The Expression Of Annexin A7 In The Brain Of Patients With Intractable Epilepsy And Pilocarpine-induced Seizures In Rats

Objectives:Epilepsy is one of the most common chronic neurological disorders characterized by recurrent spontaneous seizures, which mainly concentrated in the adolescence and the young. Most patients could be controlled effectively by the existing antiepileptic drugs. It is estimated that approximately 20%-30% of epileptic patients do not achieve adequate seizure control with the use of existing antiepileptic drugs (AEDs), even if treated with at least three AEDs. This type of epilepsy is referred to as intractable epilepsy (IE). The pathogenetic mechanisms underlying epileptic seizures, which has been the hot spot of research, are still poorly understood so far. Recent studies indicated the alternation of intracellular calcium concentration levels and the disorders of calcium homeostatic mechanisms could play an important role in the development and maintenance of IE. Annexin A7 (ANXA7) is a member of the family of annexins, which are thought to function in the regulation of calcium homeostasis and the fusion of vesicles. So our study is to investigate the expression of ANXA7 in the brain of patients with IE and pilocarpine-induced seizures in rats and to explore ANXA7's possible role in IE in order to develop new AEDs and to improve the prognosis of IE.Materials and Methods:The 38 patients with IE included in the study were selected at random from the epilepsy brain bank; all the patients involved in our study underwent the surgical resection of temporal lobe tissue. The diagnosis of the seizure type was classified according to the 1981 International Classification of Epilepsy Seizures of the International League Against Epilepsy. The 11 controls were obtained from the neurosurgery department. These samples contained temporal neocortical tissue adjacent to the lesions. All patients were diagnosed by pathology as brain trauma and had no prior history of seizures, exposure to antiepileptic drugs or other neurologic disorders. All brain tissues used in the study were obtained after informed consents. This study was approved by the National Institutes of Health and the Committee on Human Research at Chongqing Medical University. We examined the expression of ANXA7 via immunohistochemistry, double-label immunofluorescence and western blot.In order to further analysis the possible role of ANXA7 in the pathophysiology of IE, we measured the expression and localization of ANXA7 in the brain of rats Lithium-Pilocarpine model at different times (6h,24h,48h,72h,7d,15d, 1m, 2m) after the onset of status epilepticus by immunohistochemistry, double-label immunofluorescence.Result:1. Human Section1.1 Comparison of clinical characteristics: there was no significant difference in age, gender and focus localization of the studied tissue between IE and the controls (P >0.05).92.1% of patients had a history of seizure recurrence for more than 5 years, and 68.4% of patients had at least a 10-year-long clinical history.1.2 Immunohistochemistry: The mean optical density (OD) value of Anxa7 expression was determined to be 0.3641±0.04197 (x±s) in IE'brain tissue, and 0.1824±0.02123 (x±s) in the controls (P<0.05). Western blot: gray value was 0.4631±0.26610 (x±s) in drug-resistant epilepsy group, and 0.2438±0.14948 (x±s) for control group (P<0.05). The date of immunohistochemistry complied with the result of the western blot: the two-sample t-test shows significant over-expression of Anxa7 in epileptic tissue when compared to the control1.3 In order to assess the cellular localization of Anxa7, double-label immunofluorescent staining with NSE was carried out. Double-label immunofluoresence staining showed that Anxa7 positive cells coexpressed NSE. Thus, it indicated that Anxa7 exist in the neurons, especially in the cytoplasm of neurons.2. Animal Models2.1 Behavioral Observation:the epilepsy model of Lithium-Pilocarpine rats develops in 3 phases: acute phase(6-24h, recurrent seizure), silent period(4-16d,latency) and chronic phase(automatic epilepsy). All the pilocarpine-injected rats were similar in behavior, typical prone and exhibiting intense salivation, head bobbing, mastication jaw movements, forelimb clonus, running, posture loss and lateral falling. Of pilocarpine-injected animals, only those that had been observed for continuous convulsion (stage IV or V) for 30 min or more were considered status epilepticus and could be used for further analysis. About 17% of the rats that experienced status epilepticus (SE) died during 1 week after SE.2.2 Immunohistochemistry: the expression level of ANXA7 in the brain of rats Lithium-Pilocarpine model at different times (6h,24h, 48h,72h,7d,15d, 1m, 2m(n=72)) after the onset of status epilepticus were significantly higher than the control(n=9) (P<0.05), especially in the early period.2.3 To further assess the cellular localization of ANXA7, double-label immunofluorescent staining with neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and propidium iodide (PI) was performed respectively. The double-label immunofluorescent staining showed that the ANXA7 localized in the neurons but not in astrocytes and did not localize in the nucleus.Conclusion:1. The expression of ANXA7 in the brain of patients with intractable epilepsy and pilocarpine-induced seizures in rats was both higher than the control respectively.2. ANXA7 localized in the neurons but not in astrocytes and did not localize in the nucleus.3. ANXA7 may play an important role in the development and maintenance of IE.