Experimental Study Of The Nuclear Membrane Permeability Alterations Of Hippocampal Neurons In The Temporal Lobe Epileptogenesis

Objective:To study the nuclear membrane permeability of hippocampal neurons in the temporal lobe epileptogenesis.Methods:114healthy male Sprague-Dawley (SD) rats,6to8weeks old, were divided randomly into pilocarpine group (57rats) and normal group (57rats). Each group was divided into three subgroups:two hour, seven days, and sixty days after status epilepticus (SE). SE was induced by lithium-pilocarpin. Each subgroup was then divided into four small groups:all rats were used to observe the changes of behavioral and ECG; twelve rats, randomly selected six rats from the control group and the experimental group, were used to study the pathological changes; Remaining rats were used to isolate and fractionate nuclei employing sucrose density gradient centrifugation; isolation of neurons nuclear were used for immunofluorescence study to investigate the alteration of neurons nuclear membrane permeability, apoptotic nuclei, and relationship between the site of permeability alteration of neurons nuclear membrane and the nuclear pore.Results:1. After injection of lithium and pilocarpine, the success rate of modeling was70.8%, and total mortality was18.1%. 2. Spontaneous recurrent seizures (SRS) occurred in lithium-pilocarpine kindled epileptic rats, and chronic phase of epileptic rates were occurred. SRS could appear in8-22days after SE, average about14.9±6.4days.3. The slow waves took a major role in control group. The encephalogram of epileptic rats features various epileptiform waves, sharp waves predominating with occasional single spike and diffuse polyspike slow waves in acute phase. Rats mainly were slow wave, no apparent epileptic wave in latency phase, and showed sharp waves predominating with high volatility during the chronic phase.4. The pathological investigation of model groups showed cells loss in hilus, CA1, and CA3area. Disorganization was found in CA1, CA3pyramidal cell layers. In addition, cell loss and dispersion was found in dentate granule cell layer of some animals.5. After density gradient centrifugation of0.32M,2.2M,2.4M,2.6M and2.8M sucrose, the nuclei sedimenting at the2.6M-2.8M sucrose interfaces were mainly neurons nuclei and fewer astrocytes nuclei.6. Results of immunofluorescence in rat hippocampal neurons:(1) Results of nuclear membrane permeability changes:varying amounts of leaky nuclei were found in the Isolation nucleus of hippocampal neurons from rats of2h,7d and60d group after SE, which were more than those of the control group (P<0.01); Compared with group of7d after SE, the ratios of leaky nuclei in2h and60d group were higher (P<0.01).(2)Apoptotic nuclei:Isolation nucleus of hippocampal neurons from groups of2h、7d and60d after SE, includes varying amounts of apoptotic nuclei. Compared with control group or group of7d after SE, the ratios of apoptotic nuclei in2h and60d group were higher(P<0.01).(3) Verify the relationship between the site of permeability alteration of neurons nuclear membrane and the nuclear pore. nucleus of hippocampal neurons purified from groups of2h、7d and60d after SE were divided into two parts:varying amounts of leaky nuclei were found in the nuclei without incubation of nuclear pore blocking agent WGA; the nuclei incubated with WGA were not obvious nuclear membrane leakage.Conclusion:1. The nuclear pore of hippocampal neurons is impaired, which will lead to increase the permeability of their nuclear membrane after SE.2. The survival hippocampal neurons with increased nuclear envelope permeability may be involved in the epileptogenesis of temporal lobe epilepsy.