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Keywords:

  • Seizure models;
  • Epilepsy;
  • Mechanisms;
  • Convulsions;
  • Ketogenic diet

Summary

  1. Top of page
  2. Animal Screening of Antiseizure and Antiepileptic Molecules
  3. Toxicity of Putative Antiseizure and Antiepileptic Compounds
  4. Limitations of Animal Models of Epilepsy
  5. Conclusions
  6. Acknowledgment
  7. References

Animal models of human disease have been enormously important in improving our understanding of the pathophysiological basis and the development of novel therapies. In epilepsy, modeling using both in vivo and in vitro preparations has provided insight into fundamental neuronal mechanisms. Indeed, much of our understanding of seizure mechanisms comes from animal studies. The conceptual advances in understanding basic mechanisms of epilepsies have been largely validated in humans, attesting to the validity of the rationale and providing a basis for bridging the gaps between experimental and human data. While the ketogenic diet is clearly efficacious in a wide variety of seizure types and syndromes, the mechanism of action of the diet has not been established. Animal models will continue to be enormously important in furthering our understanding of how dietary therapy can help individuals with epilepsy.

Animal models have been used to design novel therapies and provide a means to evaluate molecules in regards to both efficacy and toxicity. The consequences of seizures and epilepsy on behavior, learning, and memory can also be evaluated with animal models. If used correctly, animal models can contribute to our knowledge about the efficacy, mechanisms of action, and consequences of the ketogenic diet (KD).

While it may seem self-evident, it should be remembered that an animal model must have some features of the human condition that one is modeling. For example, one major reason to use an animal model is to study the mechanism of action of the KD in suppressing seizures. If there are major differences between the way rats and humans metabolize the diet, delineating human mechanisms from rodent data may be flawed. Felbamate provides an instructive example. A highly effective antiepileptic drug in humans (Faught et al., 1993; The Felbamate Study Group in Lennox–Gastaut Syndrome, 1993), felbamate was judged to have little toxicity in rats during preclinical studies (Pellock et al., 2002). However, when the drug came onto the market, a rash of cases of aplastic anemia and hepatotoxicity were reported (Kaufman et al., 1997; Pellock & Brodie, 1997). The mechanism of toxicity is believed to be an immune reaction to a protein conjugate of the felbamate metabolite 2-phenylpropenal. Because significantly less 2-phenylpropenal is formed in rodents than in humans, rats do not develop toxicity (Popovic et al., 2004). In the case of the KD, it has been observed that there are large differences between humans and rats in their ability to produce elevated blood acetone levels on the ketogenic diet (Nylen et al., 2006). While this observation does not render the rat model invalid, investigators should be aware of possible limitations in the interpretation of data when using a rodent model.

The KD is primarily used to treat epilepsy. Fortunately, there are a large number of animal models in which the KD can be evaluated. In this review, the frequently used animal models used to evaluate efficacy and toxicity of antiseizure and antiepileptic compounds are discussed.

Animal Screening of Antiseizure and Antiepileptic Molecules

  1. Top of page
  2. Animal Screening of Antiseizure and Antiepileptic Molecules
  3. Toxicity of Putative Antiseizure and Antiepileptic Compounds
  4. Limitations of Animal Models of Epilepsy
  5. Conclusions
  6. Acknowledgment
  7. References

The first step in evaluating potential antiseizure and antiepileptic molecules, devices and dietary therapies is to determine whether the therapy will be effective in generalized or partial seizures. The most commonly used models to evaluate potential new epilepsy therapies include the maximal electroschock (MES) test, the subcutaneous pentylenetetrazol (scPTZ) test, and the electrical kindling model. The MES, scPTZ, and kindling tests have markedly different pharmacological profiles. The MES test identifies agents with activity against generalized tonic–clonic seizures while the scPTZ identifies compounds that are effective against absences and myoclonic seizures. The kindling test identifies agents that work against both partial and generalized seizures.

