The Hippocampal Pathological Characters And Sleep Phasic Alteration In Kainic Acid-induced Epilepsy Rat

Objective:The rats were induced temporal lobe epilepsy (TLE) by kainate acid (KA) injected to the center site of CA3 region of right hippocampus with the stereotaxic technology. The following chronic experiments were carried on this model. The epileptic model effects were analysesed via three levels: behavior monitoring, intracalvarium electrocorticographic recording and histological analyses. The changes of the configuration of sleep architecture were detected in epilepsic model rats.Methods:1. Grouping. All the rats in the experiments were divided randomly into the following two groups: normal saline (NS) group and KA + vehicle solution (KA) group.2. KA-induced TLE model. Adult male Wistar rats (220-260 g and clean stage) were used in the experiments. Under chloral hydrate (350 mg/kg, i.p.) anesthesia, rats were placed on the stereotaxic apparatus and 2.5μl KA (0.04μg/μl) was slowly injected (about 10 min) to CA3 region of right hippocampus (4.0 mm posterior to bregma, 4.4 mm lateral to the midline, 3.8 mm below dura). The needle was left for 3 min and the rats'scalps were sutured. The behavioral progression of KA-induced seizures was scored according to Racine's standard classification. Those rats that could reach at least the class 4 or 5 seizures were used in this study. The same volumes of normal saline to the same site were injected in the control group.3. Weight measure. The body weights of both groups were recorded on the 1st /3rd /5th /1st week/2nd week/ 4th week/8th week after KA injected.4. Behavior monitor. Two weeks after KA injection, the spontaneous seizures number of rats was recorded 5 days every week from 9:00 to 17:00 (8 h/d, 5 d/week) by video camera.5. Cortex electrodes and nuchal muscle electrodes install. After the behavior monitoring, under chloral hydrate (350 mg/kg, i.p.) anesthesia, four copper cortical electrodes were screwed into the skull (two on the left and two on the right) in order to record electroencephalogram (EEG). Two silver electrodes were placed under the nuchal muscles in order to record electromyogram (EMG). EEG/EMG electrodes were connected to a micropedestal socket. Dental cement was then used to affix all the leads and cannulaes to the skull.6. EEG record. The EEG/EMG signals were synchronously recorded by an EEG machine. In order to reduce the effect of sleep-wake cycle, the EEG record was started from 9:00 every day, and continuously recorded for 4～6 h.7. Sleep phases. The sleep-wake cycle was devided into four phases:①Wake (W) was identified by the presence of desynchronized-EEG and high-EMG activity;②Slow wave sleep (SWS) was identified by the presence of a high-amplitude slow-wave EEG and low-EMG activity, relative to that of W;③Paradoxical sleep (PS) was identified by the presence of regular theta activity on EEG, coupled with low-EMG activity relative to that of SWS and W.8. Morphological analysis. After the EEG recording, rats were deeply anesthetized by an overdose chloral hydrate and perfused transaorticly with the modified fixation procedure. The hippocampus was cut in 20μm thin for histological analyses. Nissl staining evaluated the degenerating neurons of CA1, CA3 and dentate gyrus. Timm staining evaluated mossy fiber reorganization in the inner molecular layer of dentate gyrus that accompanied epileptogenesis.9. Statistical analysis. The SPSS 13.0 software was performed in all statistical analyses. Values were expressed as mean±SEM. Results:1. Weight changes: The weight of epileptic rats lightened greatly after surgery versus the control rats. Three days after surgery, the weights of the two groups were no significant difference.2. Behavior results: In the NS group (n = 12), no rats appeared spontaneous seizures. In KA groups, the rats were appeared epileptic seizures when they sober up from anesthesia. The seizures disappeared spontaneously within 24 h after KA microinjection. The earliest spontaneous recurrent seizures were observed about 4 weeks after KA administration (n = 10).3. After 8 weeks, all of rats were performed with PSG. The SWS1 and PS decreased, while the wake was enhanced in the epileptic animal models compared with control group. The frequency of epileptic discharges in epileptic animal models was increased during sleep.4. Morphological results:4.1. Lots of neurons were lost in the CA1, CA3, and hilus region detected by Nissl staining in KA group (n = 6), compared with NS group (n = 6).4.2. Mossy fibers abnormally invaded the granule cell layer and the third of inner molecular layer detected by Timm staining in KA group (n = 6). However, neither neurons lost nor mossy fiber sprouting (MFS) appeared in NS group (n = 6).Conclusion:1. KA-induced epileptic rat model appears similar behavior changes with human.2. KA-induced epileptic rat model appears similar pathological characters in hippocampus neurons lost and MFS with human.3. Epileptic seizures can alter the configuration of sleep architecture. The main representative characters are that the wake was enhanced, SWS1 and PS were declined, and the frequency of epileptic discharges was increased.