Stable Kindled Responses and Protocol to Measure Drug Effects

Animals are kindled using 20 or 50 Hz stimulus trains, 10 s in duration, using the suprathreshold stimulus intensity (usually 400 pA) every 5 min for 6 h, for a total of 72 stimulus trains.5,7 During this initial kindling period there is not a progressive increase in afterdischarge duration and worsening of the behavioral seizures (Figure 4.2). Most of the behavioral seizures are mild limbic seizures (score 1) with occasional seizures with a score of 2. About halfway through the day a full motor seizure (score 5) will appear. Several additional class 5 seizures will appear through the rest of the day at irregular intervals. The afterdischarge duration follows a similar pattern. Periodically there is a longer afterdischarge that correlates with the worsening behavioral seizures.

Two to 3 d after the initial 1 d treatment with stimulus trains administered every 5 min for 6 h, the animals are put on an alternate day schedule (for example, Monday, Wednesday, Friday) with stimulus trains administered every 30 min. Twelve stimulus trains are given over a 6 h period. On each day of testing, the afterdischarge threshold is determined in the morning and again at the end of the day. Rats often need a "priming" stimulus at the beginning of each day to express a fully kindled motor seizure, therefore 5 min after the final determination of the afterdischarge threshold, a 400 pA stimulus train is administered. The response to this priming stimulus is not analyzed.

FIGURE 4.2

The behavioral seizure score (BSS) and afterdischarge duration (ADD) in response to each stimulus train on the day of kindling are graphed. Stimulus trains (50 Hz, 10 s, 400 pA, 1 msec biphasic) are administered every 5 min for 6 h (total 72 stimulations). (Adapted from Lothman, E. W. et al., Brain Res, 360, 83, 1988. With permission.)

FIGURE 4.2

The behavioral seizure score (BSS) and afterdischarge duration (ADD) in response to each stimulus train on the day of kindling are graphed. Stimulus trains (50 Hz, 10 s, 400 pA, 1 msec biphasic) are administered every 5 min for 6 h (total 72 stimulations). (Adapted from Lothman, E. W. et al., Brain Res, 360, 83, 1988. With permission.)

Animals are stimulated with this alternate-day protocol until a stable kindled state is reached. Here, a stable kindled state is defined as having been achieved when the animals respond to all stimulus trains with class 4 and 5 seizures and the afterdischarge durations are within 15% of each other.7 Counting the initial kindling day, it takes about 2 weeks for all of the rats in a set to reach a stable kindled state. Animals are entered into drug trials after at least three consecutive testing days (Monday, Wednesday, and Friday) of stable kindled behavioral seizures.

On the day on which the drug is to be given the protocol is modified slightly.7 After determination of the afterdischarge threshold, four suprathreshold stimulus trains are administered 30 min apart to serve as controls. The drug is then administered and the stimulus trains are administered every 30 min for an additional 6 h for a total of 16 stimulus trains (4 predrug and 12 postdrug). Figure 4.3 shows the behavioral seizure score for one animal in the drug testing protocol. Different doses of a drug can be tested in one animal or replicability can be tested by administering the same dose of a drug to an animal on different days. After drug administration the animals still receive stimulus trains on an alternate-day basis until the responses

FIGURE 4.3

Drug protocol using rapidly recurring hippocampal seizures. This figure illustrates the results from a single animal which had been kindled using the rapid kindling protocol and achieved stable kindled responses. The animal is now on an alternate-day testing protocol of stimulation every 30 min for 6 h (total 12 stimulations per day). An example of one of these days is day -2. Day 0 is the drug testing day. The drug in this case was valproic acid (300 mg/kg) and it was administered intraperitoneally after the fourth stimulus train on day 0. Day 0 is extended 2 h (four stimulations) in order to have four stimulations prior to drug administration and 6 h of data after drug administration. After day 0, the animal is continued on a Monday, Wednesday, Friday schedule of testing. Day +2 and day +5 are Friday and Monday, respectively. (A) The behavioral seizure scores (BSS) in response to each individual stimulation. (B) The averages of four behavioral seizure scores in response to consecutive stimulations (±SEM) are presented as response blocks. (Adapted from Lothman, E. W. et al., Epilepsy Res, 2, 367, 1988. With permission.)

FIGURE 4.3

Drug protocol using rapidly recurring hippocampal seizures. This figure illustrates the results from a single animal which had been kindled using the rapid kindling protocol and achieved stable kindled responses. The animal is now on an alternate-day testing protocol of stimulation every 30 min for 6 h (total 12 stimulations per day). An example of one of these days is day -2. Day 0 is the drug testing day. The drug in this case was valproic acid (300 mg/kg) and it was administered intraperitoneally after the fourth stimulus train on day 0. Day 0 is extended 2 h (four stimulations) in order to have four stimulations prior to drug administration and 6 h of data after drug administration. After day 0, the animal is continued on a Monday, Wednesday, Friday schedule of testing. Day +2 and day +5 are Friday and Monday, respectively. (A) The behavioral seizure scores (BSS) in response to each individual stimulation. (B) The averages of four behavioral seizure scores in response to consecutive stimulations (±SEM) are presented as response blocks. (Adapted from Lothman, E. W. et al., Epilepsy Res, 2, 367, 1988. With permission.)

have returned to control values. An additional 2 d are then given for recovery before the animal receives another dose of drug or a different drug. A summary of the effects of seven commonly used antiepileptic drugs is presented in Table 4.1.