In the MES test, an electric current of fixed intensity and duration is applied via ear clips or corneal electrodes, resulting in tonic extension of the hindlimbs. In the commonly used corneal model, an alternating current is administered through corneal electrodes. The time to the completion of the hindlimb tonic extensor component is recorded. In the scPTZ test, a convulsive dose of PTZ is administered and the animal is observed for clonic seizures. The test compound is evaluated for its ability to suppress the clonic seizure. In both the MES and PTZ models, various doses of the test substance are evaluated. In the 6-Hz seizure model, mice receive 6 Hz electrical stimulations delivered through corneal electrodes to induce a complex partial seizure. Typically, the seizure is characterized by a minimal clonic phase followed by stereotyped automatic behaviors. The efficacy of the test compound in suppressing the seizure is recorded. Other tests sometimes used to screen compounds include the subcutaneous bicuculline (scBIC) and picrotoxin (scPIC) tests. Bicuculline is a GABAA receptor blocker while picrotoxin blocks the chloride channel associated with the GABAA receptor. These tests are used to screen compounds that are thought to work through GABAergic mechanisms.

As shown in Table 1, there appears to be a reasonable correlation between results obtained from the common animal tests and the antiseizure profiles of antiepileptic drugs. MES-induced tonic extension seizures are blocked by drugs such as carbamazepine and phenytoin, which are known to inhibit voltage-sensitive Na+ channels, and by some drugs that enhance GABA-mediated inhibition. In the scPTZ model, seizures are blocked by antiepileptic drugs acting at the GABAA receptor (benzodiazepine, barbiturates, valproate) and by an agent that reduces T-type Ca2+ currents (ethosuximide). With the exception of ethosuximide, seizures are blocked or attenuated by most of the standard antiepileptic drugs in the kindling model. As shown in the table, the KD works in a variety of animal models, strongly suggesting that it has broad-spectrum efficacy.

Table 1.  Efficacy of antiepileptic drugs in diet in animal models of seizures and epilepsy
 MESscPTZscBICscPICAGS6 Hz
  1. +, drug or diet effective in seizure model; –, drug or diet not effective in seizure model; ±, equivocal response; MES, maximal electroshock; scPTZ, subcutaneous pentylenetetrazol; scBIC, subcutaneous bicuculline; AGS, audiogenic seizures; 6 Hz, corneal kindling model.

Carbamazepine+++±
Clonazepam+++±+
Ethosuximide++++
Felbamate++ +++
Gabapentin+++ 
Ketogenic diet+±++±+
Pregabalin+++ 
Lamotrigine++±
Levetiracetam+ +
Phenobarbital++++ +
Phenytoin++±
Oxcarbazepine+++±
Tiagabine++++
Topiramate++
Valproate+++++
Vigabatrin±± 
Zonisamide+++ +

While the models mentioned here are the most common, a wide variety of other models have also been studied, including a variety of types of electrical stimulation, chemoconvulsant agents, spontaneous mutations, transgenic and targeted gene knockout models, hyperthermia, hypoxia, trauma, intracerebral vascular occlusion, and radiation. In addition, investigators have used brain slices and cultured tissue to study molecular and cellular changes associated with seizure onset and propagation. The KD has been shown to be effective in the EL mouse model (Todorova et al., 2000), rapid kindling model (Hori et al., 1997), kainate model (Muller-Schwarze et al., 1999), flurothyl inhalation seizure model (Rho et al., 1999), norepinephrine transporter knock-out mice (Martillotti et al., 2006), and Frings audiogenic seizure-susceptible mice (Rho et al., 2002).

Toxicity of Putative Antiseizure and Antiepileptic Compounds

  1. Top of page
  2. Animal Screening of Antiseizure and Antiepileptic Molecules
  3. Toxicity of Putative Antiseizure and Antiepileptic Compounds
  4. Limitations of Animal Models of Epilepsy
  5. Conclusions
  6. Acknowledgment
  7. References

Determining the toxicity of putative drugs and dietary therapy is as important as assessing efficacy. Commonly used tests for acute toxicity include the rotorod, positional sense test, and gait and posture test. The rotorod test is an easily performed test in which the mouse or rat is placed on a rotating rod. The outcome measure is how long the rodent can remain on the rod. Rats that have toxicity will fall off the rod sooner than nontoxic or control rats. The positional sense test is a measure of motor function of the hindlimbs of rodents. The hindlimbs of the rodent are slowly lowered over the edge of a table. Normally, a rat will quickly lift its leg back to a normal position. A neurological deficit is indicated by the inability of the animal to rapidly correct such an abnormally positioned limb. In the gait and posture test, the animal is tested for stability of gait and maintenance of posture. Since the KD is typically used as a chronic therapy, animals on the diet have not been rigorously tested for signs of acute toxicity.

