarizations with bursts of actionpotential firing separated by period of electrographic silence. Staley et al. found that the duration of the seizure burst was proportional to the duration of the silent period preceding the burst, consistent with the hypothesis that the seizure burst duration depended on the renewed availability of immediately releasable glutamate. If glutamatecontaining synaptic vesicles are replaced at a steady rate, longer interburst periods allow a greater resupply of immediately releasable glutamate, and an increased duration of the subsequent electrographic seizure discharge. Interburst intervals of 2–3 s or longer were necessary to achieve the longest burst durations (up to 420 ms). Thus, as the seizure discharge develops, it consumes the supply of readily releasable glutamate needed to sustain the seizure, potentially acting as a governor on excitatory drive. As the glutamate reservoir is replenished continuously, however, additional control mechanisms are necessary to prevent reinitiation of seizure activity.

The intra- and extracellular environments

Prolonged neuronal activity during seizure discharges may also have the effect of increasing CO2 or increasing the byproducts of anaerobic metabolism, and produce extracellular acidosis or intracellular acidosis associated with extracellular alkalinosis (Chesler and Kaila, 1992). Glial cells may also contribute to acidification of the extracellular space in response to increases in the extracellular potassium concentration (Chesler and Kraig, 1987). In the hippocampal slice in vitro, acidification of the extracellular space to pH 6.7 terminated seizurelike burst firing facilitated by lowmagnesium in the artificial CSF. The attenuation of epileptiform activity began within minutes of lowering pH (Velisek et al., 1994; Velisek, 1998). The mechanisms of action – at least in part – included decreased NMDA receptor function and loss of synaptic longterm potentiation (LTP). A milder reduction of pH to 7.1 also produced milder synaptic impairment with continued loss of LTP (Velisek, 1998). Inhibition of carbonic anhydrase, which alters extracellular pH, has some anticonvulsant benefit. In humans, the carbonic anhydrase inhibitor acetozolamide has a mild anticonvulsant effect (Thiry et al., 2007). Knockout mice deficient in carbonic anhydrase are severely acidotic and are resistant to seizures produced by flurothyl gas compared to wildtype mice (Velisek et al., 1993). Intracellular acidification may also contribute to termination of seizure discharges. Spontaneous interictal spiking following focal application of bicuculline in the piriform cortex in an in vitro whole brain preparation was associated with periodic abrupt alkanization of the extracellular space followed by a slow return to baseline pH (de Curtis et al., 1998). These observations were interpreted as evidence of intracellular acidification. Application of ammonium chloride in the perfusing medium to prevent intracellular acidification increased neuronal excitability and resulted in afterdischarges following each spike, and in seizurelike discharges. The investigators hypothesized that the intracellular acidification reduced excitability by reducing gapjunction function. Application of octanol, a nonspecific gap junction blocker, abolished spontaneous interictal spiking (de Curtis et al., 1998).

Glial buffering of glutamate

Glial uptake of perisynaptic glutamate is the major mechanism forestalling accumulation of glutamate at the synapse (Benarroch, 2005). Astrocytes have an equally important role in the regulation of extracellular potassium. Astrocytic buffering of potassium maintains extracellular levels below a ceiling of 12 mM (Benarroch, 2005). In some cases, such as the epileptic brain, glia may also release glutamate, thereby prolonging postsynaptic excitation. Tian et al. (2005) recently showed that glial release of glutamate contributed to the maintenance of the paroxysmal depolarizing shift that is the hallmark of ‘epileptic’ neurons. Failure of glia to buffer extracellular glutamate, let alone glutamate release from glia, can be expected to result in prolonged excitatory drive and seizure maintenance.

Increased GABA-ergic inhibition

A basic mechanism to control focal seizure activity is GABAergic synaptic inhibition mediated by local interneurons. Seizure discharges within the seizure onset zone produce recurrent inhibition within the seizure initiation zone, thus reducing excitatory output (Kostopoulos et al., 1983; Dorn and Witte, 1995). Early investigations of the spike and wave components of ‘spikewave’ discharges showed that the spike component is associated with a burst of rapid actionpotential firing, while the wave component is associated with a pause in actionpotential firing (Dichter and Spencer, 1969). The pause in neuronal firing results from synaptic inhibition produced by local inhibitory interneurons activated by the volley of excitatory activity comprising the ‘spike’ component, an example of feedback inhibition. Feedforward inhibition is a fundamental feature of cortical processing (Swadlow, 2003). Feedforward inhibition may also play an important role; an interneuron activated by a principal cell sends inhibitory signals to principal cells outside the focus, inhibiting the propagation of the seizure (Trevelyan et al., 2007). Recent evidence indicates that a principal cell axon may synapse on the presynaptic terminal of an inhibitory interneuron, bypassing somatic activation of the interneuron altogether by causing transmitter release directly from the inhibitory synaptic terminal (Connors and Cruikshank, 2007).

Synaptic inhibition is mediated by the presynaptic release of the neurotransmitter GABA, which acts on the postsynaptic neuron via receptors located on the soma, dendrites, or presynaptic terminals. GABA receptors are present in two major varieties, GABA_A and GABA_B. GABA_B receptors are metabotropic acting through Gprotein second messengers. The preand postsynaptic distribution of GABA_B receptors, along with mixed evidence of anti and proconvulsant effects of GABAB activation, makes it difficult to determine their role in seizure termination (Chen et al., 2004). GABAA receptors are chlorideconducting membrane channels that open rapidly in response to GABA. Desensitization of GABA_A receptors during status epilepticus likely contributes to the failure of seizure termination (Chen et al., 2007). Desensitization of GABA_A receptors is also the basis of the loss of efficacy of benzodiazepine medications used to treat status epilepticus. Multiple mechanisms appear to contribute to GABA_A receptor desensitization. Increased internalization of GABA_A receptors during status epilepticus reduces the effect of GABAergic stimulation (Goodkin et al., 2007). Changes in subunit composition may also contribute to GABA_A receptor desensitization, although this process acts over many minutes to hours, and appears to affect longterm neuronal excitability and epileptogenesis rather than seizure termination. Nonsynaptic GABAA receptors, in contrast, do not desensitize and instead are capable of tonic inhibition, which produces longlasting changes in neuronal reactivity. These tonic GABA receptors typically contain particular subunits – delta and possibly gamma – that alter the properties of the receptors (Richerson, 2004). Tonic receptors are activated by micromolar levels of the extrasynaptic GABA, which arrives either by diffusion from a synapse before reuptake, or by release into the extracellular space via nonsynaptic mechanisms. Tonic GABA receptors may play an important role in epilepsy. On the one hand, reduction of tonic GABA currents (as produced experimentally by a mutation in the deltasubunit of tonic GABA receptors) is associated with generalized epilepsy. On the other hand, progesteronederived neurosteroids enhance tonic GABA currents, and may play a role in preventing seizure genesis, and potentially in terminating ongoing seizures (Stell et al., 2003). It is also clear that the contribution of extrasynaptic GABA_A receptors changes during maturation, and may contribute to changes in seizure susceptibility during development.

Introduction

Epilepsy is the most common chronic neurological condition in people with an estimated incidence of 0.05–0.1% and prevalence of 0.4–1% (Sander and Shorvon, 1996; Cowan, 2002). Despite treatment with two adequate antiepileptic drugs (AEDs), 23% of human patients continue to have seizures (Picot et al., 2008). Epilepsy has also been suggested in dogs to be the most common chronic neurological disorder (Chandler, 2006; Fluehmann et al., 2006) with an estimated prevalence of 1–2% in a referral hospital population (SchwartzPorsche, 1986) and 0.6% in first opinion practice (KearsleyFleet et al., 2013). Around 75–85% of dogs with idiopathic epilepsy will continue to have seizures (Heynold et al., 1997; Berendt et al., 2002, 2007; Arrol et al., 2012) and around 20–30% will remain poorly controlled (<50% reduction of seizure frequency) despite adequate treatment with phenobarbitone (PB) and/or potassium bromide (KBr) (SchwartzPorsche et al., 1985; Podell and Fenner, 1993; Trepanier et al., 1998). Thus, with an estimated current UK companion dog population of

9.4 million, around 55,000 dogs have idiopathic epilepsy of which 12500 will be classified as poorly controlled. Dogs with epilepsy, especially poorly controlled epilepsy with a high seizure frequency, have an increased risk of premature death, behaviour changes and a reduced quality of life (Chang et al., 2006; Berendt et al., 2007; Shihab et al., 2011; Wessmann et al., 2012). Seizures do not only affect the quality of life for the affected dogs, but also for the pet owner (Chang et al., 2006; Wessmann et al., 2012). Improving our understanding of why some patients respond to treatment while others do not is therefore of key importance.

Epilepsy is caused by a heterogeneous group of chronic conditions with seizures being its clinical manifestation. Despite the brain’s complex structure, there are only limited ways in which it can demonstrate its function and dysfunction. A wide variety of disturbances of brain structure and function can result in seizures and epilepsy. However, similar pathophysiological pathways can lead to other neurodevelopmental and neurobehavioural disorders (Johnson and Shorvon, 2011; Shihab et al., 2011). Many of these disturbances share commonalities at various levels, which can help to understand, diagnose, monitor and treat these disorders.

Investigations into why treatment could fail can therefore:

• Shed light on the mechanisms causing a refractory state to drug therapy and how to overcome them; and

• Deepen our understanding of the pathophysiology of epilepsy and its natural clinical course.

Definition

The definition of pharmacoresistant epilepsy has been a matter of debate in human medicine. Formerly, epilepsy was considered responsive to medical treatment if seizures were reduced by ³50% to one or two adequate AED which achieved therapeutic serum concentration and steady state (Regesta and Tanganelli, 1999). Most current epilepsy drug trials in veterinary medicine have used this definition to determine AED efficacy (Dewey et al., 2004, 2009; Platt et al., 2006; von Klopmann et al., 2007; Volk et al., 2008; Muñana et al., 2010, 2012b). However, this does not take into account that even a reduction of seizure frequency by more than 50% may still entail a high seizure frequency, which negatively impacts on the animal’s and the owner’s quality of life (Chang et al., 2006; Wessmann et al., 2012). A good quality of life is however best improved by seizure freedom, one of the main outcome measures nowadays in epilepsy trials in human medicine (Elsharkawy et al., 2009).

A task force of the International League against Epilepsy (ILAE) has recently agreed on a global consensus on granted outcome measures for therapeutic interventions (Level 1) and definition of pharmacoresistant epilepsy (Level 2) taking the occurrence of druginduced side effects and seizure control into account (Kwan et al., 2010):

• Level 1 categorizes the outcome of each medical treatment as either free of seizures or treatment failure, if not seizure free. The ILAE recommends a followup time of three times the longest interictal period for therapeutic trials; e.g. if the period in between seizures is 1 month then the followup time should be at least 3 months. However, as this is not always practical, seizure freedom is usually defined as an absence of seizures for a 12month period. Level 1 does not only consider efficacy, but also the occurrence of druginduced side effects, which will contribute significantly to the overall impact on the patient’s quality of life.

• Level 2 defines pharmacoresistant epilepsy as a failure to achieve freedom from seizures despite adequate trials of two (or more) welltolerated, correctly chosen and used AED regimens (whether administered as monotherapies or in combination) (Kwan et al., 2010). The number of AED trialled has been restricted to ‘two’ based on the observation that successful seizure control is very unlikely once the patient has not responded to two correctly chosen AED (Kwan and Brodie, 2000; Arts et al., 2004).

It needs to be considered that drug nonresponsiveness is not a fixed state but rather a dynamic process, which can be altered by fluctuation of the underlying pathophysiology (Kwan et al., 2010). The seizure frequency can vary over time (Berg et al., 2009; Muñana et al., 2010) and the state of drug nonresponsiveness is dependent on the timepoint of assessment (Kwan et al., 2010).

Taking into consideration the revised ILAE classification of pharmacoresistant epilepsy, a high proportion of dogs would be classified as pharmacoresistant. Spontaneous and druginduced epilepsy remission rate in people is around 63% (Kwan and Brodie, 2000), which is markedly higher than that reported in veterinary medicine, which usually ranges between 15 and 24% (Heynold et al., 1997; Berendt et al., 2007; Arrol et al., 2012). However, in a recent study comparing the efficacy and tolerability of phenobarbitone with potassium bromide as a first line treatment, seizures ceased in 85% and 52% of the treated dogs, respectively (Boothe et al., 2012). The study period was only 6 months long and thus it is likely that the percentage of dogs being seizure free would have been less with a longer followup period. Lagotto Romagnolo dogs have a documented ‘childhood’ or juvenile epilepsy, which starts when they are around 6 weeks of age (Jokinen et al., 2007). Of the affected dogs, 96% become seizure free at the age of 10 weeks generally without receiving AED treatment. Future, longterm epidemiological studies are necessary to determine prevalence of drug response more accurately in the general epileptic dog population, in addition to the effect of patient age and duration of seizure history on this response.

Risk Factors for Pharmacoresistant Epilepsy

Genetic risk factors

An increased prevalence of epilepsy has been described for many dog breeds (Jaggy et al., 1998; Kathmann et al., 1999; Berendt et al., 2002, 200; Casal et al., 2006; Gullov et al., 2011), which raises the possibility of genetic risk factors also being responsible for drugresponsiveness. The Lagotto Romagnolo, as aforementioned, has a benign childhood or juvenile epilepsy, which goes into remission in many affected dogs with age, as the function of the Lgi2 gene in cerebral synaptic remodelling becomes less important (Jokinen et al., 2007; Seppala et al., 2011). The Lgi2 gene is involved in the immediate postnatal phase of neuronal pruning, but then the Lgi1 gene has a more important role in the regulation of the later part of pruning.

Another gene mutation that has been associated with improved seizure control is located on the ABCB1 gene. The ABCB1 (multidrug resistance (MDR) 1) gene encodes a transmembrane protein, the permeabilityglycoprotein (Pgp). Pgp, an ATPdependent multidrug transporter, expressed at the blood–brain barrier (BBB), protects the brain from potential central nervous system (CNS) toxins (Schinkel et al., 1994, 1996; Schrickx and FinkGremmels, 2008), including AEDs such as PB (Potschka et al., 2002; Basic et al., 2008). Dysfunction of this neuroprotective BBB efflux pump can lead to BBB dysfunction and consequently CNS intoxication (Schinkel et al., 1994, 1996). Collies and other herding breeds can be affected by such a dysfunction of the Pgp transporter caused by a four basepair deletion (c.296_ 299del) in exon 4 of the ABCB1 gene (Mealey et al., 2001). A homozygous deletion in this region cannot only lead to CNS intoxication by drugs such as ivermectin, but also has been suggested to improve medical seizure control in affected collies (Muñana et al., 2012a).

Border collies are, however, known to have an aggressive seizure phenotype characterized by high seizure frequency, cluster seizures and poor drug responsiveness. In a recent study, 71% of Border collies were classified as pharmacoresistant (Hülsmeyer et al., 2010). PBresistant epileptic Border collies had a single nucleotide polymorphism (SNP) at intron 1 near the 5end of the ABCB1 gene, which could influence the promoter elements of this gene (Alves et al., 2011). A polymorphism at the promoter region could influence transcription activity and therefore might increase the level of Pgp expression at the BBB. The same group looked at another breed, the Australian shepherd dog, which is closely related to the Border collie and also has a severe epilepsy phenotype (Weissl et al., 2012). In this dog breed, they were not able to identify a polymorphism related to drugrefractoriness.

Another recent study looked at gene differences between PB responders and nonresponders (Kennerly et al., 2009). Five genes were suggested to have an association with PB response, although they did not reach statistical significance after adjustment for multiple comparisons (KCNQ3, SCN2A, GABRA2, EPOX HYD and ABCB4). Three of these genes encode ion channels (KCNQ3, voltage gated potassium ion channel important for postexcitatory membrane repolarization; SCN2A, sodium ion channel; GABRA2, GABA receptor), all of them are AED targets (Armijo et al., 2005). The other two genes are involved in PB metabolism (EPOX HYD) or transportation (ABCB4) (Kennerly et al., 2009).

Clinical risk factors

Apart from identifying genetic backgrounds, which can be associated with poor response to AEDs, much attention has been spent in the last decade to identify clinical risk factors that predict pharmacoresistant epilepsy. In people, it was commonly believed that seizure freedom was more likely to be achieved when a patient received AEDs immediately

after the occurrence of the first seizure. Interestingly, epidemiological studies in developing countries, where AEDs are not freely available, revealed remission rates similar to those in industrial countries (Placencia et al., 1993). Most AEDs despite having good seizure suppressing activities have little influence on the natural course of the disease (epilepsy) itself. New AEDs that were thought to also have an antiepileptogenic effect have failed in chronic epilepsy models and have not prevented the development of epilepsy (Brandt et al., 2007).

Heynold and colleagues have shown in 1997 that Labrador retrievers, which achieved freedom from seizures, received medication a longer period of time after their first seizure than those dogs that continued to seizure (Heynold et al., 1997). This implies that timing in relation to onset of seizure activity does not influence longterm seizure control in the canine patient. However, there is enough evidence in dogs, rodent models and humans that disease severity, such as high seizure frequency, cluster seizures and status epilepticus can influence drug responsiveness (Heynold et al., 1997; Kwan and Brodie, 2000; Hülsmeyer et al., 2010; Löscher and Brandt, 2010; Weissl et al., 2012). Intact male and female dogs have a higher likelihood of having cluster seizures (Monteiro et al., 2012). Seizure severity will ultimately guide clinical reasoning and the more severely affected patient will receive treatment earlier than dogs with a less severe epilepsy phenotype. It was formerly believed that very young dogs starting to have recurrent seizures have a worse outcome. As an example, Labrador retrievers had a better outcome when they developed epilepsy later in life (Heynold et al., 1997). However, a recent study from the UK could not find that the onset of seizures before the age of 1 year had any influence on survival outcome (Arrol et al., 2012). Interestingly, the same group could not find that the presence of focal seizures influenced the overall outcome, which is different to what is seen in human medicine (Kwan et al., 2011).

Pseudoresistance

Once a clinician encounters pharmacoresistant epilepsy, the first question to be asked should be about the possibility of an identifiable, underlying disease process. If seizures persist because the underlying disease has not been identified and treated incorrectly, it is termed pseudoresistance. Conditions mimicking epilepsy (see Chapter 9) need to be also considered; these include cardiac associated syncope, transient vestibular disorders, movement disorders and episodic pain. Sometimes AED treatment can aggravate such mimics (Penning et al., 2009) and so each patient presenting with assumed pharmacoresistant epilepsy needs to be thoroughly investigated with an open mind. One may also encounter failure of AED therapy if owner compliance is poor or pharmacokinetic and dynamic properties were not considered adequately.

Mechanisms of Pharmacoresistant Epilepsy

There are many possible causes of pharmacoresistant epilepsy. The mechanisms of drug resistance are likely to be variable and multifactorial (Regesta and Tanganelli, 1999; Kwan et al., 2011). Genetic factors, such as the aforementioned polymorphisms, may be relevant and help to explain why two dogs of the same breed and with the same epilepsy characteristics differ in their response to AED therapy. Diseaserelated factors are also of importance, and these include the aetiology and pathophysiology underlying the seizure disorder, the clinical course of the disease, changes in drug targets or binding sites, drug uptake into the brain and changes of the epilepsy circuit. Finally, drugrelated factors such as ineffective pharmacodynamics or kinetic properties and/or development of tolerance can all play a role in the formation of pharmacoresistance.

Most pharmacoresistant patients will often not respond to multiple AEDs despite the individual differences in their mechanisms of action (Regesta and Tanganelli, 1999; Löscher and Potschka, 2002). Patients that do not respond to the first standard AED will only have a 3% chance to respond to the subsequent chosen AEDs, which needs especially to be considered during AED trials (Kwan and Brodie, 2000). This argues against epilepsyinduced changes in specific drug targets as a major cause of drugresistant epilepsy, and makes it more likely that nonspecific mechanisms are responsible.

There are three proposed major theories for AED resistance:

1. The drug-target hypothesis: reduced drugtarget sensitivity in epileptogenic brain tissue.
2. The multidrug transporter hypothesis: clearance of antiepileptic drugs from the epileptogenic tissue through overexpression of multidrug transporters.
3. Change in the neuronal network properties.

Drug-target hypothesis

Based on the drug target hypothesis, reduced sensitivity of drug targets such as receptors or ion channels to AEDs is a key cellular mechanism that may cause drug resistance. This hypothesis is based on findings that show that in the hippocampus of human patients with pharmacoresistant temporal lobe epilepsy, the usedependent inhibition of sodium channels by carbamazepine is lost. This finding did not extend to lamotrigine, which has a pharmacologic action similar to that of carbamazepine (Remy et al., 2003). Polymorphisms in the sodium channel encoding gene SCN2A were found in humans to be responsible for AEDs acting on the sodium channel, but also for other nonsodium channel targeted AEDs (Kwan et al., 2008). As aforementioned, Kennerly et al. (2009) showed that PB nonresponders also had changes in the SCN2A gene when compared to PB responders. In addition, two other genes encoding ion channels (KCNQ3 and GABRA2) were affected (Armijo et al., 2005). In a rodent model for pharmacoresistant epilepsy it was demonstrated that there was a shift from GABA_A diazepamsensitive to GABAA diazepaminsensitive receptors in the hippocampus of PB nonresponders (Volk et al., 2006). In the same model a significant loss of neurons in the CA1, CA3c/CA4 and dentate hilus of nonresponders was found. This could lead to altered network properties, which could be responsible for refractoriness. In human medicine, altered network properties are well recognized in patients with hippocampal sclerosis. Hippocampal sclerosis has been associated with refractoriness to AEDs (Kwan et al., 2011). In dogs, the hippocampus is also involved in seizure propagation, which can result in MRI changes (Kuwabara et al., 2010). Such MRI abnormalities have not been associated with changes in seizure frequency or length in dogs but hippocampal changes visible on MRI are often associated with AED therapy failure in cats (Fatzer et al., 2000; Brini et al., 2004; Schmied et al., 2008).

A wealth of literature on human epilepsy cites evidence of autoantibodies to ion channels (GABA_B receptors, Lancaster et al., 2010; NMDAreceptors, Dalmau et al., 2008; calcium channels, McKnight et al., 2005; voltagegated potassium channels, McKnight et al., 2005). Interestingly, it also appears that cats with hippocampal changes develop autoantibodies to voltagegated potassium channels (Pakozdy et al., 2013). These patients rarely respond to standard AED and in human medicine immunomodulatory treatment has been trialled with variable results (Vincent et al., 2010). Other proposed cellular pathomechanisms of pharmacoresistant epilepsy that have been suggested include electrical coupling via gap junctions in neurons and glial cells (Voss et al., 2009) and mitochondrial oxidative stress and dysfunction (Waldbaum and Patel, 2010).

