The Relationships Among Astrocytes, Neural-Immune-Endocrine Factors And Epileptogenesis

Epilepsy is a clinical syndrome with repeated, excessive and synchronous discharge of cerebral neurons as its characteristics. There are great significance in studing the mechanism of epilepsy. It was reported by many literatures that in the process of epileptogenesis, following the marked changes of pathology of neuron, there were many morphological and functional changes in astrocytes, including the shape and quantity of cells, the activity of metabolism and enzymes, the electro- physiology , the synthesis of protein, etc. At the same time, in previous research, we have proved that the onset of epilepsy related to the imbalance of immune- neuro-endocrine network. Nowadays, the effect of astrocytes on epileptogenesis has been reported by many literatures, but they generally focus on one or two in neural, immune and endocrine factors. Whether the astrocytes could induce epilepsy by influencing the immune-neuro-endocrine network of rats, and how astrocytes participated in the adjusting the expression of Ca2+ permeable of AMPA receptors and the mechanism of inducing epilepsy, these researchs are scarce. So, this dissertation mainly studied the effects of activated astrocytes to the neural-immune-endocrine factors of rats, and the relationship of astrocytes and epilepsy, and to the NSF and Plcβ1 of neurons , in order to understand the relationship among astrocyte, neural-immune-endocrine factors and epileptogenesis.The first section of this dissertation: we choose Coriaria Lactone as the main stimulator of astrocytes as well as NF-κBp65 translocation into the nucleus as the main indicator of activation of cells in our reseach to discover the effect of CL to purifiedd cultured astrocytes, and to provide the basic for the latter researchs. The purified cultured hippocampal astrocytes were divided into 2 groups at random: 1. control group; 2. CL group, terminated culture 2,4,8,12h after treatment.â‘ Immunocytochemistry was used to assess the changes of the expression of NF-κBp65.â‘¡Radioimmunoassay was used to assess the changes of the TNFα,progestin and IL-1βof the cultured medium.â‘¢HPLC was used to assess the changes of the Glu and GABA of the cultured medium. The results:â‘ NF-κBp65 nuclear expression was induced at 2h after CL, reached the maximal levels at 8h by means of immunocytochemistry. The number and average optic density of NF-κBp65 immunoreactivity (IR) positive neucleus were remained at a high level until 12h after CL.â‘¡The concentrations of TNFαand progestin of culture medium reached the maximal levels at 8h after CL, but the concentrations of IL-1βreached the maximal levels at 2h after CL by means of radioimmunoassay.â‘¢The concentrations of Glu and GABA of culture medium reached the maximal levels at 4h after CL by means of HPLC, the differences have the marked significance compare with the control group. All of these indicated that the CL can activate and induce astrocytes to release much active substance, such as TNFα, IL-1βand Glu etc, and the activate of astrocytes reach to the peak 8h after CL.The second section of this dissertation: To study the effect of CL-activated astrocytes on the immune factor of rats. We collected the CL-activated astrocyte- conditioned medium (ACM) and injected into lateral ventricle of SD rats. Behaviour changes were observed; Immunohistochemical SABC method was used to assess the changes of the expression of TNFαand IL-1βin the cerebral cortex and hippocampus, the amount of TNFαand IL-1βwere assessed by means of radioimmunoassay in homogenate of cerebral cortex and hippocampus and cerebrospinal fluid.. The results:â‘ Seizure was observed in ACM group 30 min after injecting ACM, but return to normal 2h later.â‘¡The immunoreaction of TNF-αin hippocampus and cerebral cortex of rats were stronger than those of the control group 4h after injecting ACM(P<0.05), and the immunoreaction of IL-1βin hippocampus and cerebral cortex of rats 2h after injecting ACM was stronger than those of the control group(P<0.05).â‘¢The concentrations of TNF-αand IL-1βin homogenate of cerebral cortex and hippocampus and cerebrospinal fluid 4h after injecting ACM were higher than that of the control group(P<0.05). The results mentioned above indicated that the ACM which activated by CL can increase the expression of TNF-αand IL-1βand result in epileptogenesis.The third section of this dissertation: To study the effect of CL-activated astrocytes on the endocrine factor of rats. We collected the CL-activated astrocyte- conditioned medium (ACM) and injected into lateral ventricle of SD rats. Behaviour changes were observed; Immunohistochemical SABC method was used to observe the changes of the expression of PR in the cerebral cortex and hippocampus; The amount of progesterone was assessed by means of radioimmunoassay in homogenate of cerebral cortex and hippocampus and cerebrospinal fluid. The results:â‘ Seizure was observed in ACM group 30 min after injecting ACM, but return to normal 2h later.â‘¡By means of immunohistochemistry, the immunoreaction of PR in hippocampus and cerebral cortex of rats was weaker than those of the control group 4h after injecting ACM and returned to normal in 12h(P<0.05).