There has also been a paucity of information on the effect of the KD on higher cortical function such as visual-spatial memory. To address this question, visual-spatial memory, activity level, and emotionality were studied in rats on and off the KD following status epilepticus (Zhao et al., 2004). Rats were subjected to lithium/pilocarpine-induced status epilepticus or saline injections and were then randomized to either the ketogenic diet or a regular rat diet. One month later, rats were evaluated for visual-spatial memory in the water maze, activity level in the open field test, and emotionality with the handling test. Spontaneous recurrent seizures were also measured using videotaping. Although the authors found that rats treated with the KD appeared healthy, their weight gain was substantially lower than that in rats that received regular rat chow. The KD reduced the number of spontaneous seizures but had no discernible effect on flurothyl seizure susceptibility. KD-fed rats had significantly impaired visual-spatial learning and memory compared with rats that were fed regular diet. The KD had minimal effects on activity level and emotionality. Rats that were treated with the KD also had significantly impaired brain growth. These results are concerning; despite reducing the number of spontaneous seizures after status epilepticus, KD-treatment resulted in severe impairment in visual-spatial memory and decreased brain growth. Since in humans the KD has been shown to improve alertness and cognitive skills (Farasat et al., 2006; Pulsifer et al., 2001), the study raises the question of whether the rodent model is appropriate for assessing cognitive effects of the KD.

Limitations of Animal Models of Epilepsy

  1. Top of page
  2. Animal Screening of Antiseizure and Antiepileptic Molecules
  3. Toxicity of Putative Antiseizure and Antiepileptic Compounds
  4. Limitations of Animal Models of Epilepsy
  5. Conclusions
  6. Acknowledgment
  7. References

While animal models are very useful, they have limitations. Most antiepileptic agents have been discovered using primitive animal models that bear little resemblance to human condition. Because of interspecies differences and the large spectrum of seizures types in humans, it is difficult to mimic all of the clinical signs seen in patients with epilepsy. Putative antiepileptic agents are typically screened in adult, male rats that do not have epilepsy, using acute seizure models. While kindling is championed as a model of epileptogenesis, spontaneous seizures (epilepsy) rarely occur. More relevant models, in which the animals have epilepsy, are used infrequently because of time constraints and costs. Virtually no drug screening has been performed in young (and old) animals. Finally, studying higher level cognitive function in humans, such as language, auditory and visual processing, is not possible.

Conclusions

  1. Top of page
  2. Animal Screening of Antiseizure and Antiepileptic Molecules
  3. Toxicity of Putative Antiseizure and Antiepileptic Compounds
  4. Limitations of Animal Models of Epilepsy
  5. Conclusions
  6. Acknowledgment
  7. References

It is clear that the KD is a highly efficacious therapy for childhood epilepsy. Animal studies are no longer necessary to prove this point. However, there are many unanswered questions about the diet. Perhaps the most important question is how it works. As discussed in this supplement, a myriad of possible mechanisms of action have been identified. Further work with animal models is likely to determine which of these putative mechanisms are critical to the efficacy seen with the diet. There almost certainly will be a movement to target novel molecular targets once the mechanism or mechanisms are discovered. Animal models have served the epilepsy community well and will likely play a major role in furthering our knowledge base of dietary therapies for epilepsy.

Acknowledgment

  1. Top of page
  2. Animal Screening of Antiseizure and Antiepileptic Molecules
  3. Toxicity of Putative Antiseizure and Antiepileptic Compounds
  4. Limitations of Animal Models of Epilepsy
  5. Conclusions
  6. Acknowledgment
  7. References

I confirm that I have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Supported by NIH grants (NS0415951 and NS056170).

Disclosure: The authors declares no conflicts of interest.

References

  1. Top of page
  2. Animal Screening of Antiseizure and Antiepileptic Molecules
  3. Toxicity of Putative Antiseizure and Antiepileptic Compounds
  4. Limitations of Animal Models of Epilepsy
  5. Conclusions
  6. Acknowledgment
  7. References