Multidrug transporter hypothesis

A drug can only enter the brain via two routes, either traversing the BBB or via the ventricular system and cerebrospinal fluid (CSF). The BBB restricts the entrance of any drug or other xenobiotic substance in order to protect the CNS from toxicity. The endothelial cells in the BBB are connected by tight junctions and surrounded by a basement membrane with astrocytic foot processes covering 95% of the endothelial lining. The BBB lacks transendothelial pathways such as transcellular channels or fenestrations (Löscher and Potschka, 2002). The functional significance is that the BBB resembles continuous phospholipid membranes, which stops the diffusion of hydrophilic, large or proteinbound drugs.

Lipidsoluble drugs were thought on the other hand to diffuse easily through the BBB. Apart from the passive transport mechanism of lipophilic compounds, the BBB hosts a carriermediated transport system (Pardridge, 1999). In the last decade, multiple multidrug transporters of the ATPbinding cassette superfamily, especially Pgp and multidrug resistanceassociated protein (MRP), have been shown to be expressed physiologically on the luminal side of the endothelial cells of the BBB. Pgp and MRP are also expressed in the choroid plexus epithelial cells limiting brain entrance even further (Rao et al., 1999). These transporters appear to act as an active defence mechanism against the penetration of potential CNS toxic lipophilic compounds, therefore limiting the penetration of lipophilic drugs, such as AEDs (Fromm, 2000; Spector, 2000). AEDs which are transported by multidrug transporters include valproate, gabapentin, topiramate, phenytoin, carbamazepine, phenobarbitone, felbamate and lamotrigine (Löscher and Potschka, 2002).

In contrast to the aforementioned hypotheses of drugrefractoriness, the multidrug transporter hypothesis is based on the assumption that it is not the brain target itself but the reduced AED concentration at the target site that causes pharmacoresistance. Indeed, an increased expression of multidrug transporters, such as Pgp and MRP in brain capillary endothelial cells comprising the BBB has been demonstrated both in epileptogenic brain tissue of human pharmacoresistant patients and in animal models of pharmacoresistant epilepsy (Tishler et al., 1995; Sisodiya et al., 2002; Aronica et al., 2003; Potschka et al., 2004; Volk et al., 2004a, b; Volk and Löscher, 2005; Hoffmann et al., 2006). In pharmacoresistant epileptic tissue, Pgp is also overexpressed in astrocytic foot processes and neurons limiting the AED concentration at the target even further (Aronica et al., 2003; Volk et al., 2004a). Overexpression of the multidrug transporter Pgp was demonstrated in dogs after increased seizure activity, status epilepticus and cluster seizures (Pekcec et al., 2009b). This is an interesting finding as seizure frequency and severity are, as aforementioned, clinical risk factors for pharmacoresistant epilepsy.

More than 50 SNPs and insertion/deletion polymorphisms have been reported for ABCB1, with some of them resulting in a change of Pgp expression and/or function (Löscher and Potschka, 2005). An aforementioned study has also described a polymorphism in the promoter region of ABCB1 in Border collies associated with poor controlled epilepsy (Alves et al., 2011). This polymorphism could also have resulted in a change of function or overexpression of Pgp. However, no functional study was performed. The authors also found three other SNPs in the coding region of ABCB1, which were not associated with drug response or epilepsy. Apart from genetic influences, druginduced mechanisms will interfere with multidrug transporter expression at the BBB. The BBB efflux multidrug transporter expression adapts continuously to ensure protection and detoxification of the CNS from xenobiotic substances. Efflux transporter expression is regulated by pregnane X receptor (PXR), which reacts to xenobiotic (foreign toxic) compound exposure (Masuyama et al., 2005; Miller, 2010; Shukla et al., 2011). This results in a clearing of these compounds from the brain and/or body. In addition to the upregulation of multidrug transporter expression, PXR coregulates drug metabolizing CytP450 enzymes (Potschka, 2012). The binding domain of PXR and Pgp have many similarities and will interact with similar compounds, resulting in a dynamic process of regulating the efflux of xenobiotic compounds. AED have been reported to cause an upregulation of Pgp expression via PXR activation. However, a clear interaction of PXR with standard AEDs remains contentious (Potschka, 2012). It appears that the main driving force for Pgp overexpression is seizure activity (Potschka, 2010). Overexpression is transient and includes brain regions involved in seizure initiation and propagation (Kwan et al., 2002). A high seizure frequency therefore results in an accumulation of efflux transporters at the BBB.

Each epileptic seizure results in a glutamate release, which activates an intracellular signalling cascade in BBB endothelial cells (Bankstahl et al., 2008; Bauer et al., 2008). The glutamate binds to endothelial NmethylDaspartate (NMDA) receptors, which starts arachidonic acid signalling. Cyclooxygenase2 (COX2) processes the arachidonic acid and produces prostaglandin E2 (PGE2). PGE2 binds on the prostaglandin E receptor (EP1) resulting in Pgp expression (Pekcec et al., 2009a).

Should Pgp overexpression be one of the major reasons for drug refractoriness, blocking or reducing the expression of Pgp could reverse the lack of drug response. Several case reports in human medicine and studies in animal models of refractory epilepsy have shown an improved seizure control when Pgp inhibitors were used (Brandt et al., 2006; Potschka, 2012). However, a recent study performed in dogs using verapamil as a Pgp inhibitor did not show a significant reduction of the seizure frequency. Verapamil is not a very specific Pgp inhibitor, which could explain the negative results. Furthermore, verapamil dosage was limited due to its cardiovascular side effects. The other problem with using a Pgp inhibitor is the lack of specificity for the BBB, such that the rest of the excretory body system will be limited in function, therefore the longterm safety of this treatment approach needs to be questioned. A more promising route might be the use of COX2 inhibitors as they have been shown to decrease Pgp in rodent epilepsy studies that also resulted in a reversal of the drug refractoriness (Potschka, 2012).

Conclusion

In conclusion, our understanding of pharmacoresistance has grown significantly over the last several years. Focusing on seizure freedom and quality of life will change the approach in veterinary medicine. In conjunction with a better understanding of mechanisms involved in drug refractoriness a new era of treatment development will have to evolve, hopefully improving animals’ welfare.

A seizure (or ictus) has been defined as ’a transient occurrence of signs due to abnormal excessive or synchronous neuronal activity in the brain’ (Fisher et al., 2005). The clinical manifestations of a seizure are sudden and transient and depend on location of onset in the brain, patterns of propagation and a variety of other factors (see Chapter 1). Seizures can affect one or more of the following functions: sensory, motor, and autonomic activity, consciousness, emotional state, memory, cognition or behaviour (Fisher et al., 2005). The seizure (or ictus) may be preceded by a prodrome (or prodromal phase), which can be characterized by anxiety, restlessness, increased affection, withdrawal, aggressiveness, or vocalization, and by an aura, which is the initial manifestation of a seizure. The prodrome can occur hours to days before the seizure, the aura generally lasts seconds. The aura has been described in people as a subjective sensation, such as dizziness, tingling, and anxiety at the start of a seizure. In animals it may manifest as increased or decreased attention seeking, stereotypical sensory or motor behaviour (e.g. licking, pacing) or autonomic manifestations (e.g. salivating, vomiting, urinating). The ictus is the seizure itself and, in most cases, it lasts only a few minutes. The post-ictal period occurs soon after the seizure (or ictus) and may last seconds to days. Clinical manifestations include disorientation, aggressive behaviour, restlessness, pacing, lethargy, deep sleep, hunger, thirst, defecation, urination, ataxia, proprioceptive deficits and decreased or absent menace response with or without actual blindness.

Epilepsy has been defined as an enduring disorder of the brain that is characterized by recurrent seizures (Blume et al., 2001; Fisher et al., 2005). As there are many causes of chronic recurrent seizures, epilepsy is not a specific disease but rather a group of heterogeneous conditions. However, not all seizures are associated with epilepsy. For instance, a seizure can be the reaction of a normal brain to a transient insult, such as intoxication or metabolic disorder. If seizures no longer occur when the metabolic or toxic disorder resolves, the patient is not considered to have epilepsy.

A classification of seizures and epilepsies is important as clinical manifestations and aetiologies of seizures vary considerably. A standardized and uniform classification of seizure and epilepsy would allow consistency in the use of diagnostic terms, improve communication among clinicians and methods of evaluating treatment, and facilitate comparison of clinical cases and scientific studies.

Classification of seizures and epilepsies in veterinary medicine is largely based on its human counterpart and focuses on seizure phenomenology and aetiology (Schwartz-Porsche,

(L. De Risio and S. Platt)

1994; Podell et al., 1995; Berendt and Gram, 1999; Licht et al., 2002; Podell, 2004). The main limitations of the veterinary classification are that: (i) recognition of seizure occurrence and clinical manifestations is largely dependent on the pet-owner’s observation; (ii) electroencephalographic (EEG) data are usually not available; and (iii) no agreement has been reached for a standardized terminology in veterinary medicine. Therefore, the veterinary literature on this subject is often confusing with regard to definitions and interpretations. In addition, as in human medicine, seizure classification is an ongoing process and therefore updating is necessary.

This chapter will initially present the definitions and classifications of seizure and epilepsy in human medicine that are relevant in order to understand how terminology and classification have been developed and could evolve in veterinary medicine. Subsequently the focus will be on proposed veterinary terminology and classification.

Classification of Seizures and Epilepsies in Human Medicine

In human medicine, the International League Against Epilepsy (ILAE) has appointed a task force (Commission on Classification and Terminology) for the ongoing process of seizure and epilepsy classification and terminology. Classification systems have been developed based on ictal phenomenology, associated EEG findings and on aetiology. The Commission on Classification and Terminology of the ILAE first published its classification in 1969, which has subsequently been updated in 1981 for seizures (Commission, 1981) and in 1989 for epilepsies (Commission, 1989). Advances in molecular genetics, structural and functional neuroimaging, and neurophysiologic techniques have greatly improved the knowledge and understanding of seizures and epilepsies in humans, creating the need for a more modern system. Attempts have been made to update the 1989 and 1981 classification in 2001 and 2006 (Engel, 2001, 2006), and the most recent proposal for a revised classification has been published in 2010 (Berg et al., 2010) (Table 3.1).

Interestingly, epilepsies of unknown cause account for one-third or more of all epilepsies in humans. It is likely that several epilepsies classified as idiopathic in veterinary medicine (particularly in cats) would be more appropriately classified as unknown (or of unknown aetiology despite extensive investigations) based on the most recent ILAE classification. Future research in neuroimaging and genetics should help to further characterize and better classify these types of epilepsy (Berg et al., 2010).

In human medicine, epilepsies have also been classified as electroclinical syndromes (Berg et al., 2010). An electroclinical syndrome has been defined as a complex of clinical features, signs and symptoms that together define a distinctive, recognizable clinical disorder (Berg et al., 2010). Electroclinical syndromes are distinctive disorders identifiable on the basis of a typical age onset, specific seizure types, EEG characteristics and other factors (such as patterns of seizure occurrence with respect to sleep, provoking or triggering factors), which, when taken together, permit a specific diagnosis. The diagnosis of an electroclinical syndrome often has implications for management, treatment and prognosis (Berg et al., 2010). One of the most distinctive and clinically relevant classifications of the currently recognized electroclinical syndromes in humans is based on typical age at onset (Berg et al., 2010).

The subdivision of generalized seizures into tonic-clonic, tonic, clonic, myoclonic, atonic and absence seizures is overall unchanged since 1981. The 1981 subclassification of absence seizures has been simplified and altered. Absence seizures are characterized by a brief impairment of consciousness and may be associated with mild clonic, tonic, atonic and autonomic manifestations. Absence seizures are subclassified as typical, atypical and with special features (myoclonic absence and eyelid myoclonia) in the latest classification (Berg et al., 2010). Typical absence seizures occur in young patients with idiopathic generalized epilepsies and are usually associated with generalized 3–4 Hz spike-and-slow-wave complexes on EEG. Atypical absence seizures occur in patients with cryptogenic or structural generalized epilepsies and are often associated with slow spike-wave complexes of 1.5–2.5 Hz. Motor manifestations are more pronounced

Generalized seizures are those in which the first clinical and electroencephalographic changes indicate initial involvement of both cerebral hemispheres.

Focal seizures (previously termed as partial) are those in which the first clinical and electroencephalographic changes indicate initial activation of a system of neurons limited to a part of one cerebral hemisphere.

Idiopathic epilepsy: there is no underlying cause other than a possible hereditary predisposition. Idiopathic epilepsies are defined by age-related onset, clinical and electroencephalographic characteristics, and a presumed genetic aetiology.

Structural epilepsy: this type of epilepsy is the consequence of a known or suspected disorder of the central nervous system.

Cryptogenic (probable structural) epilepsy: this refers to a disorder whose cause is hidden or occult. Cryptogenic epilepsies are presumed to be structural.

in patients with atypical absence seizures than in those with typical absence seizures.

The 1981 classification subdivided focal seizure types into: complex (with impairment of consciousness), simple (without impairment of consciousness) and secondarily generalized (when the initially focal ictal event progresses to involvement of the entire body resulting in a generalized seizure) (Commission, 1981). The most recently proposed classification (Berg et al., 2010) recommends to abandon this terminology (simple, complex, secondarily generalized) and to describe focal seizure semiology accurately, including impairment of consciousness/awareness, when recognized, and localization and progression of ictal events.

The most recently proposed classification of seizure and epilepsies (Berg et al., 2010) has been met with considerable dissatisfaction from several expert epileptologists and therefore the previous ILAE classifications are likely to continue to be used until a more widely accepted proposal has been developed (Panayiotopoulos, 2011, 2012). Among the criticisms to the ILAE 2010 proposal (Berg et al., 2010) the comments that are most relevant to the current veterinary classification involve:

• The need to maintain, although update, a specific classification of focal seizures rather than recommend to abandon the previous classification and use only a description of the clinical manifestations (Panayiotopoulos, 2011);
• The aetiologic classification of seizures and epilepsies should remain unchanged. The terms ‘idiopathic’, ‘symptomatic’ and ‘cryptogenic’ should not be abandoned,

although their correct definition should be reiterated. The term ‘genetic’ should be introduced, not to replace ‘idiopathic’ but to represent a new category in addition to the other three in a revised classification of epilepsies (Panayiotopoulos, 2012).

Classification of Seizures and Epilepsies in Veterinary Medicine

The following classification of canine and feline seizures (Tables 3.2 and 3.3) is largely based on the ILAE classifications (mainly Commission, 1981, 1989; Engel, 2001, 2006) and on selected veterinary literature (Schwartz-Porsche, 1994; Berendt and Gram, 1999; Licht et al., 2002; Berendt, 2004; Podell, 2004; Thomas, 2010). The parallelism with the ILAE 2010 proposed new terminology (Berg et al., 2010) is indicated in parentheses where appropriate in Table 3.2, in a specific column in Table 3.3 and also discussed in the text.

Veterinary classification of seizures based on clinical manifestations

Generalized-onset seizures

The initial clinical manifestations of generalized-onset seizures indicate more than minimal involvement of both cerebral hemispheres. The motor manifestations begin bilaterally and are often symmetrical. An alteration in consciousness frequently occurs at some stage during ictus. The term ‘primary generalized’ has sometimes been used in the veterinary literature to describe generalized-onset seizures and to differentiate them from secondarily generalized focal (partial) seizures. In this case the term primary does not refer to seizure aetiology, but to the fact that the initial clinical manifestations reflect involvement of both cerebral hemispheres simultaneously from the onset of the seizure.

generalised-onset tonic-clonic seizure. The most common type of generalized-onset seizure in dogs is the tonic-clonic seizure (formerly called grand mal seizure) (Berendt and Gram, 1999; Licht et al., 2002). A prodromal phase and aura are not always recognized by the owner. Their duration and clinical manifestations are variable. The prodromal phase can last hours to days and it is commonly characterized by restlessness, anxiety or reluctance to perform normal activities. The aura may manifest as increased or decreased attention seeking, stereotypical sensory or motor behaviour (e.g. licking, pacing) or autonomic manifestations (e.g. salivating, vomiting, urinating). The ictal phase of a tonic-clonic seizure is characterized by sustained contraction of all muscles resulting in rigid extension of the limbs and opisthotonos, usually lasting 10 to 60 s (tonic phase). The animal falls on its side and often loses consciousness (defined as attentiveness or responsiveness to the owner and other external stimuli). Breathing is often irregular or absent, and

cyanosis is common. The tonic phase is followed by the clonic phase, which is characterized by rhythmic muscular contractions resulting in uncoordinated, purposeless, jerking movements of the limbs and chewing movements. Automatisms such as running or paddling movements of the limbs commonly occur. The clonic phase may alternate with tonic activity. Autonomic manifestations, such as hypersalivation, urination, defecation and mydriasis are common in dogs with generalized-onset tonic-clonic seizures although not a constant feature. The ictus usually lasts 1 to 2 min. The post-ictal phase duration and clinical manifestations are variable. The dog may rest and rapidly return to normal activity or may show confusion, disorientation, aggressive behaviour, restlessness, pacing, lethargy, deep sleep, hunger, thirst, defecation, urination, ataxia, proprioceptive deficits, and decreased or absent menace response with or without actual blindness, which can persist for 24 h or longer. The post-ictal phase duration and severity of clinical manifestations may be unrelated to the duration and severity of the ictus.

Tonic-clonic seizures also have been reported as the most common type of generalized-onset seizures in cats (Schwartz-Porsche and Kaiser, 1989; Schriefl et al., 2008). During the pre-ictal phase cats may display aggressiveness (hissing and growling), vocalization (including growling and crying), restlessness (running around erratically), anxiety, hiding or increased affection (seeking refuge with the owner), or may act as fearful. The clinical manifestations of the ictus can be violent and sometimes result in self-inflicted trauma such as excoriations, contusions, avulsion of nails and biting of the tongue (Schwartz-Porsche and Kaiser, 1989; Quesnel et al., 1997). Cats may be propelled forward, up in the air and from side to side. Facial muscle twitching is often observed before or after the tonic phase of the seizure. Autonomic manifestations such as mydriasis, salivation, piloerection and urination and occasionally defecation can also occur (Quesnel et al., 1997). The ictal phase usually lasts 30 s to 2 min. The postictal phase is similar to dogs, lasting minutes to several hours or days and is characterized by restlessness, disorientation, aimless wandering, thirst and hunger. Both cats and dogs may sleep for a few hours after the termination of the seizure.

generalized-onset tonic seizures. Tonic seizures are characterized by sustained increase in muscle contraction without clonic motor activity. Impairment of consciousness and autonomic manifestations can occur. The pre- and postictal phases are similar to those described for generalized-onset tonic-clonic seizure.

generalized-onset clonic seizures. Clonic seizures are characterized by regularly repetitive, sudden, brief, involuntary contractions, which involve the same muscle groups, and are prolonged. Impairment of consciousness and autonomic manifestations can occur. The pre- and post-ictal phases are similar to those described for generalized-onset tonic-clonic seizure.

Tonic or clonic seizures alone have been reported uncommonly in dogs and cats (Schwartz-Porsche and Kaiser, 1989; Heynold et al., 1997; Licht et al., 2002, 2007).

generalized-onsetmyoclonic seizures. Myoclonic seizures are characterized by sudden, brief, involuntary, shock-like contractions that can be generalized or confined to individual muscles or muscle groups (e.g. face, trunk, one extremity).

This type of seizure has been reported in the miniature wire-haired dachshund, beagle and basset hound dogs in association with Lafora disease (Jian et al., 1990; Fitzmaurice et al., 2001; Gredal et al., 2003). The clinical presentation is characterized by repetitive, brief myoclonic jerking of the head, neck and thoracic limbs that are frequently strong enough to cause the animal to fall backward into a sitting or lying position (Davis et al., 1990; Fitzmaurice et al., 2001; Schoeman et al., 2002). Myoclonic seizures may occur spontaneously or in response to visual (including light), tactile or auditory stimuli (Fitzmaurice et al., 2001; Webb et al., 2009). Myoclonic seizures have been reported also in cats (Schwartz-Porsche, 1989). Not all myoclonic jerks are seizures, as they can result from other causes.

generalized-onset atonic seizures. Atonic seizures are characterized by sudden loss of postural

tone of the head, one limb, or of the entire body usually lasting 1, 2 or more seconds. Consciousness may be lost. This type of seizure has been reported in dogs (Podell, 2004) and needs to be differentiated from narcolepsy/cataplexy and syncope (see Table 9.1, Chapter 9). Generalized seizures characterized by collapse, loss of consciousness and minimal limb movements have been reported in a cat with structural epilepsy (Barnes et al., 2004) and may represent a form of atonic seizure activity.

generalized-onset absence seizures. Absence seizures (formerly called petit mal seizures) are characterized by a transient and brief impairment of consciousness associated with a characteristic EEG pattern (2.5- to 4-Hz spike-and-wave complexes) (see Fig. 11.12, Chapter 11). Absence seizures have been clinically described as a transient cessation of activity with staring, unresponsiveness and ‘blanking out’ episodes. Absence seizures with myoclonic features have been reported in an 8-month-old male Chihuahua that presented with recurrent episodes of head and nose twitching associated with intermittent hind-limb jerking and suspected staring for a few seconds (Poma et al., 2010). Video-EEG documented multiple staring episodes either alone or in association with head and/or nose myoclonic jerks associated with generalized bilaterally synchronous 4 Hz spike-and-wave complexes (Poma et al., 2010). Absence seizures may occur also in cats, but they have not been reported as such or supported by EEG findings. One feline study reported a ‘mild’ form of generalized seizure activity characterized by transient cessation of activity, impaired consciousness, and bilateral facial muscle twitching for a few to several seconds (Quesnel et al., 1997). These may have been a form of absence seizures in cats. Unless absence seizures are frequent, associated with some motor manifestations or the pet-owner is very observant, they go unrecognized. Video-EEG monitoring is very helpful in the diagnosis of this type of seizure.

Focal-onset seizures

The clinical manifestations of focal-onset seizures (formerly called partial seizures) indicate initial abnormal neuronal activity localized in a particular area of a cerebral hemisphere (seizure focus). The newly proposed ILAE classification (Berg et al., 2010) introduces the concept of the seizure focus being discretely localized or more widely distributed within the affected hemisphere.

