Many animal models have been advanced to study epileptic syndromes.53 Here, we briefly review the main pharmacological tests used at the ASP to identify new anticonvulsant molecules. The qualitative and quantitative assessments of a molecule are conducted at different time points ranging from 15 min to 4 h after administration. The anticonvulsant and toxicological profiles of compounds are mainly determined by intraperitoneal (ip) or peroral (po) administration to rodents. The dosage may range from 1 to 300 mg of compounds per kg of body weight, with several animals tested at each dosage. The dose at which half of the animals are protected is termed effective dose (50%) or ED50. Similarly, the median toxic dose (50%) or TD50 is the dosage at which half of the animals display neurological impairment. The ratio of the TD50 over the ED50 is called the protective index (PI).
1.2.4.1. Maximal electroshock seizure test
The maximal electroshock seizure (MES) test consists in applying an electrical current to the animal via corneal electrodes and represents a model for generalized tonic-clonic seizures.54 The electrical signal (60 Hz current, delivered for 0.2 sec) has a high frequency/short duration nature which elicits maximal seizures generally lasting no more than 30 sec.55 A brief initial tonic flexion, and prolonged tonic extension period, followed by terminal clonus are characteristic of MES seizures.55 Protection is defined as a failure of the animal to extend hindlimbs to an angle with the trunk greater than 90°.56
1.2.4.2. 6 Hertz test
The 6 Hz test is analogous to the MES test except for the nature of the electrical signal applied. A low frequency/long duration signal (6 Hz current, delivered for 3 sec, 32 or 44 mA intensity) triggers a different type of seizures in the animal that are representative of complex partial seizures.57,58 Characteristics of 6 Hz-induced seizures are the immobility of the animal, accompanied by forelimb clonus, vibrissae (whiskers) twitching, and Straub-tail (elevated tail).59 The absence of these signs upon electrical stimulation is defined as seizure protection.
1.2.4.3. Pentylenetetrazole administration
The subcutaneous pentylenetetrazole (scPTZ) test measures the ability of a compound to raise the seizure threshold produced by a chemoconvulsant (PTZ,
Scheme 5).60 At a dose specific to the rodent model, PTZ induces clonic seizures and the test is considered a model for absence seizures.52 A compound is considered protective if it abolishes the spasms triggered by the PTZ administration.52 It is pharmacologically different from either the intravenous (iv)PTZ or the ipPTZ tests. In the iv route, the animal will start experiencing a series of myoclonic jerks (rapid contraction and relaxation of muscles) in a time-dependent manner.61 These jerks will worsen and ultimately lead to loss of righting and clonic seizure. In the ip route, defined, subconvulsive doses of PTZ are administered at given time intervals. A gradual, reproducible response can then be observed, with initial jerks and full-blown seizure each starting after a specific number of injections.62 In these tests, a molecule is considered protective if it can elevate the threshold PTZ dose necessary for convulsions and delay (iv) or increase the number of injections (ip) necessary for the onset of seizure.61,62 Molecules with efficacy in the scPTZ test may have no effect in the ipPTZ or ivPTZ tests and vice-versa.63
1.2.4.4. Pilocarpine administration
In the lithium pilocarpine-induced status epilepticus (SE) model, animals are treated ip with a solution of LiCl 20 h prior to ip injection of pilocarpine (PILO), which results in the development of SE within 30 min after administration of PILO.64 When administered alone, PILO induces secondarily generalized seizures that evolve into SE.65 A variety of chemoconvulsants such as kainic acid,66 picrotoxin,67 and bicuculline67 can be used to induce SE (Figure 4). After the SE period (8–24 h),
months).65 These seizures are representative of complex partial seizures observed in humans.20 Protection is defined by the suppression of recurring seizures.
Figure 4. Chemical structures of various chemoconvulsants used in animal testing.
Pentylenetetrazole, bicuculline and picrotoxin are GABA receptor antagonists. Kainic acid is a glutamate receptor agonist.
1.2.4.5. Audiogenic seizure animal models
The Frings mouse model is a model of generalized reflex epilepsy in which the seizure is triggered by a loud noise.68 The epileptic phenotype is caused by a mutation in the mass1 gene (monogenic audiogenic seizure susceptibility 1) which causes a premature termination of the membrane protein “very large G-protein coupled receptor 1” (VLGR1 or MASS1, 6300 amino acid residues).69,70 The characteristic response of Frings mice to an intense stimulus (100 dB, 20 s) is wild running, followed by a loss of righting reflex that occurs with forelimb and hindlimb tonic extension.52 Seizure protection is defined as the ability of a molecule to prevent tonic hindlimb extension.52 The DBA/2 (dilute brown non-agouti) mouse is another genetic model used for audiogenic seizures.71
1.2.4.6. Electrical kindling models
The rapid hippocampal kindling test is a model representative of focal seizures.52,72 The term kindling refers to repetitive, infrequent stimulations that sensitize a specific region of the brain, progressively leading to a fully kindled state.72 Once this stage is reached, stimulations that initially did not elicit a reaction will consistently trigger an enhanced response.72 In the rat hippocampal kindling model, electrodes are surgically implanted in the hippocampus and kindled seizures are produced by a 10 s train of 1 ms biphasic 200 µA pulses (50 Hz) delivered every 30 min for 6 h on alternating days for a total of 60 stimulations (5 stimulus days).52 Behavioral seizures are scored according to the Racine scale,73 that ranges from 1 (whiskers twitching) to 5 (forelimb clonus, rearing and falling). Another brain region commonly used for the kindling procedure is the amygdala.74 Compounds evaluated in this model are dosed based on their activity in other seizure tests. A molecule can be tested for its ability to stop kindled seizures (anticonvulsant activity), or its ability to delay the acquisition of the kindled state (antiepileptogenic effect).52
1.2.4.7. Neurological toxicity evaluation
The rotarod test is a measure of neurological toxicity, where a mouse is placed on a rotating rod (6 rpm).75 Control mice have the ability to maintain themselves on the rod for an extended period of time.75 Neurological impairment is defined as the inability of the mouse to remain on the rod for 1 min in three successive trials.52 In rats, the behavioral toxicity of the compound is assessed. The
neurologically impaired rat will fail to lift its leg back to a normal position.52 The gait and stance tests consists in observing symptoms of impairment in the animal, such as zigzag gait, abnormal posture, and lack of exploratory behavior or catalepsy.52