â‘¢Radioimmunoassay showed that the content of progesterone markedly increased in CSF in ACM group 2h after injecting ACM, whereas decreased in the hippocampus and cerebral cortex 4h after injecting with significant difference compared with control group (P<0.05). The results mentioned above indicated that the ACM activated by CL can decrease the expression of progesterone and PR, resulting in the attacks of epileptogenesis. The fourth section of this dissertation: To study the effect of CL-activated astrocytes on the neural factor of rats. We collected the CL-activated astrocyte- conditioned medium (ACM) and injected into lateral ventricle of normal rats. The behaviour changes were observed; Immunohistochemical SABC method was used to assess the changes of the expression of Glu and GABA in the cerebral cortex and hippocampus; The amount of Glu and GABA were assessed by means of HPLC in homogenate of cerebral cortex and hippocampus and cerebrospinal fluid. The results:â‘ Seizure was observed in ACM group 30 min after injecting ACM, but return to normal 2h later.â‘¡The immunoreaction of Glu in hippocampus and cerebral cortex of rats were stronger than those of the control group 4h after injecting ACM(P<0.05), and the immunoreaction of GABA in hippocampus and cerebral cortex of rats 2h after injecting ACM was weakener than those of the control group(P<0.05).â‘¢The concentrations of Glu in homogenate of cerebral cortex and hippocampus and cerebrospinal fluid 4h after injecting ACM were higher than that of the control group(P<0.05)but the concentrations of GABA on reverse. The results mentioned above indicated that the ACM which activated by CL can affect the expression of Glu and GABA and result in epileptogenesis.The fifth section of this dissertation: To study the effect of CL-activated astrocytes on NSF of rats and the purified cultured neurons, and on adjusting the expression of Ca2+ permeable of AMPA receptors. We collected the CL-activated astrocyte-conditioned medium (ACM) and injected into lateral ventricle of normal rats. The behaviour changes were observed; Immunohistochemical SABC method was used to assess the changes of the expression of NSF in the cerebral cortex and hippocampus; The cultured neurons were divided into 3 groups at random: 1. control group; 2. ACM group; 3. CNQX+ACM group, terminated culture 4,8,12h after treatment. Immunocytochemistry was used to assess the changes of the expression of NSF; Western blot was used to assess the changes of the content of NSF of the cultured neurons. The results:â‘ Seizure was observed in ACM group 30 min after injecting ACM, but return to normal 2h later.â‘¡The immunoreaction of NSF in hippocampus and cerebral cortex of rats were depressed than those of the control group 4h after injecting ACM(P<0.05);â‘¢The immunoreaction of NSF in cultured neurons of the ACM group were weaker than those of the control group and the CNQX+ACM group 4h, 8h after ACM(P<0.05), but The immunoreaction of NSF in cultured neurons of the control group have no marked difference with those of the CNQX+ACM group(P ?0.05);â‘£By means of Western blot , The concentrations of NSF of cultured neurons were weaker than those of the control group and the CNQX+ACM group 4h, 8h after ACM(P<0.05), but The immunoreaction of NSF in cultured neurons of the control group have no marked difference with those of the CNQX+ACM group(P ?0.05). The results mentioned above indicated that the ACM which activated by CL can reduced the expression of NSF and result in epileptogenesis. As CNQX could partly anatagonize this effect, the down-regulation of ACM of rats and cultured neurons may have relationship with AMPA receptor.The sixth section of this dissertation: To study the effect of CL-activated astrocytes on Plcβ1 of rats and the purified cultured neurons. We collected the CL-activated astrocyte-conditioned medium (ACM) and injected into lateral ventricle of normal rats. The behaviour changes were observed; Immunohistochemical SABC method was used to assess the changes of the expression of Plcβ1 in the cerebral cortex and hippocampus; The cultured neurons were divided into 2 groups at random: 1. control group; 2. ACM group, terminated culture 4,8,12h after treatment. Immunocytochemistry was used to assess the changes of the expression of Plcβ1; Western blot was used to assess the changes of the content of Plcβ1 of the cultured neurons. The results:â‘ Seizure was observed in ACM group 30 min after injecting ACM, but return to normal 2h later.â‘¡The immunoreaction of Plcβ1 in hippocampus and cerebral cortex of rats were increaseder than those of the control group 4h after injecting ACM(P<0.05);â‘¢The immunoreaction of Plcβ1 in cultured neurons of the ACM 4h group were stronger than those of the control group(P<0.05)。④By means of Western blot , The concentrations of Plcβ1 of cultured neurons were increaseder than those of the control group 4h after ACM(P<0.05). The results mentioned above indicated that the ACM which activated by CL can increase the expression of Plcβ1 and result in epileptogenesis.Summary:Coriaria Lactone could activated the astrocytes, and induced them to compose and release some active substances, such as TNFα, IL-1β,Glu and etc. The ACM which include these active substances can up-regulated the expression of TNFα, IL-1β, Glu and Plcβ1, and down-regulated the expression of progesterone and its receptor (PR) , NSF and GABA. All of these indicated that astrocytes could induce epileptogenesis maybe through breaking the balance of immune-neuro-endocrine network and influencing the Ga²⁺ permeable of AMPA receptor.