The clinical manifestations can vary considerably depending on the function of the affected cerebral area and include involuntary motor activity, autonomic signs, sensory abnormalities, alterations of consciousness and paroxysms of abnormal behaviour. These clinical manifestations may occur alone or in various combinations.

focal-onset motor seizures. Focal-onset motor seizures are characterized by involuntary, generally unilateral motor activity resulting in abnormal movements of a body part, such as turning the head to one side, flexion and/or extension of one limb, contraction of facial or masticatory muscles. Focal-onset motor seizures are presumed to arise from a seizure focus near a primary motor area in the frontal cortex contralateral to the observed involuntary motor activity.

focal-onset autonomic seizures. Focal-onset autonomic seizures result in one or more autonomic manifestation including mydriasis, hypersalivation, piloerection, lacrimation, urination, defecation, vomiting, diarrhoea and apparent abdominal pain (Breitschwerdt et al., 1979; Licht et al., 2002; Berendt et al., 2004). Phenobarbitone-responsive hypersalivation, dysphagia, salivary gland enlargement and oesophageal spasms have been reported in a few dogs and may be a form of focal autonomic seizure (Stonehewer et al., 2000; Gibbon et al., 2004).

focal-onset sensory seizures. Focal-onset sensory seizures result in abnormal sensations such as paraesthesia (numbness, tingling) limited to a defined somatosensory region of the body, or in visual hallucinations. Sensory seizures have been subclassified into somatosensory and special sensory (visual, auditory, olfactory, gustatory and vestibular) in humans (Commission, 1981). It is likely that the same sensory disturbances occur in animals, but they are difficult or impossible to identify and associate with concurrent EEG abnormalities. Therefore it can only be speculated that episodes characterized by chewing and/or licking into the air or at a specific region of the body, rubbing of the face, or biting at imaginary objects (‘fly biting’ or ‘fly catching’) are the manifestation of sensory seizures. Repetitive episodes of ‘fly biting’ could be the consequence of focal sensory seizures in the visual cortex, similar to focal visual sensory seizures that occur in humans (Cash and Blauch, 1979; Licht et al., 2002), could result from focal seizures with paroxysms of abnormal behaviour (Berendt et al., 2004), or may represent a form of compulsive behavioural disorder (see Chapter 9) (Rusbridge, 2005).

In analogy with the human classification published by the ILAE in 1981 and the veterinary medical literature, focal seizures have been referred to as complex and simple depending on whether or not consciousness is altered. However, in the context of the ILAE classification, consciousness was defined as ‘the degree of awareness and/or responsiveness of the patient to externally applied stimuli’. Responsiveness was defined as ‘the ability of the patient to carry out simple commands or willed movement’ and awareness referred ‘to the patient’s contact with events during the period in question and its recall’. These functions are difficult if not impossible to assess in veterinary patients, especially based on the pet-owner description or video documentation (Berendt et al., 2004). Therefore veterinary medicine can follow the most recent recommendation of the ILAE (Berg et al., 2010) to avoid using ictal impairment of consciousness to classify specific seizure types (e.g. simple and complex focal seizures) and to describe individual seizure phenomenology accurately including impairment of consciousness/awareness, when recognized.

Focal seizures can also occur as stereotyped paroxysms of abnormal behaviour including attention-seeking or avoidance/escaping behaviour, aimless wandering, restlessness and unprovoked aggression (Licht et al., 2002; Berendt et al., 2004). Paroxysmal abnormal behaviour may result from a manifestation of sensory seizures, involvement of the limbic system or higher cerebral activity (psychic seizures according to the 1981 ILAE classification). Episodes characterized by impaired consciousness (‘trance-like staring’), abnormal behaviour, including unprovoked aggression, extreme irrational fear, compulsive tail-chasing and fly catching, have been reported as focal seizures in eight bull terriers with interictal EEG abnormalities (multiple epileptiform spikes) and moderate to severe ventriculomegaly on computed tomography (Dodman et al., 1996).

The terms complex partial (or focal) seizures, psychomotor seizures, temporal lobe and limbic seizures or epilepsy have been used interchangeably in the veterinary literature to indicate focal seizures characterized by paroxysms of abnormal behaviour with or without some degree of impairment of consciousness.

Any type of focal-onset seizure can evolve into a generalized seizure (secondarily generalize).

The onset of ictus is characterized by clinical (usually motor) manifestations consistent with the location of the seizure focus and within seconds to minutes seizure activity spreads to involve both cerebral hemispheres resulting in bilaterally symmetrical motor disturbances (usually tonic-clonic), autonomic dysfunction and (commonly) altered consciousness. The focal onset may be subtle and the secondary generalization can occur so rapidly that the initial focal component is undetected and the seizure is misclassified as a generalized-onset seizure. Close observation is essential to recognize the focal-onset of the seizure before its secondary generalization. If the terminology recently proposed by the ILAE (Berg et al., 2010) is embraced also in veterinary medicine, the term ‘secondarily generalized’ should be abandoned and replaced by a description of localization and progression of ictal events.

Focal-onset seizures have also been reported in cats (Quesnel et al., 1997; Barnes et al., 2004; Schriefl et al., 2008; Pákozdy et al., 2010). As in dogs, clinical manifestations include motor, sensory and autonomic signs, alterations of consciousness, and paroxysms of abnormal behaviour, which can occur alone or in various combinations. Reported clinical manifestations include lack of response to

sensory stimuli, unilateral facial twitching (that can be limited to the ear, lip, or eyelids), turning the head to one side, repetitive movements of one or both limbs on one side of the body, mydriasis, hypersalivation, urination, abnormal behaviour suggestive of some form of hallucinations (unjustified hissing, growling, piloerection, attacking a real or imaginary object, unprovoked startle, running frantically, often blindly, into objects), self-chewing, biting and circling (Schwartz-Porsche and Kaiser, 1989; Quesnel et al., 1997). As in dogs, focal seizures have been reported to secondarily generalize in cats (Quesnel et al., 1997; Schriefl et al., 2008).

A peculiar type of feline focal seizures characterized by orofacial involvement, impaired consciousness, occurrence in clusters and frequent association with hippocampal pathology has been described (Pákozdy et al., 2011). Ictal signs include hypersalivation, facial twitching, lip smacking, chewing, licking, swallowing, mydriasis, motionless staring and vocalization. Secondary generalization occurs in the majority (12/15, 70%) of cats. The most common post-ictal and interictal signs are behavioural changes and aggression. Antibodies against voltage-gated potassium channel complexes may play a role in the pathogenesis of this type of feline focal seizure (Pakozdy et al., 2013).

In addition to a classification based on clinical manifestations, seizures have also been classified based on underlying aetiology (Table 3.3). The aetiology-based classification allows for a more specific differential diagnosis. Reported aetiologies of reactive and structural seizures in dogs and cats are described in detail in Chapters 4 and 5, respectively. Idiopathic epilepsy is presented in Chapter 6.

Veterinary classification of seizures and epilepsies based on underlying aetiology

Reactive seizures

Reactive seizures are the reaction of a normal brain to a systemic metabolic or nutritional disorder or exogenous toxin exposure (Podell et al., 1995). When the metabolic, nutritional or toxic disorder resolves, the animal does not have recurrent seizures, and therefore reactive seizures are not considered a form of epilepsy, per se (Jull et al., 2011). The authors feel that the term reactive seizure should continue to be used in veterinary medicine as the term ‘metabolic’ proposed by the ILAE 2010 may generate confusion by referring to metabolic disorders only leaving toxic and nutritional aetiologies poorly classified. In addition the term ‘metabolic’ has not been widely accepted by several expert epileptologists (Panayiotopoulos, 2012).

Animals with reactive seizures frequently have acute onset of bilateral symmetrical neurological deficits indicating diffuse forebrain or intracranial involvement and concurrent signs of dysfunction of other body systems. Occasionally, seizures are the only obvious clinical abnormality. Aetiologies of reactive seizures are listed in Box 3.1 and described in detail in Chapter 4.

Structural (symptomatic or secondary) epilepsy

The terms symptomatic epilepsy and secondary epilepsy have both been used to indicate recurrent seizures caused by a known and identifiable structural forebrain disorder such as vascular, inflammatory/infectious, traumatic, anomalous/developmental, neoplastic and degenerative diseases. The term structural epilepsy, introduced by the ILAE 2010 proposal, should be adopted also in veterinary medicine as its meaning indicates more clearly epilepsy resulting from structural forebrain disease. The term secondary is too generic and prone to misuse, indeed, it has sometimes been used in the veterinary literature to refer to seizures caused by structural forebrain disorders as well as metabolic or toxic disorders (Pákozdy et al., 2010) generating confusion when attempting study comparison. The term symptomatic is a truism as recurrent seizures (epilepsy) are the symptom in humans and the clinical sign in animals of an underlying disease.

Dogs and cats with structural epilepsy usually present with neurological signs (other than seizures) interictally. However, focal lesions in particular areas of the brain (‘clinically silent regions’), such as olfactory bulb and frontal lobes, can result in seizure activity

Box 3.1. Aetiologies of reactive seizures.

Metabolic

Nutritional

without any other neurological signs (Foster et al., 1988; Smith et al., 1989). Aetiologies of structural epilepsy are listed in Box 3.2 and described in detail in Chapter 5.

Classification of certain disorders may be open to debate. For example, organic acidurias such as L-2-hydroxyglutaric aciduria in Staffordshire bull terriers may be classified as structural epilepsy as they result in MRI and histological changes in the brain as well as metabolic disorders as they are caused by an error of cellular metabolism, or genetic epilepsy, as the underlying genetic mutation is known (Abramson et al., 2003; Penderis et al., 2007).

Box 3.2. Aetiologies of structural epilepsy.

Vascular

Viral

Bacterial

Rickettsial

Protozoal

Mycotic

Parasitic

Mycoplasmosis

Algal

Granulomatous meningoencephalomyelitis

Necrotizing meningoencephalitis

Hydrocephalus

Hydranencephaly

Porencephaly

Meningoencephalocele

Meningoencele

Exencephaly

Holoprosencephaly

Agenesis of the corpus callosum

Lissencephaly

Polymicrogyria

Cerebral neuronal heterotopias or dysplasias Neoplastic

Primary

Meningioma

Astrocytoma (glioblastoma multiforme)

Oligodendroglioma

Gliomatosis cerebri

Ependymoma

Choroid plexus tumours

Primary CNS lymphomas

Secondary

Haemangiosarcoma

Lymphoma

Pituitary Tumours

neuroesthesioblastoma)

Histiocytic sarcoma

Malignant melanoma

Others Degenerative

Lysosomal storage diseases

Organic acidurias

Continued

Box 3.2. Continued.

Probable symptomatic or cryptogenic epilepsy

Probable symptomatic or cryptogenic epilepsy refers to recurrent seizures caused by an underlying brain disease that is strongly suspected but cannot be identified despite extensive investigations (e.g. undetected hypoxic or vascular events, post-encephalitic changes and post-traumatic lesions that do not cause any detectable changes on brain imaging). Animals with cryptogenic epilepsy may or may not have behavioural and/or neurological abnormalities (other than seizures) interictally. The term ‘epilepsy of unknown aetiology’ has been proposed to replace the term cryptogenic epilepsy in humans (Berg et al., 2010) and although not widely accepted by expert epileptologists (Panayiotopoulos, 2012), it may be of value in veterinary medicine due to its more immediate meaning. However, in veterinary medicine, it would be important to specify when the cause of the seizures is unknown despite extensive diagnostic investigations to rule out toxic, metabolic, nutritional and structural brain disorders (see Chapter 10) rather than unknown because of minimal or no investigations.

Idiopathic or primary epilepsy

Idiopathic or primary epilepsy refers to recurrent seizures with no underlying cause other than a strongly suspected or confirmed genetic or familial basis. In this context, the term ‘idiopathic’ refers to a disorder ‘by itself’ not ‘of unknown cause’. The 1989 ILAE classification defined idiopathic epilepsies based on age at seizure onset, clinical and electroencephalographic characteristics, and a presumed genetic aetiology. The diagnosis of idiopathic epilepsy in dogs and cats is based on the age at onset (generally between 6 months and 6 years), normal interictal behaviour, physical and neurological examinations, and exclusion of metabolic, toxic and structural cerebral disorders by means of diagnostic investigations (see Chapter 6). An inherited basis, familial transmission, or a higher incidence of idiopathic epilepsy has been reported in several canine breeds (Table 6.1, Chapter 6). A genetic basis for recurrent seizures has been reported in a closed colony of laboratory cats (Kuwabara et al., 2010). The breed of these cats was not specified. The age at the time of the first seizure ranged between 4 and 12 months. General physical and neurological examinations and results of diagnostic investigations (including 1.5 Tesla MRI of the brain and CSF analysis) were all normal. All cats had focal-onset complex seizures followed by secondary generalization into tonic-clonic seizures. Based on pedigree analysis an autosomal recessive mode of inheritance was hypothesized. In the clinical setting, it is difficult to demonstrate a genetic or familial basis for recurrent seizures, particularly in cats. It is likely that several cats and some of the dogs reported to have idiopathic epilepsy in the veterinary literature actually would be better classified as having epilepsy of unknown aetiology based on the most recent ILAE proposal (Berg et al., 2010) as a genetic or familial basis was neither suspected nor confirmed in these individuals and the diagnostic investigations were not always complete. Applying the term genetic rather than idiopathic also in veterinary medicine, in analogy with the most recent ILEA proposal (Berg et al., 2010), would have the advantage of limiting its use to purebred dogs with a strongly suspected or proven genetic or familial basis for the recurrent seizures. However, this term may generate confusion as clinicians may tend to use it for purebred

dogs with the typical clinical features of idiopathic epilepsy and suspected genetic aetiology as well as dogs with known genetic mutations (e.g. Epm2b gene in miniature wirehaired dachshunds with autosomal recessive progressive myoclonic epilepsy (Lafora disease), or Lgi2 gene in Lagotto Romagnolo with benign familial juvenile epilepsy) resulting in particular types of epilepsy that do not meet the typical criteria for idiopathic epilepsy (Lohi et al., 2005; Seppala et al., 2011). Therefore it may be more appropriate to continue to use the term idiopathic to indicate dogs with clinical and diagnostic features typical for idiopathic epilepsy as well as a strongly suspected or proven genetic or familial basis for the recurrent seizures. The term genetic epilepsy could be introduced as an additional category to include disorders with known genetic mutation, as recently proposed in humans (Panayiotopoulos, 2012).

While it was originally thought that focal-onset seizures with or without secondary generalization would occur only in animals with structural brain diseases (symptomatic epilepsy), focal-onset seizures have been reported also in dogs and cats with idiopathic epilepsy (Patterson et al., 2003; Berendt et al., 2009; Kuwabara et al., 2010; Pákozdy et al., 2010). In addition, the same animal can be affected by different types of seizures (e.g. focal-onset with or without secondary generalization and generalized-onset seizures) (Quesnel et al., 1997; Licht et al., 2002; Pákozdy et al., 2010). Therefore the clinical manifestations of seizures should not be used to infer the aetiologic diagnosis.

Precipitated seizures

The majority of seizures appear to occur spontaneously, however sometimes seizures may be precipitated by a variety of environmental and internal factors. In human patients, sleep deprivation, emotional stress, menstruation, missed anti-epileptic medication and concurrent illness can result in so called ‘precipitated seizures’ (Commission, 1989). Emotional stress caused by changes in the daily routine (including moving to another place or travelling), unexpected noise, sudden awakening, or an unusual event have been reported to precipitate seizures in Labrador retrievers (Heynold et al., 1997). Another study reported that anxiety, hyperactivity or stress (e.g. working under conditions with a demand for high performance) sometimes provoked seizures in 22% (11/49) of the included Belgian shepherds (Berendt et al., 2008).

Reflex seizures

Reflex seizures are seizures that can be consistently provoked by specific sensations or perceptions (Commission, 1989). The trigger is specific and the latency between trigger and seizure is short (seconds to minutes). Reflex seizure triggers in people include flickering light (usually from a television) or other visual stimuli, immersion in hot water, reading, certain sounds and eating. These stimuli are usually limited in an individual patient to a single specific stimulus or a limited number of closely related stimuli. Reflex seizures are usually generalized (although focal seizures have also been reported in association with tactile or proprioceptive stimuli) and associated with idiopathic epilepsy in humans (Commission, 1989). Seizures consistently associated with sounds (lawnmower engine), automobile rides or veterinary offices have been observed in dogs (Thomas, 2010).

Regardless of the underlying aetiology, seizures can occur as:

• Self-limiting isolated seizures: a seizure that occurs only once in a 24-h period;
• Cluster seizures: two or more seizures within 24 h with full recovery of consciousness between seizures;
• Status epilepticus: continuous seizure activity for 5 or more minutes or two or more discrete seizures within 24 h without full recovery of consciousness between seizures.

Status epilepticus and cluster seizures are neurological emergencies described in detail in Chapters 23 and 24.

Classification of seizures and epilepsies is an ongoing process in humans and veterinary neurologists should follow its development closely. Establishing a universally accepted and standardized terminology to describe ictal phenomenology would greatly help communication among veterinary clinicians and scientists and represent the foundation of further development of the veterinary classification. The veterinary classification of seizures and epilepsies will evolve as EEG and functional MRI become more widely used, new underlying aetiologies are detected, and breed-related epileptic syndromes with specific genetic mutations are identified.

Reactive seizures are the reaction of a normal brain to a systemic metabolic, nutritional or exogenous toxic disorder (Podell, 1995). Reported prevalence of reactive seizures in dogs and cats varies among studies ranging from 7 to 32% in dogs and from 4 to 28% in cats (Bateman and Parent, 1999; Platt and Haag, 2002; Rusbridge, 2005; Pákozdy et al., 2008, 2010; Zimmermann et al., 2009; Brauer et al., 2011; Steinmetz et al., 2013) (see Chapters 7 and 8). In a recent study the most frequent cause of reactive seizures were intoxications (37/96, 39% of dogs) and hypoglycaemia (31/96, 32% of dogs) (see Fig. 7.2, Chapter 7); 49% (47/96) of dogs had generalized tonicclonic seizures with loss of consciousness and 41% (39/96) of dogs were presented in status epilepticus (Brauer et al., 2011). Dogs with reactive seizures caused by exogenous toxicity have a significantly higher risk to develop status epilepticus, particularly as first manifestation of a seizure disorder, than dogs with other seizure aetiologies (Zimmermann et al., 2009). In cats with reactive seizures and structural epilepsy, status epilepticus is significantly more common than in cats with idiopathic epilepsy (see Schriefl et al., 2008; Pákozdy et al., 2010; Chapter 3).

Clinical presentation in animals with systemic metabolic, nutritional and toxic disorders is variable depending on the underlying aetiology. Toxic disorders generally have an acute (less than 24 h) onset and neurological signs may be preceded or accompanied by gastrointestinal, cardiovascular or respiratory signs. However, chronic lead intoxication may result in recurrent seizures. Metabolic and nutritional disorders can present with an acute, subacute, or chronic onset and may be progressive or relapsing remitting. The neurological examination generally reveals diffuse, bilateral and often symmetrical neurological deficits, however seizures can sometimes be the only neurological abnormality. Diagnostic investigations (see Chapter 10) are aimed at identifying the underlying aetiology of the seizures and, if present, of the other clinical signs. Treatment is aimed at the underlying aetiology and seizure control with anti-epileptic medications (AEMs).

This chapter describes disorders commonly resulting in reactive seizures. For detailed information on specific AEMs, as well as management of cluster seizures and status epilepticus, the reader is referred to Chapters 12 to 24.

Systemic Metabolic Disorders Causing Seizures

Metabolic diseases can cause seizures by interfering with energy metabolism, altering osmolality, acid-base status, or producing endogenous toxins (O’Brien, 1998). The metabolic disorders most commonly reported to cause seizures in the veterinary literature are described below.

Hypoglycaemia

Overview

The blood glucose concentration is of prime importance for normal neuronal metabolism. Glucose is the single most important source of energy in the brain and carbohydrate storage in neural tissue is limited (Hess, 2010). Therefore cerebral function depends on a continuous supply of glucose. Glucose enters the brain by noninsulin dependent facilitated transport mechanisms, which requires a minimum blood glucose level to operate effectively. Persistent hypoglycaemia results in neuronal adenosine triphosphate (ATP) depletion, Na-K-ATPase dysfunction, cytotoxic oedema, excitatory neurotransmitter release (especially glutamate), activation of glutamate receptors, increased intracellular calcium, zinc, and nitric oxide synthase activity, reactive oxygen species generation, lipid peroxidation, DNA damage and cellular necrosis.

Clinical presentation

Hypoglycaemia at glucose concentrations of less than 60 mg/dl (<3 mmol/l) can result in neurologic signs including seizures. Clinical signs vary depending on the rate of decrease, magnitude and duration of hypoglycaemia and include behavioural changes, altered mental status, nervousness, tremors, generalized weakness and seizures. Severe brain damage, coma and death may occur with glucose concentrations of less than 18 mg/dl (<1 mmol/l). As with other homeostatically regulated parameters, the onset and severity of neurological signs may depend more on the rate of decrease than the actual concentration of glucose. Clinical signs may be triggered by fasting or exercise. There are multiple causes of hypoglycaemia (Box 4.1).

Diagnosis

The diagnosis of hypoglycaemic encephalopathy is based on documenting hypoglycaemia

Box 4.1. Underlying causes of hypoglycaemia.

Overproduction of endogenous insulin or insulin-like substances (e.g. insulin-like growth factor):

• Pancreatic insulinoma;
• Islet cell hyperplasia;
• Extrapancreatic neoplasms including intestinal

smooth-muscle tumours, hepatic tumours, or lymphoma.

Exogenous insulin overdose in animals with

diabetes mellitus (especially in cats).

Decreased glucose production:

• Severe hypoadrenocorticism;
• Hypopituitarism;
• Growth hormone deficiency;
• Hepatic failure;
• Glycogen storage disease;
• Glycogen depletion in young puppies (usually of

toy or miniature breeds) and kittens.

Excess glucose consumption:

• Sepsis;
• Hunting dog hypoglycaemia syndrome associated with inadequate caloric intake and

strenuous exercise.

Drug associated:

• Xylitol toxicity

in an animal with neurological signs that improve or resolve following normalization of blood glucose concentration. Specific diagnosis of the underlying disorder requires additional investigations. The reader is referred to internal medicine textbooks for further details on diagnosis and treatment of underlying aetiologies of hypoglycaemia. When hypoglycaemia is suspected in animals presenting with seizures or other neurological signs a blood sample should be taken. Falsely low glucose concentrations may result from use of human point-of-care glucometers in haemo-concentrated animals or from delayed separation of serum from blood cells (which continue to consume glucose).