It is possible to use this protocol for longer periods of time.7 Criteria for kindled behavioral seizures are met for up to 18 h, but after this the motor responsiveness decreases. In addition, after prolonged testing there is suppression of normal responses to stimulation for up to a week after the prolonged test day. If the test procedure is carried out every day instead of the alternate-day schedule, there is a gradual decrease in responsiveness on days 4 and 5. Both the behavioral seizure score decreases and the afterdischarge duration shortens. As with the prolonged

TABLE 4.1

Effect of Antiepileptic Drugs Against Rapidly Recurring Hippocampal

Seizures in Rats

Effect of Antiepileptic Drugs Against Rapidly Recurring Hippocampal

Seizures in Rats

Suppression of

Suppression of

Dose

kindled motor

limbic

Shortening of

Drug

(mg/kg i.p.)

seizures0

seizures13

afterdischargec

Carbamazepine

30

+

+

++

50

+

+

++

Phenytoin

80

+

+

NE

Phenobarbital

30

+

+

+

60

+

+

++

Primidone

150

+

+

+

Valproic acid

200

+

+

+

300

+

+

++

Diazepam

5

+

+

+

10

+

+

+

Ethosuximide

300

NE

NE

NE

a For motor seizures, + indicates the drug reduced seizures to < class 3 seizure.

b For limbic seizures, + indicates the drug reduced seizures to < class 1 seizure.

c For afterdischarge duration, + indicates the drug reduced afterdischarge duration at least 15%; ++ indicates the drug reduced afterdischarge duration at least 50%.

NE is no effect.

Data adapted from Reference 8.

a For motor seizures, + indicates the drug reduced seizures to < class 3 seizure.

b For limbic seizures, + indicates the drug reduced seizures to < class 1 seizure.

c For afterdischarge duration, + indicates the drug reduced afterdischarge duration at least 15%; ++ indicates the drug reduced afterdischarge duration at least 50%.

NE is no effect.

Data adapted from Reference 8.

testing on one day, it takes about a week to recover normal responses after a week of every-day testing.

For data presentation and analysis, it is convenient to average the responses (both behavioral seizure scores and afterdischarge durations) to four consecutive stimulus trains (Figure 4.3B). Thus, results are presented as response blocks. This presentation has proven quite useful for studying the effects of antiepileptic drugs.8 Values are presented as means ± standard errors of the means and statistical analysis of the animals within one group can be done, as well as comparisons between groups of animals.

Toxicity of drugs can also be determined with some simple observations. Sedation can be tested by observing the animal's reaction to sudden noises or to touch. Motor dysfunction can be tested by observation of the gait and testing of the righting reflex and muscle tone. More complicated toxicity tests (as described in Chapter 8) can be performed within the limitations of having the animal's head attached to a recording cable and the time limits (30 min) between stimulations.

The protocol described above can be used to test the effects of drugs on kindled seizures. The above protocol cannot be used to examine drug effects on the kindling process. However, a protocol of stimulating every 30 min can be utilized to examine the kindling process (Figure 4.4).9 After implantation of the electrodes and determination of the afterdischarge threshold, suprathreshold stimulus trains of 20 Hz for

FIGURE 4.4

Protocol to test kindling acquisition. These graphs present the behavioral seizure score (BSS) and afterdischarge duration (ADD) for an animal that received 20 Hz stimulation (10 s, 400 pA) every 30 min over the course of two consecutive days (6 h each day). Notice the relatively steady increase in seizure score and afterdischarge duration. (Adapted from Lothman, E. W. and Williamson, J. M., Epilepsy Res., 14, 209, 1993. With permission.)

FIGURE 4.4

Protocol to test kindling acquisition. These graphs present the behavioral seizure score (BSS) and afterdischarge duration (ADD) for an animal that received 20 Hz stimulation (10 s, 400 pA) every 30 min over the course of two consecutive days (6 h each day). Notice the relatively steady increase in seizure score and afterdischarge duration. (Adapted from Lothman, E. W. and Williamson, J. M., Epilepsy Res., 14, 209, 1993. With permission.)

2 or 10 s are administered every 30 min on consecutive days, for up to 4 d. This procedure results in a gradual and steady increase in the behavioral seizure score, until the rat is consistently having class 5 seizures in response to each stimulus train. The afterdischarge duration gradually increases over the first 2 d and then plateaus at an increased level.

0 0

Post a comment