Hippocampal Kindled Rat Model

Brief Description

Ability of a test compound to block behavioral seizures and/or decrease the ADD in a hippocampal kindled rat model of focal seizures.

Introduction

In recent years, the kindling model has been a useful adjunct to the more traditional anticonvulsant tests employed by the ETSP to identify a test substance’s potential utility for treating complex partial seizures. Importantly, the hippocampal kindled rat provides an experimental model of focal seizures that become secondarily generalized. This rat model is useful for not only identifying compounds effective against partial seizures, but also allows for the investigation of complex brain networks that may contribute to seizure spread and generalization from a focus [1, 2]. Moreover, of the various electrical kindling paradigms described in the literature, the hippocampal kindling model described by Lothman and colleagues [1] offers several distinct advantages for the routine screening and evaluation of potential anticonvulsant compounds. First, this model provides a temporal framework for assessing drug efficacy in a focal seizure model. Specifically, the refractory period of individual animals is sufficiently short to permit repeated stimulations over a short time span. Second, this kindled rat model can be employed to assess the ability of an investigational compound to block fully kindled seizures evoked by an electrical stimulus. Finally, the hippocampal kindled rat model can also be used to assess the ability of an investigational compound to elevate threshold to focal firing. In the ETSP screening paradigms, the threshold test is likely to be more sensitive to modulation by an investigational drug. Therefore, compounds with a subtle effect on focal seizure activity in the kindled rat are not likely to be overlooked in the threshold test, thereby making the hippocampal kindled rat a useful screen at the ETSP.

Methods

Animals are first surgically prepared for kindling acquisition. Adult, male Sprague-Dawley rats (275-300g) are surgically implanted with bipolar electrodes [1]. Briefly, a bipolar electrode is stereotaxically implanted into the ventral hippocampus (AP -3.6, ML -4.9, VD -5.0 from dura, incisor bar +5.0) under ketamine-xylazine anesthesia. Animals are allowed to recover for one week before the kindling regimen begins. The rapid hippocampal kindling paradigm consists of applying a repeated stimulation regimen on alternating days for a total of 5 stimulus days, as described previously [3]. During the stimulation regimen, a 50 Hz, 10 sec train of 1 ms biphasic 200 µA pulses is delivered every 30 min for 6 hours, thereby giving 12 stimulations per stimulus day. Once animals are kindled to consistently present with a Stage 5 behavioral seizure, the test compound is evaluated for its ability to modify the fully expressed kindled seizure and ADD after a one-week, stimulation-free period. Unlike other acute seizure tests conducted by the ETSP, each kindled rat is allowed at least 5 days between tests to “washout” any investigational compound after testing.

Focal Seizures in Hippocampal Kindled Rats
Candidate substances are evaluated for their ability to block fully kindled secondarily generalized seizures after kindling acquisition [1]. The BSS are rated according to the following criteria [4]:
Stage 1 - mouth and facial clonus
Stage 2 - stage 1 plus head nodding
Stage 3 - stage 2 plus forelimb clonus
Stage 4 - stage 3 plus rearing
Stage 5 - stage 4 plus repeated rearing and falling

For all following tests, the mean values and S.E.M. are calculated for the ADD and p values will be determined by Student's t-test. Significant differences in BSS from control and treated groups are determined by the non-parametric Mann-Whitney U test, with p<0.05 determined to be statistically significant.

Test 11: Identification of neurotoxicity and ability to block hippocampal-kindled focal seizures. In preparation for the hippocampal kindled rat test, an appropriate dose (devoid of MMI is identified. For the MMI study, three groups of 2 rats per time point are administered the test compound at 300, 100, and 30 mg/kg and MMI is monitored at 1/4, 1/2, 1, 2, and 4 hr in an open field. Based on the results from the above MMI study, two kindled rats are then administered a non-toxic dose of the test drug and given the kindled stimulation at 15, 45, 75, 105, 135, 165, and 195 min post-drug administration. The effect of the treatment on their BSS and ADD is recorded. If the results from this initial screen suggest that a drug possesses activity against the fully expressed kindled seizure, the group size is increased to 8 rats (Test 12 & 13).

Test 12: [Effect on Afterdischarge Threshold in Hippocampal Kindled Rats (only performed upon special request of the COR)]: In contrast to the effect of an investigational drug on kindled seizures induced by supra-threshold stimulation, this study evaluates the ability of a drug to increase the Afterdischarge Threshold (ADT) in the fully kindled rat. The initial stimulation is conducted at an intensity of 20 µA. Stimulus intensity is increased in 10 µA increments every 1 - 2 min until an afterdischarge is elicited. Fifteen minutes after the pre-drug threshold determination, a single dose of the test substance is administered to 2 animals. In this way each animal serves as its own control. The individual rat ADT is then re-determined at varying times i.e., 1/4, 1, 2, and 4 hours after drug administration. Both BSS and ADD are recorded at each of the time points tested. The seizure score and ADD are also recorded at the ADT. Behavioral seizures are scored according to the criteria described above [4]. Individual seizure scores, ADDs, and ADTs are recorded. In the case of an active compound, 4 or more animals are used, and the results are then averaged and the group mean and S.E.M. are calculated.

Test 13: Quantitation of a drug's ability to block focal seizures based on BSS. Separate groups of kindled rats (n = 6-8) receive escalating doses of the candidate substance based off the results from Test 11. Rats are then tested at 15, 45, 75, 105, 135, 165, and 195 min after drug administration. The BSS and ADD of each rat are noted, with mean and S.E.M. calculated. Animals displaying a seizure score of 3 or less are considered protected. These data are used to establish a dose-response curve and subsequent ED50. Significant changes in ADD are also recorded. When an investigational compound is observed to significantly lower seizure score (3 or lower) and ADD, a dose-response study is initiated. The BSS and ADD for each dose are averaged at the TPE, and the mean and the S.E.M. compared to control by the non-parametric Mann-Whitney U test. The ability of a candidate substance to reduce seizure severity is quantitated and an ED50 is determined by probit analysis.

Results

Test 11: For each time group, results are expressed as the total number of animals protected out of the number of animals tested over time (i.e., 2 out of 2 protected). Test compounds that produce anticonvulsant effects (i.e., at least 1/2 protected at two or more time points) will then be rescreened with an increased n (e.g., n=6-8). Active compounds may then become candidates for other models of pharmacoresistance, including the 6 Hz test and the lamotrigine-resistant amygdala kindled rat test (Test 25).

Test 13: The BSS and ADD for each dose are averaged at the TPE, the S.E.M. calculated, and compared to control values. The ability of a candidate substance to reduce seizure severity is quantitated by results collected from several doses demonstrating protection (BSS≤3) and an ED50 is determined by probit analysis.

Discussion

Kindled seizures not only provide an experimental model of focal seizures, but also provide a means of studying complex brain networks that may contribute to seizure spread and generalization from a seizure focus [1]. Additionally, kindling models the changes in neuronal plasticity that occur during epileptogenesis [3]. The rapid hippocampal kindling model allows for the rapid routine screening and evaluation of new anticonvulsant substances undergoing evaluation by the ETSP. Animals can be quickly and reproducibly kindled to Stage 5 seizures in the course of two weeks [5] and this model demonstrates both aberrant behavioral and electrographic seizure activity, giving two useful dependent measures in the screening of investigational compounds. Finally, the threshold to focal firing can also be altered by an investigational compound, thus making the hippocampal kindled rat a useful screening model of focal seizures at the ETSP.