Management

Once hypoglycaemia is confirmed, emergency treatment of hypoglycaemia-induced seizures involves slow (over 15 min) intravenous administration of 0.5–1 ml/kg of 50% dextrose, diluted 1:2 or 1:4 with 0.9% saline or sterile water. This dosage may need to be repeated and a continuous infusion of 5% dextrose (e.g. 50 ml of 50% dextrose in 500 ml of 0.9% saline or sterile water) may be necessary to maintain normoglycaemia. If intravenous therapy is difficult to perform, oral administration of a rapidly absorbed source of sugar (such as honey, Karo corn syrup or maple syrup) can be a useful substitute. Feeding frequent small meals can also help maintain normoglycaemia. Supportive care of the seizuring animal should be performed as described in Chapter 24. Specific treatment for the underlying cause of hypoglycaemia should be provided.

Insulinoma

Overview

Insulin secreting pancreatic b-islet cell neoplasia is the most common cause of hypoglycaemiainduced seizures in adult dogs. Feline insulinoma is rare. These tumours are associated with excess insulin secretion independent of the negative feedback effects caused by hypoglycaemia. Hyperinsulinaemia produces hypoglycaemia through suppression of glucose release and production rather than increased glucose utilization (Goutal et al., 2012). Most canine insulinomas are malignant. Metastatic lesions have been detected in 45% to 64% of dogs in different studies and most dogs have involvement of regional lymph nodes with or without distant metastases (commonly in the liver) by the time insulinoma is diagnosed (Hess, 2010). Insulinoma has been reported predominantly in adult to old dogs (mean age of 9 years) and in medium- to large-breed dogs.

Clinical presentation

Clinical signs are related to insulin-induced neuroglycopenia (seizures, generalized weakness, collapse, ataxia, obtundation and impaired vision) and/or to hypoglycaemia-induced catecholamine release (tremors, nervousness and hunger) (Goutal et al., 2012). Clinical signs can occur intermittently (Hess, 2010).

Fasting, excitement or exercise can precipitate or worsen clinical signs by decreasing blood glucose concentration. Feeding can result in either alleviation (when restoring normoglycaemia) or exacerbation (by stimulating insulin secretion) of clinical signs (Hess, 2010). A peripheral polyneuropathy has been described in dogs with insulinoma and may be caused by an immune mediated paraneoplastic process or by glucose-mediated metabolic disturbances in the lower motor neurons.

Diagnosis

Neurological signs consistent with hypoglycaemia (blood glucose concentrations below 60 mg/dl or 3 mmol/l) and concurrent hyperinsulinaemia (serum insulin concentration greater than 20 mU/ml) are suggestive of insulinoma. When a dog suspected of having an insulinoma is normoglycaemic, it should be fasted under close observation and blood glucose concentration should be measured every 1 to 2 h. Most dogs with insulinoma would develop hypoglycaemia within 12 h of fasting. When hypoglycaemia (blood glucose concentration <60 mg/dl or <3mmol/l) is detected, blood should be collected for measurement of insulin concentration and the dog should then be fed (Hess, 2010). Some dogs require repeated insulin measurements to confirm the suspicion of insulinoma. In one study hyperinsulinaemia was detected in 76% of dogs with insulinoma when insulin was measured once and in 91% of dogs when insulin was measured twice (Leifer et al., 1986). Fructosamine concentration can help to detect chronic hypoglycaemia as this parameter reflects the blood glucose concentrations over the previous 1–2 weeks and it has been reported to be significantly decreased in dogs with insulinomas. The clinical diagnosis of insulinoma can be further supported by identification of a pancreatic mass with abdominal ultrasonography or contrast-enhanced computed tomography (Figs 4.1, 4.2a, b). The sensitivity of abdominal ultrasonography in detecting insulinoma ranges from 28% to 75% (Goutal et al., 2012). Contrast-enhanced ultrasonography may increase the diagnostic yield of ultrasound. Ultrasound-guided fine-needle aspirate cytology represents a relatively noninvasive tool to

Management

Resolution of clinical signs may be difficult to achieve or to maintain in some dogs as the dextrose bolus may induce further insulin release from the tumour, leading to worsening of the hypoglycaemia. In such cases, repetitive dextrose boluses are not effective as a single strategy and either alternative or adjunctive therapies should be considered. These include: dexamethasone 0.1–0.5 mg/kg intravenously every 12 h, and glucagon at initial infusion rate of 5 ng/kg/min and subsequently adjusted based on blood glucose values up to 13 ng/kg/min. AEMs (see Chapters 12 and 24) may be necessary in severely affected cases. Frequent feeding of small meals should be initiated as soon as the animal can eat. Long-term treatment involves medical management (pre-and post-operatively or in dogs in which surgery cannot be performed) and surgical resection of the pancreatic mass and gross metastases. Whenever possible, surgical resection is considered the treatment of choice as despite being rarely curative, it offers the greatest chance of both durable control of clinical signs and prolonged survival time in dogs with insulinomas. However, outcome is affected by tumour staging. Some dogs can develop diabetes mellitus (transient or permanent) post-operatively. Medical management of insulinoma involves small frequent meals (every 4–6 h) of a diet rich in proteins, fat and complex carbohydrates, prednisolone

0.5 mg/kg/day orally (up to 4 mg/kg/day in refractory cases), and exercise restriction and avoidance of excitement. Additional medical therapy to relieve the hypoglycaemia may be required in some dogs and involves diazoxide 5 mg/kg every 12 h orally, which can be increased gradually without exceeding 60 mg/kg/day, or synthetic somatostatin such as octreotide (Goutal et al., 2012). Streptozotocin, a nitrosurea chemotherapeutic agent, can be used to selectively destroy beta cells in the pancreas or metastatic sites, but it can be nephrotoxic and emetogenic and its use in dogs warrants further investigations. At home immediate management of hypoglycaemic crisis involves oral administration of honey, corn syrup or maple syrup. Median survival time 12 to 14 months (range 0 days to 5 years) in dogs undergoing surgery in different studies. Whereas median survival ranges from 74 to 196 days in dogs undergoing medical treatment only. Combination of medical and surgical treatment resulted in a median survival time of 1316 days (44 months) in a recent study (Goutal et al., 2012).

Hepatic encephalopathy

Overview

Hepatic encephalopathy (HE) is a biochemical disorder of the brain secondary to various hepatic disorders such as congenital and acquired portosystemic shunt, micro-vascular dysplasia, congenital urea-cycle enzyme deficiencies, and acute or chronic severe parenchymal liver damage associated with cirrhosis, neoplasia, chronic active hepatitis, liver steatosis (lipidosis) in cats or toxicosis (Hardy, 1990). HE is more common in dogs than in cats.

Congenital portosystemic shunts represent the most common cause of HE. They generally occur as single vessels that provide direct vascular communication between the portal venous supply and the systemic venous circulation (caudal vena cava or azygous vein), bypassing the liver, and are not associated with portal hypertension. Approximately 25% to 33% of congenital portosystemic shunts are intrahepatic and the remainder are extrahepatic in dogs and cats (Berent and Tobias, 2009). The majority of intrahepatic portosystemic shunts occur in large or giant breed dogs, whereas most extrahepatic portosystemic shunts are seen in small and toy breed dogs (Berent and Tobias, 2009). In animals with congenital portosystemic shunts, the liver has been deprived of growth factors from birth and is therefore abnormally small with a hypofunctional parenchymal mass.

Acquired portosystemic shunts are usually multiple, extrahepatic and occur in animals with chronic portal hypertension secondary to hepatic arteriovenous malformations, noncirrhotic portal hypertension, or chronic hepatitis and cirrhosis (Berent and Tobias, 2009). Portosystemic shunts (congenital or acquired) occur far more commonly in dogs than in cats.

Hepatic microvascular dysplasia is a microscopic malformation of the hepatic microvasculature resulting in shunting of portal blood into the systemic circulation. Micro-vascular dysplasia can occur as an isolated disease or in association with macroscopic portosystemic shunts and can occur with or without concurrent portal hypertension. Hepatic microvascular dysplasia has been reported most commonly in the cairn terrier and Yorkshire terrier (Christiansen et al., 1995).

Urea-cycle enzyme deficiencies are rare congenital errors of metabolism in which one of the enzymes involved in ammonia metabolism fails.

Parenchymal hepatic diseases severely decrease the capacity of the liver to remove toxic products of intestinal metabolism and synthesize factors necessary for normal cerebral function.

The pathophysiology of HE is complex, multifactorial and incompletely understood (Box 4.2). The reader is referred to the cited references for a more detailed description of HE pathophysiology.

Clinical presentation

Neurological signs of HE are mainly consistent with forebrain involvement and include alterations in behaviour or personality, obtundation that may progress to stupor and coma, continuous pacing, aimless wandering, circling, head pressing, hypersalivation (particularly in cats), blindness and seizures. The neurological signs are often intermittent. Several factors can precipitate or exacerbate neurologic signs of HE (Box 4.3).

Non-neurological signs of HE vary depending on the underlying hepatic disease and include polyuria-polydipsia, vomiting, diarrhoea, weight loss, decreased endurance, ascites, icterus (rarely) and, in case of congenital portosystemic shunt, retarded or insufficient growth and signs of lower urinary tract disease (stranguria, pollakiuria, hematuria, dysuria) due to ammonium biurate crystalluria. Copper-coloured irises unusual for the breed have been reported in cats with congenital portosystemic shunts (Plate 1).

Diagnosis

Haematological changes include mild to moderate microcytic, normochromic nonregenerative anaemia. Serum biochemistry abnormalities

Box 4.3. Factors that can precipitate or exacerbate neurologic signs of HE.

• Feeding (particularly food rich in protein and fatty acids);
• Bacterial production of ammonia in large intestine (e.g. following constipation);
• Gastrointestinal haemorrhage;
• Hypokalaemia (due to diarrhoea, anorexia, vomiting, salivation, ascites);
• Hypovolaemia;
• Alkalosis;
• Fever;
• Infection;
• Renal disease resulting in reduced excretion of ammonia;
• Administration of CNS depressant undergoing

hepatic metabolism.

include decreased albumin, urea, glucose and cholesterol levels. In animals with parenchymal hepatic disease, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin are often elevated and vitamin K-dependent clotting factor levels may be decreased. Ammonium biurate crystals can be identified in the urine sediment in approximately half of the dogs with HE (Rothuizen, 2009).

The diagnosis of hepatobiliary disease can be confirmed by the presence of increased levels of fasting plasma ammonia and fasting

(e.g. 12-h) and post-prandial (e.g. 2-h) total serum bile acid concentrations. The sensitivity and specificity of fasting ammonia in the diagnosis of portosystemic shunts has been reported as 91% and 84% in dogs and 83% and 86% in cats, respectively (Ruland et al., 2010). The sensitivity and specificity of serum bile acids in the diagnosis of portosystemic shunts has been reported as 78% and 87% in dogs and 100% and 84% in cats, respectively (Ruland et al., 2010). Dogs and cats with congenital urea-cycle enzyme deficiencies have hyperammonaemia and low bile acid concentrations (Rothuizen, 2009).

Definitive diagnosis of portal vascular anomalies requires ultrasonography (Figs 4.3a–c), portovenography, scintigraphy (transcolonic or trans-splenic), computed tomographic angiography or magnetic resonance angiography (d’Anjou et al., 2004; Sura et al., 2007; Berent and Tobias, 2009; Mai and Weisse, 2011; Nelson and Nelson, 2011). Liver biopsy is required for confirmation of parenchymal hepatic disease. MRI of the brain of dogs and cats with portosystemic shunts may reveal cerebral cortical atrophy characterized by widened sulci and hyperintensity of the lentiform nuclei on T1-weighted images (Torisu et al., 2005). In addition, bilateral diffuse hyperintensity predominately of the parietal and occipital cortex grey matter on T2-weighted MR images has been reported in a dog with portosystemic shunt. The authors suggested that the signal changes represented cerebral oedema associated with cortical laminar necrosis caused by acute hyperammonaemia (Moon et al., 2012).

Management

The management of HE depends on the underlying cause, the degree of hepatic dysfunction and the severity of clinical signs. The aims of HE treatment are: (i) stop seizure activity; (ii) reduce serum levels of neurotoxic agents; and (iii) treat the underlying aetiology.

Seizures should be controlled with AEMs that undergo minimal or no hepatic metabolism such as levetiracetam (LEV) (see Chapter 16). LEV can be administered in dogs and cats at 60 mg/kg intravenously or orally as an initial loading dose, which can be followed (8h later) by a maintenance dose of 20 mg/kg every 8 h. Alternatively or in addition to LEV, bromide can be administered at a loading dose in dogs (see Chapter 14). Intravenous propofol as boluses (1–2 mg/kg) or constant rate infusion (0.1–0.6 mg/kg/min titrated to effect or up to 6 mg/kg/h) may be required in animals with persistent seizure activity (see Chapter 24). Oro-tracheal intubation, ventilation, haemodynamic support and intensive care monitoring may be required.

The production of ammonia in the gastrointestinal tract can be reduced by administration of:

• Lactulose (1–3 ml/kg/day orally divided into two to three doses) to decrease intestinal transit time, prevent constipation, alter the intestinal flora, and create an acid environment in order to trap ammonia as ammonium;

• Antibiotics such as ampicillin (22 mg/kg orally every 8 h), metronidazole (7.5 mg/ kg orally every 12 h), or neomycin (20 mg/ kg orally every 8 h), to reduce the colonic bacterial flora producing ammonia;

• Low-level high-quality protein diet.

In animals unable to take oral medications (e.g. propofol infusion, severe post-ictal phase, stupor or coma) lactulose can be administered by retention enema (20 ml/kg, made of three parts of lactulose diluted in seven parts warm water, every 4–6 h) and the antibiotic can be administered parenterally. Feline liver steatosis (lipidosis) should not be treated by dietary protein reduction but instead by forced feeding of amino acid-rich nutrients (Rothuizen, 2009). Protein intake should not be restricted excessively in growing young animals. A balanced rather than excessively restricted protein intake is currently preferred in people with HE (Bismuth et al., 2011). Administration of high doses of probiotics containing urease negative bacteria (such as Lactobacillus acidophilus or Enterococcus faecium) may help to modify the intestinal flora and decrease ammonia production. In addition, it has been suggested that administration of L-carnitine may reduce ammonia level by increasing energy metabolism (Bismuth et al., 2011). Intravenous fluids should be administered to animals with fluid and electrolyte abnormalities. Conditions that aggravate HE (see Box 4.3) should be avoided. Proton pump inhibitors such as omeprazole should be administered to animals with proximal gastrointestinal haemorrhage. Supportive nutraceutical therapy such as S-adenosyolL-methionine (SAMe) has been recommended for a variety of liver diseases; however, it is usually unnecessary in portovascular anomalies that can be treated surgically (Berent and Tobias, 2009). Congenital portosystemic shunts can be completely or partially ligated with nonabsorbable sutures or gradually attenuated with an ameroid constrictor, cellophane band, or hydraulic occluder. Gradual attenuation is preferred to reduce the risk of post- operative complications (Berent and Tobias, 2009). Acute complications of shunt attenuation include intraoperative haemorrhage, prolonged anaesthetic recovery, seizures and other forebrain neurological signs, portal hypertension and refractory hypoglycaemia. Severe persistent neurologic signs (including blindness and seizures) represent the most common cause of death after portosystemic shunts attenuation (Berent and Tobias, 2009). Central blindness has been reported in up to 44% of cats following portosystemic shunt surgery and usually resolves within 2 months after surgery (Kyles et al., 2002). Post-operative seizures have been reported in 3% to 7% of dogs and 8% to 22% of cats after shunt attenuation (Tisdall et al., 2000; Kyles et al., 2002; Frankel et al., 2006; Lee et al., 2006; Lipscomb et al., 2007). Seizures may occur up to 80 h after surgery, frequently progress to status epilepticus and may be difficult to control. These seizures are not associated with hypoglycaemia, hyperammonaemia, or attenuation technique (Berent and Tobias, 2009). Post-operative seizures can be treated with intravenous levetiracetam or propofol (see Chapters 16 and 24). Levetiracetam administered at 20 mg/kg orally every 8 h for a minimum of 24 h before surgery significantly decreased the risk of post-operative seizures and death in dogs undergoing surgical attenuation of extrahepatic congenital portosystemic shunts with ameroid ring constrictors (Fryer et al., 2011).

Renal-associated encephalopathy

Overview

Renal-associated encephalopathy can occur in animals with acute or end-stage chronic renal failure (uraemic encephalopathy), following haemodialysis (dialysis disequilibrium syndrome) or renal transplantation (post-renal transplantation encephalopathy). As for hepatic encephalopathy the pathogenesis of renal-associated encephalopathy is complex and incompletely understood. Neurological signs may result from accumulation of uraemic toxins, altered balance between excitatory and inhibitory neurotransmitters, as well as acid-base, fluid and electrolyte abnormalities. Parathyroid hormone (PTH) disturbance may also play a role in the pathogenesis of uraemic encephalopathy. In addition, hypertension secondary to renal disease may lead to intracranial cerebrovascular accidents (O’Brien, 1998). Dialysis disequilibrium syndrome is thought to be caused by an osmotic gradient between the brain cells and the extracellular fluid due to excessively rapid haemodialysis, which results in cellular swelling and cerebral oedema. Post-renal transplantation encephalopathy (resulting in seizures, stupor, ataxia and central blindness) has been reported in cats and it is most likely associated with uncontrolled hypertension (Kyles et al., 1999). Uraemic encephalopathy has been infrequently reported in dogs with renal failure (Wolf, 1980; Fenner, 1995).

Clinical presentation

Neurological signs of uraemic encephalopathy include obtunded mental status that can progress to stupor, muscle fasciculations (especially in facial muscles), head bobbing, generalized seizures and generalized weakness. In one retrospective study including 29 dogs, altered mental status was more common in dogs with chronic renal disease, whereas seizures were more common in dogs with acute renal failure (Fenner, 1995). Other clinical signs such as polyuria, polydipsia, vomiting and dehydration are reflective of renal failure.

Diagnosis

Diagnosis of renal-associated encephalopathy is based on signs of cerebral (mainly forebrain) dysfunction in an animal with renal failure or soon after haemodialysis or renal transplantation and no other cause of brain disease. Serum biochemistry shows very severe azotaemia, hyperkalaemia, and hyperphosphataemia with or without hypocalcaemia. Urinalysis performed before fluid therapy shows isosthenuria.

Management

Treatment should be aimed at the causes of the renal failure, if possible, as well as symptomatic and supportive. Fluid, electrolyte and acid-base imbalances should be corrected. Hypertension, if present, should be treated. Seizures can be treated as described in Chapters 12 and 24, however dosages of AEMs may need to be decreased in case of impaired renal excretion.

Hyponatraemia

Overview

Hyponatraemia occurs when plasma sodium concentration is below reference levels (usually 140 mEq/l or mmol/l in dogs, and 149 mEq/l or mmol/l in cats) (Benitah, 2010). Occurrence of clinical signs depends more on rapidity of onset of hyponatraemia than to magnitude of change. Neurologic signs may occur with sodium concentrations less than 120 mEq/l or mmol/l in dogs and less than 130 mEq/l or mmol/l in cats (de Morais and DiBartola, 2008a). Serum sodium concentration reflects the amount of sodium relative to the volume of water in the body and not total body sodium content. Hyponatraemic animals may have decreased, increased or normal total body sodium content and therefore volemic status also needs to be considered when investigating serum hyponatraemia (de Morais and DiBartola, 2008a). Hyponatraemia may result primarily from increased water gain, administration of fluids low in sodium (e.g. 5% dextrose in water, 0.45% sodium chloride), hypertonic solution without sodium

(e.g. mannitol, glucose) and an excessive sodium loss (e.g. loop diuretics, thiazides). Causes of hyponatraemia classified based on plasma osmolality and hydration status are listed in Box 4.4.

Clinical presentation

Clinical signs of hyponatraemia include lethargy, anorexia, vomiting, generalized weakness, ataxia, obtunded mental status progressing to stupor and coma, and seizures. The severity of neurologic signs is greater

Box 4.4. Causes of hyponatraemia classified

based on plasma osmolality and hydration status (modified from de Morais and DiBartola,

2008a).

With plasma hyperosmolality (>310 mOsm/kg):

• Hyperglycaemia;
• Mannitol infusion.
• With normal plasma osmolality (290–310 mOsm/kg):
• Hyperglycaemia;
• Severe hyperproteinaemia.
• With plasma hypo-osmolality (<290 mOsm/kg):
• Hypervolaemia:
• Severe liver disease causing ascites;
• Congestive heart failure;
• Advanced renal failure;
• Nephrotic syndrome;
• Normovolaemia:
• Psychogenic polydipsia;
• Hypotonic fluid infusion;
• Syndrome of inappropriate antidiuretic hormone secretion;
• Antidiuretic medications (e.g. narcotics,

nonsteroidal anti-inflammatory drugs,

vincristine);

• Myxedaema coma due to severe hypothyroidism;

• Hypovolaemia:

• Gastrointestinal loss (diarrhoea with or without vomiting);
• Third-space loss (pancreatitis, peritonitis,

uroabdomen, cavitary effusion);

• Cutaneous burns;
• Renal loss (hypoadrenocorticism, diuretic

administration).

when hyponatraemia develops rapidly. In acute hyponatraemia, water flows down its concentration gradient and enters brain cells producing cerebral oedema and increased intracranial pressure. In addition, hypervolemic animals may be presented with ascites, peripheral or pulmonary oedema, and jugular distension; whereas hypovolemic animals may present with signs of dehydration including decreased skin turgor, dry mucous membranes, delayed capillary refill time, tachycardia, hypotension, increased packed cell volume (PCV) and total protein concentration, and high urine specific gravity. Determination of the animal’s volume status helps to identify the underlying cause of the hyponatraemia and initiate correct treatment.

Diagnostic investigation

In hypovolemic and hyponatraemic animals calculation of the fractional excretion of sodium (FE_Na) can be used to determine if the kidneys are the source of excessive sodium loss (see equation below). The FE_Na should be less than 1% for nonrenal sources of sodium loss and 1% or greater if sodium is being lost by the kidneys (de Morais and DiBartola, 2008a).

Calculation of the fractional excretion of sodium is:

U /S

Na Na

FE_Na = ´ 100 (4.1)

U /S

Cr Cr

where FENa = fractional excretion of sodium, U_Na = urine concentration of sodium (mEq/l), S_Na = serum concentration of sodium (mEq/l), UCr = urine concentration of creatinine (mg/dl) and S_Cr = serum concentration of creatinine (mg/dl).

Additional diagnostic investigations including laboratory analysis and imaging will vary depending on the suspected underlying aetiologies of hyponatraemia (see Box 4.4). The reader is referred to internal medicine textbooks for further details on diagnosis and treatment of each condition.

Management

Treatment is aimed at increasing serum sodium levels and treating the underlying cause of the hyponatraemia. Isotonic (0.9%) saline or balanced electrolytes solutions can be administered to hypovolemic animals, while water restriction (i.e. limiting water intake to less than urine output) can be performed for animals with normovolaemia or hypervolaemia associated with excessive water intake or renal retention. A loop diuretic and dietary sodium restriction can be considered in hypervolaemic animals. To avoid life-threatening neurologic complications such as brain stem myelinolysis, recommended rates of correction for chronic (>2 days) hyponatraemia are 10–12 mEq/l/day or approximately 0.5 mEq/l/h (0.5 mmol/l/h) (Benitah, 2010). When hyponatraemia is corrected too rapidly, the increasing osmolality of the extracellular space results in water movement from the intracellular to the extra-cellular space, and consequent cellular dehydration and shrinkage. This cellular shrinkage can separate the neurons from their myelin sheaths leading to myelinolysis. Mental status, hydration and electrolytes (particularly serum sodium concentration) need to be monitored frequently (e.g. every 4–6 h) during correction of hyponatraemia. Seizure can be treated as described in Chapters 12 and 24, however dosage and choice of AEMs will vary depending on the underlying aetiology of hyponatraemia.

Hypernatraemia

Overview

Hypernatraemia occurs when plasma sodium concentration is above reference levels (usually 155 mEq/l or mmol/l in dogs, and 162 mEq/l or mmol/l in cats) (Benitah, 2010). Occurrence of clinical signs depends more on rapidity of onset of hypernatraemia than to magnitude of change. Neurological signs generally occur when serum sodium levels exceed 170 mEq/l or mmol/l in dogs and 175 mEq/l or mmol/l in cats (>350 mOsm/kg) (de Morais and DiBartola, 2008b). Hypernatraemia can result from water or hypotonic fluid loss, or excessive sodium gain (Box 4.5).

Sodium and its attendant anions account for approximately 95% of the osmotically active

Box 4.5. Causes of hypernatraemia (modified from de Morais and DiBartola, 2008b).

Pure water deficit (normovolaemic hyper

natraemia):

• Inadequate water intake;

• Animal unable to drink or no access to water;
• Primary hypodipsia;

• Diabetes insipidus (central or nephrogenic).

Hypotonic fluid loss (hypovolaemic

hypernatraemia):

• Gastrointestinal;
• Vomiting;
• Diarrhoea;
• Small intestinal obstruction;
• Renal:

• Osmotic diuresis (mannitol infusion,

hyperglycaemia);

• Non-osmotic diuresis (furosemide administration);
• Chronic renal failure;
• Non-oliguric renal failure;
• Post-obstructive diuresis;

• Third-space loss;
• Pancreatitis;
• Peritonitis;
• Cutaneous;

• Burns.

Excessive sodium gain (hypervolaemic

hypernatraemia):

• Hypertonic fluid administration (intravenous

hypertonic saline, sodium bicarbonate, sodium

phosphate enema);

• Hyperaldosteronism;
• Hyperadrenocorticism;
• Excessive sodium chloride intake (e.g. salt

poisoning).

substances in the extracellular water. Therefore hypernatraemia is associated with hyperosmolality (de Morais and DiBartola, 2008b).

Clinical presentation

Clinical signs of hypernatraemia are mainly neurological and include anorexia, lethargy, vomiting, behavioural changes, head pressing, ataxia, generalized muscular weakness, muscle fasciculations, seizures, blindness, obtunded mental status progressing to stupor and coma, and death in severe cases. The severity of the neurological signs depends more on the rate of increase in sodium concentration than on the degree of hypernatraemia. With acute hypernatraemia water moves out of cells into the hyperosmolar extracellular space producing neuronal dehydration. The resulting decrease in cerebral volume may cause stretching and tearing of small cerebral vessels, leading to intracranial haemorrhage (subarachnoid, subdural, and/or intraparenchymal). Both cellular dehydration and intracranial haemorrhage may contribute to cerebral dysfunction in the acutely hypernatraemic animal (Benitah, 2010). When hypernatraemia develops slowly and gradually (e.g. <1 mEq/l/h or <1 mmol/l/h), the cerebral neurons compensate by increasing intracellular osmolality by movement of sodium, potassium, chloride and glucose intracellularly and by producing osmotically active solutes, called idiogenic osmoles (such as taurine, sorbitol and inositol) to adapt to the hypertonicity and minimize cerebral cellular dehydration. Clinical signs may be minimal or absent with slowly developing hypernatraemia. In addition to the clinical signs caused by the hypernatraemia and associated hyperosmolality, clinical signs of hypovolaemia (decreased skin turgor, dry mucous membranes, delayed capillary refill time, tachycardia, hypotension, increased PCV and total protein concentration, and high urine specific gravity) or hypervolaemia (ascites, peripheral or pulmonary oedema and jugular distension) may be present.

Diagnosis

Diagnostic investigations including laboratory analysis and imaging will vary depending on the suspected underlying aetiologies of hypernatraemia (see Box 4.5). The reader is referred to internal medicine textbooks for further details on diagnosis and treatment of each condition.

Management

Treatment is aimed at restoring normal extra-cellular fluid volume, decreasing sodium serum levels and treating the underlying cause of the hypernatraemia. Serum sodium concentration should be corrected at a rate of less than 0.5 mEq/l/h (0.5 mmol/l/h) to minimize the risk of cerebral cellular swelling, cerebral oedema and increased intracranial pressure. Free water deficit can be calculated based on the formula in equation 4.2 at bottom of page.

Oral water administration is the preferred method to correct water deficits in normovolaemic animals. Isotonic intravenous fluids should be used in normovolaemic animals that cannot drink and in hypovolaemic animals. Once the extracellular fluid volume has been restored, hypotonic fluids can be administered as maintenance treatment. Normovolaemic animals with pure water deficits can be administered 5% dextrose in water intravenously. Hypernatraemia secondary to excessive sodium gain can be treated with 5% dextrose in water intravenously and a loop diuretic to promote natriuresis (Benitah, 2010). Mental status, hydration and electrolytes (particularly serum sodium concentration) need to be monitored frequently (e.g. every 4–6 h) during correction of hypernatraemia. Seizures can be treated as described in Chapters 12 and 24; however, dosage and choice of AEMs will vary depending on the underlying aetiology of hypernatraemia.

Hypocalcaemia

Overview

Hypocalcaemia results in tetany (intermittent contraction of extensor muscles) and seizures when serum ionized calcium concentration is equal or lower than 0.8 mmol/l (3.2 mg/dl) or total calcium concentration is below 1.5 mmol/l (6 mg/dl) (Drobatz and Casey, 2000; Brauer et al., 2011). To convert mmol/l to mg/dl, the value in mmol/l has to be multiplied by 4 (Schenck and Chew, 2008). Total serum calcium is approximately 50% ionized, 40% protein bound (especially to albumin), and 10% chelated with anions such as citrate or phosphate. Only ionized calcium is biologically active. The proportion of ionized calcium is affected by serum protein level, acid-base status (ionized calcium levels are decreased by alkalosis and increased by acidosis) and the presence of anions that act as chelators (e.g. Mg⁺⁺ or phosphate). Calcium homeostasis is regulated by parathyroid hormone (PTH), calcitriol (1,25- dihydroxyvitamin D) and calcitonin. The main organs involved in calcium metabolism are bone, kidney and small intestine.

Low calcium concentrations increase neuronal membrane permeability to sodium ions, resulting in neuronal hyperexcitability in the peripheral and central nervous system.

Clinical presentation

Clinical signs include nervousness, behavioural abnormalities (aggression, vocalization), decreased activity, panting, pacing, muscle stiffness, fasciculations, cramping and tetany, shifting limb lameness, stiff gait, hyperthermia, facial rubbing and biting at feet (probably due to paraesthesia) and seizures. Neurological signs can be intermittent and sometimes can be triggered by external stimuli or exercise. Differential diagnoses for underlying aetiologies of hypocalcaemia in dogs and cats are listed in Box 4.6. The reader is referred to internal medicine textbooks for further details on diagnosis and treatment of each condition.

Diagnosis

Diagnosis of hypocalcaemia is based on characteristic clinical signs and serum-ionized calcium concentration equal or lower than 0.8 mmol/l (3.2 mg/dl). Decreased serum total calcium should prompt assessment of serum ionized calcium. The use of corrective formulae to estimate ionized calcium concentration based on serum total calcium and total protein or albumin concentration is not recommended. These formulae do not accurately predict ionized calcium concentration and should not be used to make therapeutic decisions. Serum

Box 4.6. Causes of hypocalcaemia (modified from Schenck and Chew, 2008).

• Puerperal tetany (eclampsia);
• Renal failure (acute and chronic);
• Protein-losing enteropathies (hypoalbuminaemia);
• Acute pancreatitis;
• Ethylene glycol toxicity;
• Phosphate enema;
• Hypoparathyroidism:
• Primary;
• Idiopathic or spontaneous;
• Post-operative bilateral thyroidectomy;
• After sudden reversal of chronic hypercalcaemia;
• Secondary to magnesium depletion or excess;
• Nutritional secondary hyperparathyroidism;
• Citrate anticoagulant overdose with blood transfusion;
• Hypovitaminosis D.

ionized calcium concentration is typically higher than ionized calcium concentration in heparinized plasma or whole blood (measured by means of a blood gas analyser) due to dilution with heparin (Schenck and Chew, 2008). Calcium concentrations should not be assessed on EDTA plasma as EDTA chelates calcium, giving artificially low calcium concentrations. Additional laboratory investigations (such as measurement of plasma PTH, magnesium, vitamin D metabolites, urinalysis) and diagnostic imaging are indicated depending on the suspected underlying cause of hypocalcaemia in individual patients.

Management

Diazepam (0.5 to 1.0 mg/kg intravenously, up to a maximum total dose of 20 mg, in dogs and cats, repeated to effect or twice within 2 h; see Chapters 21 and 24) can be used to control the tetany and seizures initially while the diagnosis of hypocalcaemia is confirmed. Hypocalcaemia is treated with 10% calcium gluconate at a dose of 0.5–

1.5 ml/kg (providing 5–15 mg/kg of calcium) administered slowly IV (over 10 to 30 min) to effect as individual requirements vary. Clinical improvement is generally obvious within minutes of initiating the infusion. Electrocardiographic monitoring is advisable because of the risk of cardiotoxicity. If bradycardia, premature ventricular complexes, increased P-R interval, prolonged QRS complex or shortening of the Q-T interval is observed, the IV infusion should be briefly discontinued. Maintenance therapy of hypocalcaemia involves calcium gluconate (at a dosage equal to the one used IV for the control of tetany), diluted in an equal volume of saline, administered SC every 6 to 8 h. Serum calcium concentration should be monitored every 1–3 days to adjust the calcium gluconate dose. Generally, after serum calcium concentrations have been maintained within reference range for 48 h, the administration of calcium gluconate can be gradually tapered off to every 8–12 h. Long-term maintenance treatment with oral calcium (25 mg/kg every 8–12 h) and vitamin D supplementation may be required. Specific treatment for the underlying aetiology of hypocalcaemia should be instituted as promptly as possible.

Nutritional Disorders Causing Seizures

Thiamine (vitamin B1) deficiency

Overview

Thiamine (vitamin B1) deficiency has been reported in dogs and cats fed thiamine-deficient diets including commercial canned pet food (Marks et al., 2011; Markovich et al., 2014). Thiamine can be destroyed by heat during cooking or processing, preservatives such as sulfur dioxide, sulfate trace minerals, ultraviolet and gamma irradiation, and thiaminase enzyme activity, which is found predominantly in shellfish, fish viscera and some bacteria. In addition, thiamine deficiency can result from decreased intake due to anorexia or vomiting, decreased intestinal absorption (e.g. diarrhoea), abnormal utilization

(e.g. liver dysfunction) or increased requirements (e.g. fever, infection). The metabolically active form of thiamine, thiamine pyrophosphate, plays an essential role in three enzyme systems (pyruvate dehydrogenase, alpha Tissues dependent on glucose or lactateketoglutarate dehydrogenase, and transketo-pyruvate for energy, such as the brain and lase), which are essential for complete oxida-heart, are particularly compromised in thiation of glucose through the Krebs cycle. mine deficiency.

Fig. 4.5. Transverse T2-weighted MR image of the brain of a domestic short hair cat with thiamine (vitamin B1) deficiency. Note the bilaterally symmetrical hyperintensities localized to the lateral geniculate nuclei. See Plate 2 for the histology showing focal haemorrhagic necrotic lesions localized to the lateral geniculate nuclei.

Clinical presentation

Clinical signs include anorexia, vomiting, abnormal mentation, seizures, dilated and unresponsive pupils, absent menace response bilaterally, opisthotonos with increased extensor tone of all four limbs, ataxia, tetraparesis, postural reaction deficits and vestibular dysfunction (Garosi et al., 2003; Penderis et al., 2007; Palus et al., 2010). In addition, cervical ventroflexion and hyperesthesia to stimuli have been reported in thiamine-deficient cats. Seizures can progress to coma and death if the thiamine deficiency is not treated.

Diagnosis

A presumptive diagnosis of thiamine (vitamin B1) deficiency is based on dietary history, clinical and MRI findings and response to therapy. MRI findings include bilaterally symmetrical hyperintensity on T2-weighted and FLAIR images localized to the red nuclei, caudal colliculi, vestibular nuclei and cerebellar nodulus in dogs (Figs 4.4a–e) (Garosi, 2003) and to the lateral geniculate nuclei, caudal colliculi, periaqueductal grey matter, medial vestibular nuclei, cerebellar nodulus and facial nuclei in cats (Fig. 4.5; Plate 2) (Penderis et al., 2007; Palus et al., 2010). These MRI changes have been reported to resolve following thiamine supplementation (Garosi et al., 2003; Palus et al., 2010). Bilaterally symmetrical hyperintense lesions on T2-weighted and FLAIR images have been reported to affect also the cerebral cortex (parietal, occipital, hippocampal lobe) in cats with thiamine deficiency (Marks et al., 2011). Diagnosis can be confirmed by determining whole blood thiamine concentration by high-performance liquid chromatography. This test is now commercially available in many countries and has replaced the erythrocyte transketolase activity assay because of its superior sensitivity and specificity for thiamine status (Marks et al., 2011). Diet samples can also be submitted for thiamine analysis. Pathologic changes include bilaterally symmetrical spongiosis, necrosis and haemorrhage of upper brainstem nuclei including caudal colliculus, lateral geniculate (Plate 2), medial vestibular and oculomotor nuclei.

Management

Treatment should be instituted immediately for any animal suspected of having thiamine deficiency. Thiamine should be administered at 50–100 mg per dog and 25–50 mg per cat IM, SC or PO every 12–24 h until a response is obtained or another diagnosis is established. Emergency management of seizures can be performed as described in Chapter 24. The underlying cause of thiamine deficiency should be identified and managed. The majority of affected dogs and cats respond rapidly to thiamine supplementation and diet change.

Exogenous Toxic Disorders Causing Seizures

Overview

Numerous exogenous toxins can induce seizures (see Box 3.1, Chapter 3) through different mechanisms including increased excitation, decreased inhibition, and interference with energy metabolism (O’Brien, 1998). Toxins may affect the nervous system directly or indirectly, by affecting other organs whose dysfunction secondarily affects the brain (e.g. HE due to toxin-induced hepatic failure, xylitol-induced hypoglycaemia). This chapter focuses predominantly on toxins with direct effect on the nervous system.

Clinical presentation

The suspicion of toxin exposure is often based on the history and the onset of acute neurological signs (including excitation and hyperactivity or obtundation, stupor, coma, muscle tremors and fasciculations, seizures and ataxia) often associated with vomiting, diarrhoea, salivation, bronchoconstriction, bradycardia or tachycardia and hyperthermia. The source of intoxication is not always obvious to the pet owner, and therefore veterinarians should be proactive in asking questions and mentioning possible sources of intoxication any time the clinical presentation raises the suspicion of toxin exposure. Seizure can occur in clusters or as status epilepticus (e.g. organophosphates,

strychnine, mycotoxins) or less commonly may be isolated and recurrent (e.g. lead) (O’Brien, 1998).

Diagnosis

The presumptive diagnosis is often based on the history of toxin ingestion and clinical signs. Definitive diagnosis is made by identification of the toxin in feed or suspect bait material, gastric content from vomitus or lavage fluids, water, blood or urine (for urinary excreted toxins).

Management

Emergency treatment of neurotoxicity involves multiple simultaneous steps, including:

• Systemic stabilization (airway patency, ventilation, oxygenation, normalization of blood pressure, correction of any fluid, electrolyte or acid-base imbalances, management of cardiac arrhythmias);
• Seizure control (see Table 4.1 and Chapters 12 and 24);
• Excessive skeletal muscle tremor control (Table 4.1);
• Decontamination and prevention of further absorption of toxin (Table 4.1);
• Control of body temperature: convective whole body cooling (e.g. wetting the fur, placing a fan near the animal) in hyperthermic (³40°C, 104°F) animals, or gradual warming in hypothermic animals. Body temperature should be closely monitored to avoid inducing hypo- or hyperthermia;
• Administration of an antidote when available;
• Nursing care.

Decontamination

The methods of decontamination and prevention of further toxin absorption depend on the route of entry of the toxin and its metabolic profile.

Cutaneous absorbed toxins

Bathing is the standard method of decontamination for cutaneous exposure to most toxic substances (Rosendale, 2002). The patient should be stabilized prior to bathing. The stimulation of bathing may trigger or exacerbate neurological and cardiovascular signs. People handling the animal should wear protective gloves and aprons. Liquid handdishwashing detergents are usually recommended over shampoo as they are more effective at removing lipid soluble substances. Sometimes bathing must be repeated to completely remove the toxin. Clipping may be the best way to remove adhesive substances.

Gastrointestinal absorbed toxins

When the toxin has been ingested, decontamination involves:

• Induction of emesis within 4–6 h of toxin ingestion (e.g. apomorphine in dogs and xylazine in cats, see Table 4.1), gastric lavage within 2–6 h of toxin ingestion or colonic lavage;
• Administration of activated charcoal with a cathartic (e.g. sodium sulphate or sorbitol; see Table 4.1);
• Providing demulscents (milk, kaolin-pectin) for any gastrointestinal irritation.

Emetics that stimulate vomiting by direct irritation of the pharyngeal or gastric mucosa should not be used. Induction of emesis is contraindicated in case of ingestion of caustics or volatile substance, if vomiting has already occurred, and in animals with seizures, impaired mental status or abnormal gag reflex due to the risk of aspiration pneumonia (Rosendale, 2002).

Gastric lavage is performed in the anaesthetized animal by gastric intubation and irrigation of the stomach with warm water. A cuffed endotracheal tube must be in place during the procedure to minimize the risk of aspiration of stomach contents. Gastric lavage is indicated when induction of emesis is contraindicated (e.g. profound CNS depression or seizures) or when a large amount of toxin is likely to still be present in the stomach. Gastric lavage is contraindicated in the case of ingestion of: caustics, small volume of toxin (which can be decontaminated with activated charcoal) and moderate amount of toxin more than 2 h previously.

Indication Medication Dosage

The risks of general anaesthesia and of aspiration pneumonia need to be considered. The animal must be monitored continuously during recovery from general anaesthesia from gastric lavage.

Colonic lavage is indicated for toxins than may be absorbed from the colon when ingestion has occurred more than 4–6 h before presentation. Colonic lavage may be beneficial when performed earlier than 4 h post-ingestion of organophosphates and carbamates. Colonic lavage is performed by inserting a lubricated narrow non-rigid tube from the anus into the rectum and transverse colon and instillation of warm water under gravity flow. Animals with decreased consciousness should be endotracheally intubated during the procedure as colonic distension can stimulate emesis.

Activated charcoal can be administered by stomach tube after gastric lavage or syringe-fed to animals that can swallow. Activated charcoal is indicated for adsorption of most toxins when the toxic substance is likely to still be present in the gastrointestinal tract, particularly with toxins with slow gastrointestinal release and adsorption or toxins undergoing enterohepatic recirculation. In these cases repeated dosing of activated charcoal (without sorbitol) 1–4 g/kg PO every 6–8 h for 24 h is indicated. Serum sodium should be closely monitored for patients receiving repeated charcoal doses due to potential of development of hypernatraemia. Constipation can be prevented by maintaining the animal well hydrated. Strong acids or alkalis, dissociable salts and metals, and alcohols are not adsorbed by activated charcoal. Activated charcoal is contraindicated in animals with high risk of aspiration pneumonia or hypernatraemia. Cathartics are often administered in association with activated charcoal to minimize toxin absorption by reducing intestinal transit time. Repeated dosing is not recommended due to the risk of osmotic diarrhoea and hypernatraemia. Cathartics are contraindicated in dehydrated or hypovolaemic animals.

Urinary excreted toxins

With urinary excreted toxins, diuresis and/or modification of the urine pH can enhance toxin excretion (O’Brien, 1998). Urine acidification can be achieved by administering ammonium chloride, 100 mg/kg PO for dogs and 20 mg/ kg PO for cats. Ammonium chloride should not be used in acidotic animals and overuse may result in ammonia toxicosis. Urine alkalinization can be achieved by administering sodium bicarbonate at 0.5 to 2 mEq/kg IV every 4 h. Possible adverse effects of systemic pH modulation need to be considered. The acid-base status of the animal must be monitored.

Intravenous lipid emulsion infusion

Intravenous lipid emulsion (IVLE) (e.g. Intralipid) infusion has been increasingly used as antidotal treatment of toxicosis from various lipophilic agents (Fernandez et al., 2011; Gwaltney-Brant and Meadows, 2012; Kaplan and Whelan, 2012). IVLE is composed of neutral, medium to long-chain triglycerides derived from plant oils (e.g. soybean, safflower), egg phosphatides and glycerine, formulated primarily as a source of essential fatty acids for patients requiring parenteral nutrition (Gwaltney-Brant and Meadows, 2012). The exact mechanism of antidotal action of IVLE is unknown. The sequestration effect theory proposes that IVLE acts as pharmacological ‘sink’ for lipid soluble compounds decreasing their tissue availability and increasing their clearance (Kaplan and Whelan, 2012). IVLE expands the plasma lipid phase creating a discrete compartment that sequesters lipophilic agents and prevents them from reaching their sites of action. In addition, the expanded plasma lipid phase creates a concentration gradient that facilitates the passage of the lipophilic compounds from the interstitial space into the intravascular space. Potential adverse effects of IVLE infusions include: interference with lipophilic medications (i.e. methocarbamol, diazepam, phenobarbitone, propofol) administered for symptomatic or supportive care; pancreatitis due to persistent lipaemia; and hypersensitivity due to IVLE components (Gwaltney-Brant and Meadows, 2012). Based on growing number of case reports in veterinary medicine, IVLE infusion shows promise in the management of toxicosis from a variety of lipophilic agents, including macrocyclic lactones and pyrethrin compounds (Pritchard, 2010; Clarke et al., 2011; Haworth and Smart, 2012; Epstein and Hollingsworth, 2013). More studies are needed to determine optimum time of initiation, dosing regimens and margin of safety of IVLE as antidotal treatment for different lipid soluble toxicities. Treatment protocols are likely to be affected by the degree of lipid solubility and half-life of the toxin. The protocol recommended in Table 4.1 is based on the human literature and veterinary case reports. Care must be taken to use aseptic technique when handling and administering any lipid emulsions as they can promote bacterial growth (Kaplan and Whelan, 2012).

Insecticides

Pyrethrin and pyrethroid (permethrin)

Overview

Pyrethrins are natural insecticides obtained from Chrysanthemum cinerariaefolium, while pyrethroids (e.g. permethrin and fenvalerate) are synthetic analogues of pyrethrins classified as type I (no alpha-cyano-3-phenoxybenzyl group) or type II (with alpha-cyano-3phenoxybenzyl group) (Hansen, 2006; Wismer and Means, 2012). Etofenprox is a nonester pyrethroid-like insecticide. Permethrin toxicosis is one of the most commonly reported poisonings in the USA and the UK in small animals. Pyrethrins and pyrethroids can be absorbed dermally, orally (e.g. grooming in cats) and via inhalation. Many pyrethrin and pyrethroid formulations are registered for topical use on dogs and/or cats (as spot-on, flea collars, medicated shampoo) and in the household for flea and tick control. Generally, most products registered for use on dogs and cats are safe when used according to label directions in healthy pets. Cats are more sensitive than most other species to pyrethrins and pyrethroids probably due to deficiencies in glucoronyl transferase resulting in slower hepatic metabolism of these compounds. Intoxication results from administration to cats of products labelled for use on dogs, overdose or repeated over-application (Hansen, 2006). Secondary exposure may occur in cats that are in contact with dogs or treated environments. Pyrethrins and pyrethroids are highly lipophilic and rapidly distribute to adipose tissue and the central and peripheral nervous system (Wismer and Means, 2012).

Mechanism of action

Type I and II pyrethroid and pyrethrin compounds can slow both opening and closure of voltage-gated sodium channels, causing prolonged neuronal depolarization or repetitive discharges of motor and sensory nerve fibres. Type II pyrethroids also inhibit binding of GABA to the GABA_A receptor, which prevents influx of chloride. This causes further membrane depolarization, blockade of action potential and failure of membrane repolarization.

Clinical presentation

Clinical signs of toxicosis may occur within 3 to 72 h of exposure and include muscle fasciculations and tremors, hypersalivation, ataxia, vomiting, diarrhoea, obtundation, mydriasis, hyperexcitability, hyperactivity, paresthesia, hyperesthesia, seizures and dyspnoea. Type I pyrethroids tend to cause tremors and seizures, whereas type II pyrethroids cause depolarizing conduction blocks with weakness and paralysis (Wismer and Means, 2012). Death may occur but is uncommon.

Diagnosis

Clinical diagnosis is based on history of exposure and clinical presentation. The pet-owners should be specifically questioned on recent ectoparasite treatment either directly on the affected animal or other pets in the house.

Management

Treatment is symptomatic and supportive, including dermal decontamination by bathing with a mild hand-dishwashing detergent and water (in case of dermal exposure to spot-on preparations), methocarbamol (Table 4.1), AEMs (see Table 4.1 and Chapters 12 and 24), fluid therapy and oxygenation (if necessary). Activated charcoal (Table 4.1) can be used in case of oral exposure. The use of intravenous lipid emulsion (Table 4.1) as adjunctive treatment to reduce tissue concentrations of permethrin has produced encouraging results in cats with permethrin toxicosis (Haworth and Smart, 2012; Kuo and Odunayo 2013).

Paresthesia to spot-on preparations may be treated by rubbing vitamin E, corn or olive oil on the application area (Wismer and Means, 2012).

Prognosis

Clinical signs generally resolve within 72 h following appropriate treatment (Hansen, 2006). However, recovery may take a week or longer in some cases. Delayed treatment and generalized seizures are associated with a less favourable prognosis and increased probability of death. Mortality rates vary between 5% and 45% in cats.

Organophosphates and carbamates

Overview

Organophosphates (chlorpyrifos, diazinon, dichlorvos, fenthion) and carbamates (aldicarb, methomyl, carbofuran, carbaryl) are widely used for insect and nematode control in dogs and cats and for insect control in the household and garden. They are available as sprays, pour-ons, oral anthelminthics, baits, collars, dips, dusts and granules (Wismer and Means, 2012). Exposure commonly results from accidental cutaneous overdose or ingestion (Dorman and Fikes, 1993). Organophosphate toxicity may result in acute (<7 h), intermediate (7–96 h) and delayed (1–4 weeks) syndromes.

Mechanism of action

Organophosphates are irreversible inhibitors of acetylcholine esterase (AChE) and recovery depends upon synthesis of new AChE. Carbamates are reversible inhibitors of AChE with restoration of AChE activity when the carbamate insecticide and enzyme separate. Organophosphate and carbamate intoxication results in accumulation of the neurotransmitter acetylcholine in the synaptic cleft and activation of muscarinic, nicotinic and CNS cholinergic synapses.

Clinical presentation

Clinical signs of acute organophosphate toxicity develop within minutes to hours depending on dose, route and toxicity of the compound and include:

• Muscarinic signs (associated with parasympathetic stimulation) such as hypersalivation, lacrimation, urination, increased gastrointestinal motility, defecation, bradycardia, dyspnoea and miosis;
• Nicotinic signs (associated with skeletal muscle stimulation) such as muscle fasciculations and tremors, which may result in a rigid stance and gait, and eventually weakness and paralysis;
• CNS signs including anxiety, restlessness, hyperactivity, obtundation to coma and generalized seizures.

All of the above clinical signs are not necessarily seen in every case and variability depends on toxicant type, dosage, formulation, route of exposure, poisoned species and stage of intoxication. Cats are generally more susceptible to AChE inhibitors than dogs. Death from either organophosphates or carbamates is associated with respiratory dysfunction resulting from respiratory tract secretions, bronchiolar constriction, intercostal and diaphragm muscle paralysis and CNS-mediated respiratory paralysis.

The intermediate and chronic (also named organophosphate-induced delayed neuropathy) syndromes are characterized by generalized neuromuscular weakness. The reader is referred to other textbooks for further information on these syndromes that are not characterized by seizures.

Diagnosis

Clinical diagnosis is based on history of ingestion, clinical presentation and improvement or resolution of muscarinic signs after atropine administration. If the diagnosis is uncertain, a test dose of atropine (0.02 mg/kg IV) can be administered after evaluating the baseline heart rate. If the heart rate increases, the pupils dilate and hypersalivation stops in 10–15 min, the animal is unlikely to have organophosphate or carbamate intoxication as it takes approximately 10 times this test dose to resolve clinical signs caused by these compounds.

Laboratory findings of heparinized whole blood cholinesterase (ChE) activity reduced by 50% of normal (based on the normal range for that laboratory) suggest exposure, whereas ChE activity less than 25% of normal indicates toxicosis in animals with characteristic clinical signs (Wismer, 2012). ChE activity of heparinized whole blood is a combination of true AChE activity of RBCs and pseudo-ChE activity of serum. Packed cell volume should be checked on blood samples for AChE testing as anaemia can result in decreased AChE activity. Blood should be kept refrigerated to prevent the loss of enzyme activity. ChE activity can remain decreased for 6 to 8 weeks following organophosphate exposure, whereas it may be normal in animals with carbamate intoxication. Carbamates bind reversibly with AChE in the body and they may also dissociate from AChE or pseudo-ChE in a blood tube or other specimen during transit. Stomach content, vomitus, hair, or suspected baits can be submitted to the laboratory for an organophosphate or carbamate residue screen.

Management

Treatment includes administration of atropine

(0.2 to 0.4 mg/kg, one-fourth of the initial dose slowly IV and the rest IM or SQ) to counteract the muscarinic signs, decontamination and prevention of further toxin adsorption (Table 4.1), skeletal muscle relaxants (Table 4.1), AEMs (see Table 4.1 and Chapters 12 and 24) and supportive care. Dramatic cessation of parasympathetic signs is usually observed within 3 to 5 min after administration of atropine IV. Repeated administration of atropine, IV, IM or SC, at one-half of the initial dose is often required, especially in cats with organophosphate toxicity. Glycopyrrolate (0.01–0.02 mg/ kg IV) may also be used to control the muscarinic signs. Cardiac activity should be monitored. In animals with dermal exposure, decontamination is performed by bathing with a mild hand-dishwashing detergent and water. The individuals bathing the animals should wear protective gloves and aprons. In animals with oral exposure, decontamination is performed by induction of emesis (in asymptomatic animals) or gastric lavage and administration of activated charcoal with a saline cathartic or sorbitol (Table 4.1). Fluid therapy should be performed to correct possible dehydration, electrolyte imbalances and acidosis. Endotracheal intubation and ventilation may be necessary in cases of respiratory paralysis.

Pralidoxime chloride (2-PAM) is an AChEreactivating oxime that acts specifically on the organophosphate-enzyme complex and counteracts the nicotinic cholinergic signs (Clemmons, 1990). Administration of 2-PAM should begin within 24 to 48 h of organophosphate intoxication as after this time the toxic compounds are irreversibly bound to AChE. The recommended dose is 10–20 mg/kg slowly IV with fluids over 30 min; or IM or SC. If nicotinic signs persist, the same dose can be repeated every 8–12 h, for 24 to 48 h. 2-PAM should be discontinued after three or four treatments if there is no response or nicotinic signs worsen. 2-PAM produces better results when atropine has already been administered; the atropine dose can be reduced when 2-PAM is used. Signs of muscle weakness and fasciculations usually disappear within 30 min. 2-PAM is not beneficial in treating carbamate toxicosis.

If it is uncertain whether the toxicant is an organophosphate or a carbamate, 2-PAM should be used unless it is likely that the toxicant is carbaryl, in which case 2-PAM may be harmful. Administration of diphenhydramine (1 to 4 mg/kg PO every 8 h) has been recommended by one author to counteract the nicotinic signs 24 to 48 h after intoxication and to prevent signs of subacute intoxication (Clemmons et al., 1984; Clemmons, 1990). However, its use is controversial and other authors consider diphenhydramine contraindicated in animals with organophosphate and carbamate intoxication (Ellenhorn et al., 1997).

Prognosis

Prognosis depends on the dose and duration of exposure to the insecticide and promptness of adequate treatment (Blodgett, 2006). Generally, prognosis is considered good unless the animal shows signs of respiratory dysfunction or seizures (Wismer and Means, 2012).

Chlorinated hydrocarbons

Overview

Chlorinated hydrocarbons, also named organochlorines, have been used for prevention and control of insect infestations around farms, homes and on animals from the 1950s through the 1970s. Chlorinated hydrocarbons include endrin, aldrin, dieldrin, heptachlor, lindane, DDT and endosulfane. Most of these insecticides have been banned because of accumulating tissue residues and environmental persistence. Contaminated soils or leakage from old dump sites are possible sources of exposure for wildlife and domestic carnivores. The most likely source of exposure in dogs and cats is old stockpiles of insecticides and improper waste disposal. In addition, a few of these compounds may still be legal for ectoparasite control in dogs in certain countries. Exposure in dogs and cats may occur by ingestion, inhalation (less likely), or cutaneous absorption when the insecticide is applied topically and accidentally overdosed (Raisbeck, 2006).

Mechanism of action

The mechanism of action of most chlorinated hydrocarbons is poorly understood and they are considered to be nonspecific stimulants of the central nervous system (Hatch, 1988). Persistent opening of neuronal sodium channels and GABA inhibition are possible mechanisms.

Clinical presentation

Clinical diagnosis is based on history of exposure and clinical presentation. Clinical signs include hypersensitivity, nervousness, muscle tremors, spastic gait, ataxia, mydriasis, salivation, vomiting and severe generalized tonic-clonic seizures, which may be precipitated by external stimuli and may last for 2–3 days. Hyperthermia occurs as a result of the seizures. Death may occur within minutes or hours or after several days.

Diagnosis

Confirming the diagnosis is quite difficult as chlorinated hydrocarbon residues may be found in blood or tissue of normal animals due their persistence in tissues (particularly fat) (Raisbeck, 2006). Tissue samples should be submitted in glass or metal containers rather than plastic ones.

Management

Treatment is symptomatic since there is no known antidote and involves dermal

(e.g. bathing) or gastrointestinal (e.g. emesis or gastric lavage, repeated administrations of activated charcoal due to enterohepatic recirculation) (Table 4.1) decontamination, skeletal muscle relaxants (Table 4.1), AEMs (see Table 4.1 and Chapters 12 and 24) and supportive care (including ventilator support in severely affected animals). Forced diuresis with 5% mannitol in 0.9% sodium chloride can enhance urinary excretion.

Prognosis

Signs of acute toxicosis usually abate in 1 to 2 days. Complete recovery may take weeks. The prognosis is guarded to good, depending on the dose of exposure, severity of neurological dysfunction and promptness of treatment.

Molluscicides

Metaldehyde

Overview

Metaldehyde is a cyclic tetramer of acetaldehyde included in a variety of snail and slug baits, most commonly in the form of green granules, but also as liquid, powder or pellets (Yas-Natan et al., 2007).

Protein-rich material, such as bran or grain, is usually added to the bait to make it more attractive to slugs and snails, causing this type of bait to be palatable to dogs as well. Baits are sometimes mixed with other pesticides, most commonly with carbamate insecticides. In some countries, metaldehyde is also used as a fuel in small heating systems, such as camping stoves and lamps. Metaldehyde is degraded to various aldehydes in the stomach resulting in a formaldehyde odour of the gastric contents.

Mechanism of action

The exact mechanism of metaldehyde toxicity is currently unclear and may be associated with an increase in monoamine oxidase activity and a decrease in gamma-aminobutyric acid (GABA), norepinephrine and serotonin concentrations.

Clinical presentation

Clinical signs occur within 20 min to 3 h from ingestion and include tachycardia, tachypnea, hypersalivation, muscle tremors, vomiting, hyperesthesia, nystagmus (especially in cats), ataxia, opisthotonus, seizures, hyperthermia, diarrhoea and obtunded mental status to coma (Yas-Natan et al., 2007). The seizures are tonic, similar to those secondary to strychnine intoxication, but generally they do not worsen with stimuli in dogs. However, in cats seizures have been reported to be triggered or exacerbated by auditory, visual or tactile stimuli. Metabolic acidosis is common. Death may occur from respiratory failure 4 to 24 h after ingestion or from liver failure 3 to 4 days after ingestion in dogs. Post-metaldehyde intoxication liver disease has not been reported in cats.

Diagnosis

Diagnosis can be reached by laboratory analysis of gastric contents, serum, urine and liver. Samples must be kept frozen for analysis.

Management

Treatment includes induction of emesis (in mildly affected animals with no seizures and toxin ingestion <3 h), gastric lavage (toxin ingestion >3 h, ingestion of large volumes), activated charcoal (Table 4.1), methocarbamol for muscle tremors, AEMs (see Table 4.1 and Chapters 12 and 24), fluid therapy to correct metabolic acidosis, convective whole body cooling (e.g. wetting the fur, fan) and respiratory support. General anaesthesia may be necessary in animals that do not respond to AEMs (see Chapter 24). After the acute clinical signs have been controlled, treatment must focus on minimizing possible liver damage in dogs.

Prognosis

Prognosis is generally good in animals treated promptly and aggressively. A retrospective study including 18 dogs with metaldehyde intoxication reported a recovery rate of 83% (Yas-Natan et al., 2007). Prognosis is guarded in animals presenting with severe hyperthermia (>41.6°C or 107°F). Prolonged status epilepticus following metaldehyde intoxication has not been associated with spontaneous recurrent seizures in dogs (Jull et al., 2011). Recovery may take approximately 2 weeks in cats (Puschner, 2006).

Rodenticides

Strychnine

Overview

Strychnine has been used as pest control of rodents and other animal species worldwide.

Its sale has been restricted or banned without special permits in several countries. However, malicious and less commonly accidental or secondary (from ingestion of strychnine-poisoned rodents) poisonings of companion animals still occurs (Berny et al., 2010). The toxic dose in most animals ranges from 0.3 to

1.0 mg/kg with the lethal dose being 2.0 mg/kg in cats.

Mechanism of action

Strychnine antagonizes stereochemically and competitively the motor inhibitory neurotransmitter glycine at the brainstem and spinal cord level and inhibits glycine release from Renshaw cells. Some supraspinal signs may also be associated with strychnine inhibition of GABA.

Clinical presentation

Clinical signs generally occur within 10 min to 2 h after toxin ingestion and typically begin with nervousness and restlessness, rapidly progressing to increased tone of both extensor and flexor muscles resulting in a stiff gait. All skeletal muscles including the appendicular, epaxial, facial, abdominal and respiratory muscles have tetanic spasms. Auditory

(e.g. loud noise), visual (e.g. bright light) and tactile stimuli exacerbate the tetanic muscle spasms and can trigger tonic seizures. This feature, however, is not specific to strychnine and has been observed in other types of poisonings including metaldehyde, penitrem A, roquefortine and chlorinated hydrocarbons. Hyperthermia (secondary to the severe muscle contractions) is commonly observed. The animal remains conscious during seizures unless respiratory paralysis occurs. Apnea can lead to cerebral anoxia, loss of consciousness and death 30 min to 2 h following the onset of neurologic signs, if the animal is untreated (Murphy, 2002).

Diagnosis

Baits may have specific colours in some countries, which can help recognition during gastric decontamination. Definitive diagnosis is based on toxicologic analysis of stomach contents, urine, blood or hepatic and renal tissue. Ante-mortem analysis of stomach contents and urine is most likely to provide a diagnosis. The dimethoxy derivative of strychnine, brucine (2,3-dimethoxystrychnidin-10-one), may be detected in serum.

Management

Treatment includes decontamination (induction of emesis in asymptomatic animals or gastric lavage followed by activated charcoal and cathartic administration in animals showing clinical signs of intoxication) (Table 4.1), promoting toxin excretion, controlling the tetany and seizures (Table 4.1, see Chapters 12 and 24), convective whole body cooling, adequate oxygenation (Murphy, 2002) and supportive care. Sedation or general anaesthesia for 24–72 h may be required. Forced diuresis with 5% mannitol in isotonic saline and acidification of the urine will enhance urinary elimination of strychnine. Animals with respiratory failure should be administered oxygen and if needed intubated and ventilated. Non-anesthetized animals should be kept in a dimly lit, quiet area and all forms of sensory stimulation should be minimized.

Prognosis

Prognosis is fair to guarded, depending on the amount of toxin ingested and promptness of treatment. If the animal survives the first 24 h post-toxin ingestion, prognosis for complete recovery is good.

Bromethalin

Overview

Bromethalin is a rodenticide that is sometimes implicated in accidental or malicious poisonings of small animals. It is available in pelleted forms such as place packs, blocks or bars of bait and baited worms (DeClementi and Sobczak, 2012). Exposure mainly occurs by ingestion of bromethalin bites. Secondary (or relay) poisoning may occasionally occur by ingestion of bromethalin-poisoned rodents. Bromethalin is readily absorbed from the gastrointestinal tract and reaches peak plasma levels within several hours after ingestion.

It is highly lipophilic and therefore reaches high concentration in the brain.

Mechanism of action

Bromethalin and its active metabolite, desmethyl bromethalin, uncouple oxidative phosphorylation resulting in decreased cellular adenosine triphosphate (ATP) concentrations and reduced activity of ATP-dependent sodium and potassium ion channel pumps. This produces an increase in intracellular sodium concentrations, intracellular movement of water and consequent cerebral oedema, vacuolization of myelin and increased intracranial pressure.

Clinical presentation

Onset and type or clinical signs are dose-dependent. Dogs ingesting 2.5 to 5 mg/kg of bromethalin develop clinical signs in 1 to 4 days (Murphy, 2002), whereas dogs ingesting more than 5 mg/kg of bromethalin may develop clinical signs within 2–24 h after ingestion. Low doses (e.g. 2.5 mg/kg in dogs) result in pelvic limb ataxia and/or paresis/paralysis with extensor rigidity and obtunded mental status. High doses (e.g. 5 to 6.5 mg/kg in dogs) produce hyperexcitability, tremors, focal motor and generalized seizures, hyperthermia, obtunded mental status, which can progress to stupor, coma, decerebrate posture and death secondary to respiratory arrest (Dorman et al., 1990a, b). Cats ingesting 0.54 mg/kg develop signs (including ataxia, focal motor seizures, vocalization, obtunded mental status and stupor) 2 to 7 days after exposure (Dorman et al., 1990c). Most recently clinical signs and death have been reported following exposure to bromethalin doses as low as 0.46 mg/kg in dogs and 0.24 mg/kg in cats (DeClementi and Sobczak, 2012).

Diagnosis

The ante-mortem diagnosis is most often made based on the history of bait ingestion (stools may have a green discoloration) and the development of clinical signs. Definitive diagnosis can be reached only post-mortem by detecting bromethalin or its metabolites in kidney, liver, fat or brain samples using gas chromatography with electron capture (Dorman et al., 1990a, b). Samples should be submitted frozen and protected from light.

Management

There is no antidote to bromethalin. Treatment involves decontamination and prevention of further toxin adsorption (induction of emesis or gastric lavage followed by repeated administration of activated charcoal and administration of a cathartic with the initial dose of activated charcoal) (Table 4.1), AEMs (see Table 4.1 and Chapters 12 and 24) in seizuring animals, methocarbamol or diazepam in animals with excessive tremors (Table 4.1), and supportive care. The repeated and prolonged administration of activated charcoal (1–5 g/kg every 6–8 h for up to 2–4 days in animals ingesting high dosage of bromethalin) is indicated due to the enterohepatic recirculation of bromethalin. Cerebral oedema can be treated with mannitol. Serum sodium levels should be closely monitored due to the potential for hypernatraemia of repeated administration of activated charcoal as well as mannitol.

Prognosis

Prognosis is fair with prompt and prolonged treatment in animals with mild clinical signs. Animals with obtundation and ataxia may recover over a period of 2 to 4 weeks. Prognosis is guarded in animals with severe neurological signs such as tremors, seizures, coma or paralysis.

Zinc phosphide

Overview

As with other rodenticides, zinc phosphide poisoning can be accidental or malicious in small animals. Secondary (or relay) poisoning has been reported in dogs feeding on the dead rodents and other animals poisoned by zinc phosphide. When zinc phosphide reaches the stomach, on exposure to moisture and an acidic environment, it hydrolyses to phosphine gas which is rapidly absorbed across the gastric mucosa and distributed systemically where it exerts its toxic effect (Proudfoot, 2009).

Mechanism of action

The corrosive action of zinc phosphide accounts for the early, acute and generally haemorrhagic emetic effect on the gastric mucosa. The systemic toxicity of zinc phosphide is caused by phosphine. Postulated mechanisms of action of phosphine include inhibition of cytochrome C oxidase with mitochondrial dysfunction and interruption of cellular respiration, inhibition of serum acetyl cholinesterase activity resulting in cholinergic overdrive and formation of reactive oxygen species (ROS) with resultant oxidative stress, damage to cell lipids, proteins and nucleic acids and cell death (Proudfoot, 2009).

Clinical presentation

Clinical signs occur within 15 min to 4 h after ingestion (but may be delayed by 12 to 18 h) and include vomiting (often with frank or dark blood clots), aimless pacing or running, vocalization, anxiety, discomfort, generalized muscle fasciculations, tremors, exaggerated response to external stimuli and tonic-clonic or tonic seizures. In addition, the production of phosphine gas within the stomach may lead to gastric or abdominal distension resulting in abdominal pain and potentially gastric dilatation-volvulus. Death can occur (Murphy, 2002; Proudfoot, 2009).

Diagnosis

The odour of phosphine is similar to rotten fish or acetylene gas and may be detected on the breath or in the stomach contents of intoxicated animals. However, some types of zinc phosphide may also be odourless. Owners should be warned about the risk of phosphine gas inhalational exposure during transit to the veterinary hospital and adequate precautions should be taken to minimize veterinary and support personnel exposure. The presence of zinc phosphide (phosphine gas) can be detected in stomach contents, vomitus, or suspect bait by laboratory analysis (gas chromatography-mass spectrometry or Dräger detector tube test) (Murphy, 2002; Proudfoot, 2009). The sample should be frozen in an airtight container soon after collection to prevent loss of phosphine gas.

Management

No specific antidote exists. Treatment involves decontamination (gastric lavage and activated charcoal), reducing phosphine production by decreasing the acidity of the gastric lumen through administration of a liquid antacid

(e.g. magnesium or aluminium hydroxide and calcium carbonate) or 2–5% solution of sodium bicarbonate orally or through gastric lavage tube, symptomatic and supportive care (intravenous fluids, oxygen supplementation, gastro protectants, analgesia and hepatic supportive agents) and AEMs (see Table 4.1 and Chapters 12 and 24). Adequate room ventilation is imperative during gastrointenstinal decontamination (Gray et al., 2011).

Prognosis

Prognosis can be favourable in animals treated promptly and effectively (Murphy, 2002; Gray et al., 2011).

Sodium monofluoroacetate (Compound 1080)

Overview

Sodium monofluoroacetate was introduced as a rodenticide in the USA in 1946 and subsequently used as pest control particularly of non-native species such as the fox and possum in Australia and New Zealand, respectively. Sodium monofluoroacetate is one of the most toxic pesticides and its use is restricted to trained, licensed applicators. However, accidental or malicious poisoning of domestic animals can occur in baiting areas. Accidental or malicious poisoning of companion animals can occur directly from bait ingestion or secondarily following the ingestion of a poisoned carcass. Sodium monofluoroacetate can be absorbed from the gastrointestinal and respiratory tracts as well as across mucous membranes and abraded skin. Dogs are more susceptible than cats to this toxicant.

with oxaloacetate to produce fluorocitrate. Fluorocitrate is converted to 4-hydroxytrans-aconitate, which binds and inactivates aconitase resulting in inhibition of citrate oxidation. This results in inhibition of the tricarboxylic acid (TCA) or Kreb’s cycle, cellular energy depletion, citric acid and lactic acid accumulation, a decrease in blood pH, and interference with cellular respiration and metabolism of carbohydrates, lipids and proteins. Organs with cells with a high metabolic rate, such as the heart, brain and kidneys, are most susceptible to dysfunction (Goh et al., 2005; Parton, 2006; Proudfoot et al., 2006). In addition to blockade of the TCA cycle, citrate accumulates within blood to toxic concentrations and binds to calcium resulting in serum ionized hypocalcaemia.

Clinical presentation

Clinical signs occur 30 min to 2 h after ingestion, depending on the dose, and include restlessness, hyperirritability, hyperesthesia, running, barking and howling episodes, vomiting, salivation, defecation, diarrhoea, urination, tremors, tonic-clonic seizures, hyperthermia (in dogs) and eventually coma and death 2 to 12 h after the onset of clinical signs. Hypothermia, vocalization, cardiac arrhythmias and episodes of bradycardia between seizures have been reported in cats.

Diagnosis

Diagnosis of sodium monofluoroacetate poisoning is usually based on characteristic clinical signs in conjunction with known access to the poison. Clinical pathological changes include metabolic acidosis, serum-ionized hypocalcaemia and elevations in serum citrate concentrations over two to three times greater than the reference range. Analysis of gastric content from vomitus or lavage fluids can confirm the diagnosis. The sample should be kept frozen until analysis to avoid bacterial breakdown of the toxin.

gastric and colonic lavage in symptomatic animals, administration of activated charcoal with a cathartic) (Table 4.1), methocarbamol or diazepam in animals with excessive tremors (Table 4.1), AEMs (see Table 4.1 and Chapters 12 and 24) in seizuring animals, symptomatic and supportive care. Calcium gluconate (see hypocalcaemia) should be administered if serum-ionized calcium concentration is equal or lower than 0.8 mmol/l (3.2 mg/dl). In addition, administration of sodium bicarbonate or acetamide has shown promising results (O’Hagan, 2004; Parton, 2006). The recommended dosage of sodium bicarbonate is 300 mg/kg (3.6 ml/kg of 8.4% solution) IV over 15–30 min. Alternatively, half of the calculated dose may be given as a bolus and the remainder infused slowly. Administration of sodium bicarbonate may worsen hypocalcaemia and cause hypokalaemia and hypernatraemia. Serum-ionized calcium, sodium and potassium levels should be monitored regularly in order to implement fluid therapy as well as calcium and potassium supplementations as required.

The recommended dosage of acetamide (15 g of acetamide granules dissolved in 1 l of warmed 5% dextrose) in dogs is 10 to 25 ml/kg IV (infused though a filter following sterilization) over a 60-min period followed by approximately 5 ml/kg/h IV for the next 12 to 18 h until resolution of clinical signs. In cats with fluoroacetate poisoning, the acetamide dose should be reduced by at least 75% (Parton, 2006). Electrolytes should be closely monitored as hyponatraemia may develop due to the large volume of administered free water.

Other treatment modalities are being investigated in Australia and New Zealand due to the higher prevalence of sodium monofluoroacetate poisoning than other countries.

Prognosis

The prognosis is poor to grave, depending on the amount of sodium monofluoroacetate ingested and the severity of clinical signs at initial evaluation (Goh et al., 2005). Early acetamide or sodium bicarbonate treatment and good supportive care can improve survival (O’Hagan, 2004; Parton, 2006).

Automotive Products

Ethylene glycol

Overview

Ethylene glycol is a commercial antifreeze automotive product whose metabolites, including glycolic acid, are extremely toxic to dogs and cats. Common sources of exposure include container spill, engine flush or engine leak (Khan et al., 1999). Intoxication is most commonly accidental but can also be malicious.

Mechanism of action

Ethylene glycol is biotransformed to glycolic acid, which is metabolized to formic acid, oxalic acid and oxalate. These metabolites are highly toxic and result in severe metabolic acidosis and acute renal failure. The oxalate combines with calcium to form oxalate crystals in renal tubules (especially proximal), urine and within the lumen or perivascular space of cerebral capillaries (Dial et al., 1994a).

Hypocalcaemia secondary to calcium oxalate deposition may contribute to CNS signs, although the concurrent metabolic acidosis shifts calcium to the ionized active state, reducing the chances of hypocalcaemia-associated clinical signs. Acidosis may also contribute to cerebral damage.

Clinical presentation

Clinical signs usually occur within 30 to 60 min of exposure and include obtundation, vomiting, ataxia, seizures, hypothermia, severe metabolic acidosis, serum hyperosmolality, polydipsia, polyuria, calcium oxalate monohydrate and dihydrate crystalluria, isosthenuria and eventually renal failure (Dial et al., 1994a).

Diagnosis

Ethylene glycol colorimetric spot tests are available for use with urine and serum. However, these tests can give false negatives in cats as the ethylene glycol toxic dose in cats can be below the detectable level of the

can occur in animals administered medications containing propylene glycol (diazepam, activated charcoal). A quantitative test kit has recently been evaluated and may aid in timely diagnosis of ethylene glycol exposure (Scherk et al., 2013). Laboratory tests for rapid analysis of serum, plasma or urine for ethylene glycol and glycolic acid also have been reported (Smith and Lang, 2000; Van Hee et al., 2004). Birefringent crystals may be detected in urine 3 h (in cats) and 5 h (in dogs) after ingestion. Anion gaps greater than 40–50 mEq/l are also suggestive of ethylene glycol intoxication. Signs of acute renal failure (azotaemia, hyperkalaemia, hyperphosphataemia) are usually seen approximately 12–48 h post-ethylene glycol ingestion. Moderate to severe hypocalcaemia is frequently present. Serum osmolality as high as 450 mosm/kg serum and an osmole gap as high as 150 mosm/kg serum may be detected 3 h after ethylene glycol ingestion. Both the gap and the measured osmolality may remain elevated for approximately 18 h after ingestion. Ultrasonographic changes vary from mild to marked increases in renal cortical echogenicity. Another diagnostic procedure that may help in the early diagnosis of ethylene glycol intoxication is examination of the oral cavity, face, paws, vomitus and urine with a Wood’s lamp to determine whether they appear fluorescent. Many antifreeze solutions contain sodium fluorescein, a fluorescent dye that aids in the detection of leaks in vehicle coolant systems (Thrall et al., 2006).

Treatment should be instituted promptly even if the results of confirmatory tests are not available yet. Treatment is aimed at preventing absorption, increasing excretion and preventing metabolism of ethylene glycol using a chemical antidote such as fomepizole or ethanol. Although therapeutic recommendations have traditionally included induction of vomiting, gastric lavage and administration of activated charcoal, it is likely that these procedures are not beneficial because of the rapidity of ethylene glycol absorption (Thrall et al., 2006). In addition, absorption of ethanol is inhibited by charcoal. Fomepizole (4-methylpyrazole), a competitive inhibitor of alcohol dehydrogenase, is considered safe and effective for dogs if started within 8 h of exposure (Table 4.2) (Dial et al., 1994a; Connally et al., 1996). Fomepizole can be used also in cats, although a much higher dosage is required (Dial et al., 1994b). If Fomepizole is not available, 20% ethanol can be used (Table 4.2). It acts as a competitive substrate for the enzyme alcohol dehydrogenase. Although effective in the treatment of ethylene glycol toxicity, ethanol may result in CNS and respiratory depression. Supportive and symptomatic care includes intravenous fluids to correct dehydration, acid-base and electrolyte imbalances and to promote diuresis, and AEMs in seizuring animals (see Table 4.1 and Chapters 12 and 24).

Prognosis

The mortality rate in dogs is reported to range from 59% to 70% and is thought to be even

Medication Species Dosage

higher in cats. However, prognosis has been reported to be favourable in dogs and cats treated within 8 and 3 h following ingestion, respectively (Thrall et al., 2006).

Detergents and Disinfectants

Hexachlorophene

Overview

Hexachlorophene is used as a germicide in soaps, shampoos and disinfectant solutions. Exposure may result from both topical contact and ingestion (Bath, 1978; Thompson et al., 1987). Nursing puppies have been poisoned following hexachlorophene application to the mammary glands of the bitch (Ward et al., 1973).

Clinical presentation

Clinical signs in dogs are usually characterized by acute onset of tremors, especially of the head, which may increase with excitement and disappear during rest or sleep. Opisthotonus, severe seizures and death have also been reported. Clinical signs in cats include mydriasis, vomiting, weakness, ataxia, spastic or flaccid paralysis and hypovolaemic shock (Thompson et al., 1987).

Management

Treatment involves decontamination, supportive care, skeletal muscle relaxants and AEMs (see Table 4.1 and Chapters 12 and 24).

Prognosis

Clinical recovery may take 4 to 6 weeks.

Heavy Metals

Lead

Overview

Lead is the most common heavy metal causing toxicosis in animals. Sources of lead intoxication include lead-based paints, linoleum, putty, roofing felt, golf balls, bullets or pellets, fishing weights, old car batteries, wheel weights, improperly glazed ceramic water bowls and toys (Morgan, 1994). The most commonly identified source of exposure in dogs and cats is lead-based paint chips or dust, usually from home renovation. Cats can ingest old paint dust and chips contaminating the coat while grooming (Knight and Kumar, 2003). High-fat low-calcium diets may facilitate absorption of lead from the alimentary tract.

Mechanism of action

The toxic mechanism of lead is caused predominantly by its ability to substitute for other polyvalent cations (particularly divalent cations, such as calcium (Ca²⁺) and zinc (Zn²⁺) ) in multiple molecular processes, and subsequently affect metal transport, energy metabolism, membrane ionic channel conduction, inter- and intracellular signalling, diverse enzymatic processes (including sulfhydryl-containing enzymes involved in haem synthesis), protein maturation, cell adhesion and regulation of gene transcription. The effects of lead neurotoxicity include decreased cellular energy metabolism, impaired haem biosynthesis (resulting in increased erythrocytes fragility, basophilic stippling and circulating nucleated erythrocytes), oxidative stress, lipid peroxidation, altered activity of second messenger systems, altered neurotransmitter release (e.g. decreased GABA neurotransmission) and neurotransmitter receptor density, excitotoxicity, apoptosis impaired development and function of oligodendrocytes, abnormal myelin formation, abnormal neurotrophic factor expression, abnormal dendritic branching patterns and disruption of the blood-brain barrier (Lidsky and Scneider, 2003; Garza et al., 2006).

Clinical presentation

Clinical presentation is variable depending on duration and degree of exposure. Gastrointestinal signs (including vomiting, anorexia, diarrhoea and sometimes also abdominal pain) precede or accompany neurological signs (including seizures, blindness, obtundation, hyperexcitability, behavioural changes, head pressing, ataxia and tremors) (Zook et al., 1967; Knecht et al., 1979; Morgan, 1994). Clinical signs in cats may be more vague than in dogs and most commonly include anorexia, vomiting and seizures. Central vestibular abnormalities, including vertical nystagmus and ataxia, have also been reported in cats with lead poisoning (Knight et al., 2001). Clinical signs can be acute or chronic. In cases of chronic exposure,the seizures are intermittent(Bratton and Kowalczyk, 1989).

Diagnosis

Haematology and serum biochemistry may reveal one or more of the following abnormalities: nucleated erythrocytes and basophilic stippling in red blood cells with no or mild anaemia, increased packed cell volume, leukocytosis, elevated hepatic enzymes, hyperglycaemia and hypercholesterolaemia. Radiography may allow identification of metallic material in the gastrointestinal tract (e.g. golf balls or toys) or subcutaneously (e.g. pellets). Blood lead levels of 40 mg/dl or higher are considered diagnostic of lead poisoning (Morgan, 1994). However, blood lead concentration fluctuates and due to sequestration in other organs does not necessarily correlate with total body burden of lead or with clinical signs (Bratton and Kowalczyk, 1989; Knight and Kumar, 2003). If the blood lead values are in the high normal range and lead poisoning is suspected clinically, treatment followed by measurement of urine lead levels can be diagnostic. Electroencephalographic changes in non-sedated dogs are characterized by intermittent high-amplitude slow wave activity (Knecht et al., 1979). A post-mortem diagnosis can be made by analysing lead concentration in kidneys, liver, brain and bones (Knight and Kumar, 2003). Levels of 3.6 to 10 mg/g by wet weight of liver tissue are considered diagnostic of lead toxicosis (Bratton and Kowalczyk, 1989).

Management

Treatment of lead toxicosis involves prevention of further exposure, decontamination of the individual (e.g. bathing in case of lead-laden dust coat exposure, administration of cathartics and/or enemas, endoscopic or surgical removal of lead-containing foreign bodies), decontamination of the environment, supportive care and chelation therapy to remove lead from the blood and soft tissues. The cathartic magnesium sulfate (250–500 mg/kg PO in dogs and 200 mg/kg PO in cats) can help to decrease lead absorption by forming insoluble lead sulfate. Treatment with the chelating agent, calcium disodium ethylene diamine tetraacetate (CaNa₂EDTA), using a dose of 25 mg/kg intravenously or orally, four times a day for 2 to 5 days, has resulted in recovery within 36 to 48 h. However, in some animals, CaNa2EDTA has initially worsened neurologic signs. The most recently available chelator, succimer (meso-2,3-dimercaptosuccinic acid), administered at 10 mg/kg of body weight, orally, every 8 h, for 10 days, has been reported to be safe and effective in the treatment of lead poisoning in dogs and cats (Ramsey et al., 1996; Knight et al., 2001). Succimer may also be administered rectally as a solution in patients that are vomiting or that are unable to take oral medications. Chelation therapy with calcium EDTA (27 mg/kg SC for 5 days) alone or in association with D-penicillamine (33–55 mg/kg/day divided every 6–8 h) has also been reported. Seizures should be treated promptly with diazepam and/or other AEMs (see Table 4.1, and Chapters 12 and 24). Thiamine supplementation (1–2 mg/kg IM or 2 mg/kg PO every 24 h) can contribute to neurological improvement.

Prognosis

Clinical response to therapy is the best prognostic indicator. Prognosis is favourable in the majority of lead-poisoning cases treated with chelating agents (Morgan, 1994). Continuous or uncontrolled seizures may be associated with a less favourable prognosis.

Poisonous Plants

Seizures have been reported following exposure to the plants listed in Box 4.7 (Barr, 2006).

Several of these plants cause vomiting. The gastric content from vomitus or lavage fluids can be submitted for plant material

Box 4.7. Poisonous plants.

Amaryllis (Amaryllis spp.) Angel’s trumpet (Brugmansia spp.) Aralia, balfour aralia, dinner plate aralia, ming

aralia, geranium-leaf aralia, wild coffee, coffee tree (Polyscias spp.) Black locust (Robinia pseudoacacia)

Bleeding heart, Dutchman’s breeches, squirrel

corn, staggerweed (Dicentra spp.) Box, common box, boxwood (Buxus sempervirens) Broom (Cytisus spp.)

Candelabra cactus, false cactus, mottled

spurge, dragon bones (Euphorbia lactea) Castor bean (Ricinus communis) Chinaberry tree (Melia azedarach) Croton (Croton tiglium) English ivy, Irish ivy, common ivy (Hedera helix) Euphorbium (Euphorbia resinifera) Golden chain tree (Laburnum anagyroides) Golden corydalis, bulbous corydalis, scrambled

eggs, fitweed, fumitory (Corydalis spp.) Meadow saffron, autumn crocus (Colchicum autumnale) Mediterranean thistle (Atractylis gummifera) Milkweed, butterfly weed (Asclepias spp.) Mimosa, silk tree (Albizia julibrissin)

Pencil tree, milkbush, Indian tree, rubber euphorbia,

finger tree, naked lady (Euphorbia tirucalli)

Persian violet, alpine violet, sowbread

(Cyclamen spp.)

Sago palm, leatherleaf palm, Japanese fern

palm (Cycas revoluta, Cycas spp.) Sandbox tree, monkey pistol (Hura crepitans) Schefflera, umbrella tree, rubber tree, starleaf

(Schefflera spp.) Sea onion (Urginea maritima) Spurge, creeping spurge, donkey tail (Euphorbia myrisinites)

Squill, starry hyacinth, autumn scilla, hyacinth scilla, Cuban lily, Peruvian jacinth, hyacinth of Peru, bluebell (Scilla spp.) Snowdrop (Galanthus nivalis) Tobacco, tree tobacco (Nicotiana spp.) Tung oil tree (Aleurites spp.) Water hemlock, cowbane (Cicuta spp.) White cedar (Thuja occidentalis)

Yellow jessamine, Carolina jessamine

(Gelsemium sempervirens) Yesterday today and tomorrow (Brunfelsia spp.) Zamia (Macrozamia spp.) Zulu potato, climbing onion (Bowiea volubilis)

information on geographic distribution of these plants and clinical presentation, treatment and prognosis following exposure is beyond the scope of this book and can be found elsewhere (Barr, 2006).

Blue-green algae (cyanobacteria)

Overview

Blue-green algae (cyanobacteria) are commonly found growing in fresh and salt water in temperate areas worldwide. Under certain climatic and nutritional conditions cyanobacteria reproduce explosively, resulting in algae blooms that accumulate at the water surface and sometimes produce hepatotoxins and/or neurotoxins. Dogs can be exposed by drinking or ingesting contaminated water while swimming (Edwards et al., 1992; Gugger et al., 2005; Puschner et al., 2010; Elford et al., 2012; Faassen et al., 2012). Cats seem to be affected less commonly than dogs.

Mechanism of action

Blue-green algae Anabaena, Aphanizomenon, Lyngbya and Oscillatoria can produce the neurotoxins anatoxin-a (a potent postsynaptic depolarizing neuromuscular blocking agent) and anatoxin-as (a potent acetylcholinesterase inhibitor) (Hooser and Talcott, 2006).

Clinical presentation

Clinical signs often occur very rapidly (10 min to a few hours) after oral exposure and include salivation, lacrimation, urination and defecation, muscle rigidity, muscle tremors, seizures, appendicular paralysis, respiratory paralysis and death within 30 to 60 min of the onset of clinical signs.

Diagnosis

Management

Treatment includes decontamination (bathing if the animal went into the contaminated water, gastric lavage, activated charcoal and/ or a cathartic) (Table 4.1), AEMs in seizuring animals (see Table 4.1 and Chapters 12 and 24) and aggressive supportive care including intravenous fluid therapy, atropine (in animals exposed to anatoxin-as neurotoxin) and respiratory support (Hooser and Talcott, 2006).

Prognosis

The prognosis for animals that show clinical signs is guarded to poor.

Mycotoxins

Penitrem A and roquefortine

Overview

The mycotoxins penitrem A (produced by Penicillium crustorum) and roquefortine (produced predominately by Penicillium roqueforti as well as other Penicillium species including

P. crustorum) can be found in mould-contaminated cheese, bread, rice, walnuts and decaying organic matter such as silage, garbage and compost. Absorption after oral ingestion is rapid. Dogs are more commonly affected than cats, probably because of their tendency to scavenge and ingest rotting material (Barker et al., 2013).

Mechanism of action

The exact mechanism of action of these mycotoxins is unknown; however, results of in vitro studies indicate that penitrem A may antagonize the release or action of glycine in the CNS and interfere with the release of glutamate, aspartate and gamma-aminobutyric acid in central and peripheral synapses (Young et al., 2003).

generalized muscle tremors (generally 2–3 h following ingestion), seizures, ataxia, tachycardia and hyperthermia (Lowes et al., 1992; Young et al., 2003; Eriksen et al., 2010). Seizures can sometimes be triggered by auditory (e.g. loud noise), visual (e.g. bright light) and tactile stimuli. Status epilepticus can occur.

Diagnosis

The presumptive diagnosis is based on the history of ingestion of mouldy food or compost and exclusion of other aetiologies. Definitive diagnosis is made by identification of the mycotoxins by means of laboratory analysis of samples of the ingested material, gastric content, serum or urine. Liquid chromatography-mass spectrometry can be used to screen for the mycotoxins penitrem A and roquefortine in serum and urine samples.

Management

Treatment involves reducing toxin absorption (by induction of emesis and/or gastric lavage and administration of activated charcoal and an osmotic cathartic) (Table 4.1), methocarbamol to control the muscle tremors and AEMs (see Table 4.1 and Chapters 12 and 24) and supportive care (intravenous fluid therapy, convective whole body cooling and, in severely affected animals, intubation and ventilation). Emesis should be induced in asymptomatic animals that present within 15–30 min after suspected or confirmed ingestion of mouldy food or non-corrosive waste material (Barker et al., 2013). Administration of activated charcoal should be repeated over 1–3 days due to entero-hepatic recirculation of mycotoxins. Seizures may not respond to diazepam administration and it has been suggested that poor or no response to diazepam is suggestive of mycotoxicosis (Barker et al., 2013). If there is inadequate response after the first administration of diazepam, alternative AEMs such as levetiracetam, phenobarbital, propofol and ketamine (see Table 4.1 and Chapter 24) should be used.

several months to achieve complete resolution of neurological signs (Eriksen, 2010).

Animal-related Poisoning

Neurological signs including seizures can occur following exposure to venom of several animal species including insects (bee, wasp), spiders, reptiles and amphibians.

Toad

Overview

There are more than 200 species of Bufo toads in the world and they all have parotid glands on their dorsum that release toxic substances when the toad is attacked or threatened. These include bufotoxins and bufagenins, bufotenine, dopamine, epinephrine, norepinephrine, serotonin and indolealkylamines. Most small animals do not consume Bufo toads. Toxicosis occurs in animals that masticate or hold the toad instead of just biting and then releasing it. The toxins are rapidly absorbed through the oral mucosa and enter the systemic circulation. The toxins can also be absorbed through the gastric mucosa following ingestion, through open skin wounds and across the conjunctiva (Peterson and Roberts, 2006). Toad intoxication has been diagnosed in dogs and cats, however, dogs are more commonly affected (Roberts et al., 2000). Chances of exposure are higher when toads are most active (i.e. in the evening after high rainfall or with high temperatures).

Mechanism of action

Bufotoxins and bufagenins are digitalis-like compounds, which result in cardiac arrhythmias. In addition, bufotoxins cause vasoconstriction. Bufotenins are hallucinogenic. Epinephrine, norepinephrine and dopamine cause tachycardia, hypertension and seizures.

Clinical presentation

The toxins produced by the Bufo marinus toad (Fig. 4.6) (which is found in Florida, USA and north and north-east Australia) have been reported to result in neurological signs including seizures, ataxia, nystagmus, extensor rigidity, opisthotonos and stupor in dogs (Roberts et al., 2000). The neurological signs occur within minutes to 1 h after exposure and are preceded by oral mucous membrane hyperaemia, hypersalivation and pawing at the mouth. Cardiac arrhythmias often occur. Severity of signs is directly proportional to toxin dose. Death may occur following prolonged seizure activity and cerebral oedema or cardiac arrest.

The European Bufo vulgaris (common toad) and the Asian Bufo gargarizans are related species to the Bufo marinus toad and can also cause poisonings, although seizures have not been described (Peterson et al., 2006).

Diagnosis

No true confirmatory tests exist for Bufo toad intoxication. Clinical diagnosis is based on history of exposure and presenting signs. Digoxin serum immunoassays can identify the digitalis-like compounds produced by the toad parotid glands in the serum of the intoxicated animal.

Management

Treatment involves decontamination by thorough flushing of the oral cavity with running water for at least 5 min, AEMs (see Table 4.1; Chapters 12 and 24), detection and management of cardiac arrhythmias, intravenous fluids and diuretics to promote urinary excretion of the toxin. Emesis, gastric lavage or endoscopic removal is indicated in the rare event of toad ingestion.

Prognosis

Overall mortality is low in animals treated within a few hours of exposure (Roberts et al., 2000; Reeves, 2004).

Therapeutic Agents and Supplements

Metronidazole

Overview

Metronidazole is a nitroimidazole antibacterial and antiprotozoal agent used in the treatment of giardiasis, anaerobic infections and inflammatory bowel disease (Evans et al., 2003). Neurotoxicity from metronidazole has been reported in dogs receiving as low as 67 mg/ kg/day for an average of 3–14 days (Dow, 1989) or 60.3 mg/kg/day for 44.9 days (Evans et al., 2003), and in cats receiving 111 mg/kg/ day for 9 weeks or 58 mg/kg/day for 6 months (Caylor and Cassimatis, 2001).

Mechanism of action

The neurotoxic mechanism of metronidazole has not been identified. Proposed mechanisms include thiamine antagonism and inhibition of neuronal protein synthesis.

Clinical presentation

Neurological signs in cats include disorientation, ataxia, seizures and blindness (Caylor and Cassimatis, 2001). Neurological signs in dogs are characterized by cerebellar and central vestibular dysfunction (Dow et al., 1989; Evans et al., 2003). Neurologic signs may be preceded or associated with anorexia, vomiting and/or diarrhoea.

Diagnosis

Diagnosis is based on the history of metronidazole administration, clinical signs and resolution of signs following metronidazole discontinuation.

Management

Treatment involves metronidazole discontinuation, symptomatic and supportive care. In dogs, diazepam has been reported to markedly improved recovery times of animals with metronidazole toxicosis. Diazepam was administered at an initial dose of 0.2–0.5 mg/kg IV followed by 0.3–0.5 mg/kg PO every 8 h for 3 days (Evans et al., 2003). Mean response time (defined as the time to resolution of the debilitating clinical signs) was 4.25 days for untreated dogs to 13.4 h for treated dogs, and mean recovery time (defined as the time to resolution of all residual clinical signs) was 11 days for untreated dogs and 38.8 h for treated dogs (Evans et al., 2003).

Prognosis

Prognosis is generally excellent, with most animals recovering within 2 weeks. However, sometimes recovery can be prolonged in severely affected animals.

Ivermectin and other macrocyclic lactones

Overview

The macrocyclic lactones, including avermectins (abamectin, ivermectin, eprinomectin, doramectin and selamectin) and milbemycins (moxidectin, milbemycin and nemadectin),

are parasiticides able to kill a wide variety of arthropods and nematodes. Ivermectin is commonly used as heartworm preventative in dogs and cats, as ear miticide in cats, as treatment of sarcoptic and demodectic mange in dogs, and as anthelminthic in ruminants, swine and horses. Intoxication can occur when ivermectin is inadvertently overdosed by the owner or veterinarian or when dogs or cats are exposed to products intended for large animals (including by ingestion of contaminated faeces), which contain a higher concentration of ivermectin than small animal products. In addition, subpopulations of herding type breeds, primarily collies as well as Shetland sheepdogs and Australian shepherds, old English sheepdogs, German shepherds and some mixes of these breeds have a unique sensitivity to macrocyclic lactones and other drugs due to an autosomal recessive mutation in the ATP-binding cassette (ABC) B1 (ABCB1) gene (formerly named multidrug resistance 1 (MDR1) gene). This mutation results in a lack of functional permeability glycoprotein (P-gp) (Nelson et al., 2003; Merola and Eubig, 2012). P-gp is a member of the ATP-binding cassette (ABC) superfamily of transporters and represents an important neuroprotective component of the blood-brain barrier as it limits the entry of macro-cyclic lactones and other xenobiotics into the CNS. The defective P-gp results in accumulation of relatively high concentrations of P-gp-substrate drugs in the CNS, even when relatively low doses of drug are administered.

Mechanism of action

Ivermectin and other macrocyclic lactones cause toxicity in mammals by acting as GABA_A-receptor agonists in the CNS. Low CNS concentrations of macrocyclic lactones produce stimulatory CNS effects (e.g. tremors, seizures), whereas higher concentrations result in inhibitory CNS effects (e.g. ataxia, obtundation, stupor, coma).

Clinical presentation

Clinical signs of ivermectin toxicity have been reported at dosages ranging from 0.1 to 0.4 mg/kg PO in ABCB1 gene mutation-susceptible canine breeds, 0.2–2.5 mg/kg PO in non-susceptible canine breeds and of 0.3 mg/kg SC in cats (Merola and Eubig, 2012). Clinical signs include obtundation, disorientation, hypersalivation, muscle tremors, ataxia, blindness, mydriasis, seizures, stupor and coma (Hopper et al., 2002; Nelson et al., 2003; Kenny et al., 2008; Merola et al., 2009). Due to the long half-life of macro-cyclic lactones (from 2 days for ivermectin to 19 days for moxidectin), clinical signs of toxicosis may persist for days to weeks, depending on the agent, dose and the breed involved. Death may occur from respiratory arrest if no therapeutic intervention takes place.

Diagnosis

The clinical diagnosis is based on the history of exposure or over-dosage and clinical signs. A genetic test for the ABCB1-1D mutation is commercially available and can help to identify susceptible dogs. Plasma or stomach contents can be submitted to a veterinary diagnostic laboratory for quantification of ivermectin. Post-mortem diagnosis can be reached by analysis of ivermectin concentration in frozen brain, liver and fat.

Management

There is no specific antidote for ivermectin toxicity. Treatment involves decontamination including multiple doses of activated charcoal (Table 4.1) to interrupt the enterohepatic recirculation of ivermectin; a saline cathartic (Table 4.1), supportive care (intravenous fluids, ventilator support, cardiovascular, respiratory and neurologic monitoring, nursing care), AEMs (see Table 4.1 and Chapters 12 and 24) and myorelaxants in animals with tremors. Induction of emesis (Table 4.1) can be considered in animals with no neurologic signs and recent oral exposure. Historically, GABA agonists AEMs (such as benzodiazepines and barbiturates) were not recommended in animals with ivermectin-induced seizures as ivermectin is also a GABA agonist and exacerbation of clinical signs was a concern. However, this no longer seems to be the case. AEMs with a different mode of action such as levetiracetam (see Table 4.1 and Chapter 16) may be used.

Intravenous lipid emulsion (IVLE) administration has been suggested as a treatment that may shorten the duration of clinical signs of macrocyclic lactones and other lipophilic compound toxicosis. It is hypothesized that the IVLE acts as a ‘lipid sink’ and draws lipophilic compounds into the plasma lipid phase, thereby removing the compound from the target tissues and promoting its elimination (Merola and Eubig, 2012). Intralipid 20% preparation for intravenous infusion administered at 1.5 ml/kg as initial slow bolus followed by a constant rate infusion of 0.25 ml/kg/min over a 30–60 min period has been reported to result in rapid recovery in dogs and one cat with ivermectin toxicosis (Pritchard, 2010; Clarke et al., 2011; Bates et al., 2013; Epstein and Hollingsworth, 2013). IVLE does not seem beneficial to dogs that are homozygus for the ABCB1-1D mutation, possibly because of pre-existent high brain tissue concentration of ivermectin (Wright et al., 2011). Physostigmine and flumazenil may also be beneficial in the treatment of ivermectin toxicity. In severely affected animals intensive nursing care including respiratory, cardiovascular and nutritional support are necessary for several days.

Prognosis

Recovery may occur, however it is often prolonged (>3 weeks). Blind animals may recover vision (Kenny et al., 2008). Exposure to ivermectin doses >5 mg/kg carries a guarded prognosis. Outcome is likely to be more favour-able when decontamination measures are instituted soon after exposure and good supportive care is provided.

Levamisole

Overview

Levamisole is an anthelmintic, microfilaricide and immunostimulant (Bradley, 1976; Vandevelde et al., 1978). Levamisole toxicity has been reported with accidental overdosing (about four times the therapeutic dose) in companion animals.

Mechanism of action

Levamisole is a nicotine-like stimulant producing both muscarinic and nicotinic effects at cholinergic receptors.

Clinical presentation

Clinical signs of levamisole toxicosis include nausea, vomiting, hypersalivation, frequent urination and defecation, muscle tremors, ataxia, anxiety, hyperesthesia with irritability, clonic seizures, obtunded mental status, dyspnoea and cardiac arrhythmias.

Management

Treatment includes gastrointestinal decontamination, AEMs (see Table 4.1 and Chapters 12 and 24) and supportive care (including fluid therapy and oxygenation).

Methylxanthines

Overview

Methylxanthine compounds such as caffeine, theophylline, aminophylline and theobromine are CNS stimulants that are sometimes involved in accidental or malicious poisoning of companion animals (O’Brien, 1998). Intoxication most commonly occurs following ingestion of chocolate, caffeine-based tablets or elixirs. Dark chocolate and cocoa beans have much higher theobromine content than milk or white chocolate. Animals ingesting 20 mg/kg of caffeine or theobromine have mild clinical signs. Ingestion of 60 mg/kg causes seizures.

Mechanism of action

Methylxanthines enhance catecholamine effects both directly and indirectly through phosphodiesterase inhibition, elevation in intracellular cyclic AMP (AMPc) and increased calcium influx into neurons (O’Brien, 1998). This results in increased neuromuscular excitability and a positive inotropic effect. Competitive inhibition of cellular adenosine receptors further increases intracellular AMPc and results in CNS stimulation.

Clinical presentation

Clinical signs generally occur within 1–2 h post-ingestion and include restlessness, hyperactivity, vomiting, diarrhoea, tachycardia, cardiac arrhythmias, tachypnoea, polydipsia/ polyuria, ataxia, muscle tremors, tonic seizures, hyperthermia, cyanosis, coma and death (Glauberg and Blumenthal, 1983; Tawde et al., 2012).

Diagnosis

Diagnosis is based on the history of methylxanthine (generally theobromine, which is present in chocolate) ingestion, clinical signs and presence of chocolate (or other source of methylxanthine) in gastric content from vomitus or lavage fluids. Methylxanthine concentrations in gastric content, serum, plasma or urine can be analysed.

Management

Treatment is symptomatic and supportive, including induction of emesis (in asymptomatic animals within 2–4 h post-ingestion), activated charcoal with a cathartic (Table 4.1), intravenous fluids to promote methylxanthine excretion, antiarrythmic agents, skeletal muscle relaxants, AEMs (see Table 4.1 and Chapters 12 and 24) and supportive care. Removal of urine though an indwelling catheter prevents reabsorption of the toxin across the bladder mucosa (Hooser and Beasley, 1986).

Prognosis

The prognosis is generally favourable for animals with mild to moderate clinical signs that are treated promptly and aggressively.

Amphetamine and amphetamine-like compounds

Overview

Amphetamine and amphetamine-like compounds such as such as methylphenidate (Ritalin, Concerta), pemoline (Cylert) and dextroamphetamine/amphetamine combinations (Adderall, Dexedrine) are used to treat behaviour disorders in people, including attention deficit disorders in children and adults (Albretsen, 2002). Pets may be exposed by accidental ingestion of their owner’s medications. Toxic doses of amphetamines in dogs and cats can be as low as 1 mg/kg. Pemoline is known to cause clinical signs in dogs at oral doses greater than 2.8 mg/kg (Albretsen, 2002).

Mechanism of action

Amphetamine and amphetamine-like compounds stimulate the release of catecholamine and/or serotonin and prevent their reuptake and metabolism with consequent CNS stimulation (O’Brien, 1998; Stern and Schell, 2012).

Clinical presentation

Clinical signs include hyperactivity, restlessness, irritability or other behavioural changes, mydriasis, muscle tremors, tachycardia or other cardiac arrhythmias, hypertension, tachypnea, hyperthermia, vomiting, diarrhoea and, rarely, seizures (O’Brien, 1998; Albretsen, 2002; Stern and Schell, 2012). Coma and death (attributed to DIC secondary to hyperthermia and respiratory failure) can occur in severe intoxication.

Diagnosis

Diagnosis is based on the history of ingestion, clinical signs, and recovery of pills or capsules in the gastric content from vomitus or lavage fluids. Amphetamines can be detected in urine or plasma by laboratory analysis.

Management

There is no specific antidote for amphetamines or amphetamine-like drug intoxication. Treatment involves decontamination (induction of emesis in asymptomatic animals or gastric lavage followed by the administration of activated charcoal and a cathartic) (Table 4.1), supportive care (including fluid therapy and convective whole body cooling), methocarbamol in animals with tremors and AEMs in seizuring animals (see Table 4.1 and Chapters 12 and 24). Diazepam administration has been reported to increase excitability in some dogs

with amphetamine toxicosis and it is therefore avoided to control hyperactivity, restlessness, irritability or other excitatory behavioural changes; however, it can be used in the acute management of seizures (Albretsen, 2002; Stern and Schell, 2012). Phenothiazine tranquilizers have been recommended to control hyperactivity, restlessness, irritability or other behavioural changes (Stern and Schell, 2012). Acepromazine can initially be administered at 0.05 mg/kg IV and titrated to effect. The dose can be gradually increased to 0.1 to

1.0 mg/kg if clinical signs do not resolve with lower doses. Alternatively, chlorpromazine can initially be administered at 0.5 mg/kg IV and subsequently titrated up to effect. Blood pressure should be monitored at higher doses of phenothiazine to ensure that hypotension does not occur. Animals should be kept in a dark and quiet area and stimulation should be minimized (Stern and Schell, 2012). Treatment of tachycardia or other cardiac arrhythmias may sometimes be required. Urinary acidification has been recommended (to a pH of between 4.5 and 5.5) to enhance the elimination of amphetamines. This can be achieved with ammonium chloride administration at 100 to 200 mg/kg/day PO divided four times daily or ascorbic acid 20–30 mg/kg PO, SQ, IM or IV. Urinary acidification should not be attempted if the animal is acidotic, if acid-base status cannot be monitored regularly, or in case of rhabdomyolosis or acute renal failure (Albretsen, 2002; Stern and Schell, 2012).

Prognosis

Prognosis is generally good with animals receiving prompt and appropriate treatment (Stern and Schell, 2012).

Selective serotonin reuptake inhibitors

Overview

Selective serotonin reuptake inhibitors (SSRIs) such as sertraline (Zoloft), fluoxetine (Prozac), paroxetine (Paxil), and fluvoxamine (Luvox) are often used to treat depression, obsessive-compulsive disorders and other behavioural problems in people (Albretsen, 2002).

Fluoxetine has been used in dogs for the treatment of fear or anxiety, acral lick dermatitis and narcolepsy. Paroxetine has been used to treat urine-spraying problems in cats.

Mechanism of action

As suggested by their name, SSRIs inhibit the reuptake of serotonin at the presynaptic membrane resulting in increased synaptic serotonin levels and overstimulation of serotonin receptors.

Clinical presentation

Clinical signs include obtundation, stupor or coma, anorexia, vomiting, diarrhoea, hyper-salivation, abdominal pain, arrhythmias, hypo- or hypertension, tachypnea, dyspnoea, hyperactivity, hyperaesthesia, tremors, seizures (rarely) and hyperthermia. Signs consistent with CNS depression (e.g. obtundation, stupor or coma) are more common than signs of CNS stimulation (e.g. agitation, aggression, tremors or seizures) (Thomas et al., 2012).

Diagnosis

Diagnosis is based on the history of SSRI ingestion, clinical signs and recovery of pills or capsules in the gastric content from vomitus or lavage fluids.

Management

There are no antidotes available for the treatment of SSRI toxicosis. Treatment is symptomatic and supportive, including induction of emesis (in asymptomatic animals), gastric lavage, activated charcoal with a cathartic (in animals without diarrhoea) (Table 4.1), antiarrythmic agents, skeletal muscle relaxants, AEMs (see Table 4.1 and Chapters 12 and 24) and supportive care. In animals with severe clinical signs, serotonin receptor antagonists such as cyproheptadine at 1.1 mg/ kg PO in dogs and 2.4 mg per cat PO can be administered. The medication can be crushed, mixed with saline and administered rectally to animals unable to take oral medications (Albretsen, 2002).

Prognosis

The overall prognosis for animals with SSRI toxicosis is excellent with prompt and appropriate treatment (Thomas et al., 2012).

5-Hydroxytryptophan

Overview

5-Hydroxytryptophan (5-HTP), sometimes called Griffonia seed extract, is sold as an over-the-counter dietary supplement. It has been used to help with depression, chronic headaches, obesity and insomnia in people.

Mechanism of action

5-HTP is rapidly converted to serotonin within target cells of the CNS and cardiovascular system, resulting in overstimulation of serotonin receptors.

Clinical presentation

Clinical signs occur from 10 min to 4 h after ingestion and include vomiting, diarrhoea, abdominal pain, hypersalivation, mydriasis, seizures, hyperthermia, obtunded mental status, tremors, hyperesthesia, ataxia, tachycardia and tachypnea (Gwaltney-Brant et al., 2000). In a study including 21 dogs with evidence of accidental 5-HTP ingestion, the minimum toxic dose reported was 23.6 mg/kg and the minimum lethal dose was 128 mg/kg (Gwaltney-Brant et al., 2000).

Diagnosis

Diagnosis is based on history of exposure and clinical signs.

Management

Treatment includes decontamination (by induction of emesis and/or gastric lavage, administration of activated charcoal), AEMs (see Table 4.1 and Chapters 12 and 24) and supportive care including intravenous fluid therapy and convective whole body cooling.

Cyproheptadine, a serotonin antagonist, has been used at 1.1 mg/kg, PO or rectally, every 1 to 4 h until signs resolve. Rectal administration may be preferred when severe vomiting or recent administration of activated charcoal make the oral route unfeasible (Gwaltney-Brant et al., 2000).

Prognosis

The majority of dogs that receive prompt and aggressive treatment recovered within 36 h after exposure (Gwaltney-Brant et al., 2000).

Guarana (Paullinia cupana) and ma huang (Ephedra sinica)

Overview

Guarana (Paullinia cupana), a natural source of caffeine, and ma huang (Ephedra sinica), a natural source of ephedrine, are the main components of an herbal medicine marketed as a weight loss and energy supplement.

Mechanism of action