Excitotoxicity Of TNFα Derived From KA Activated Microglia On Hippocampal Neurons In Vivo And In Vitro

Microglia constitute 20% of the total glial cell population and belong to macrophages cell line. They are sensors for pathological events in the CNS. Sustained activation and proliferation of microglia were observed during the pathological progressions of injuries, inflammation and epilepsy in CNS.Activated microglia secret a variety of proinflammatory cytokine and toxic molecular, include IL1β, TNFa and NO et al. Those over secreted substance have excito-toxic effects on neurons and which are associated with neurpnal loss in neurodegeneration disease such as epilepsy. So activated MG are generally recognized as cytotoxic effecter cells.TNFa is an important inflammatory mediator which is mainly derived from MG at the site of injured neurons in the brain. Over expression of brain TNFa in transgenic mice is associated with the occurrence of age-dependent neurodegenerative changes and sporadic spontaneous seizures. It have been found that High dose of TNFa during anti-tumor therapy in clinic could induce seizures in human. However, antagonisms of TNFa using a monoclonal antibody or inhibition of MG activation decreases the seizure susceptibility of neurons and improves their survival rate..Although the evidence provided by above materials indicates that the activation state of MG and the high level of TNFa in the brain may have relationship with the seizure-induced neuronal injury, fewer detailed studies have demonstrated that the TNFa derived from activated microglia may play an important role in the event of excitotoxicity of hippocampal neurons. Therefore, in present study, KA was used as an activator and minocycline was used as a tool drug to inhibit the activation of microglia. This study is divided into three parts to explore the above matter through in vivo and in vitro experiments.To confirm that KA can activate rat's MG and promote its secretion, MG from neonatal rat cerebral cortex were separated, purified and cultured.100μM KA was used to activate MG and minocycline pretreatment was used to inhibit the activation of MG. Then OX-42 immunostaining was performed to observing the morphological changes of MG undergoing above different treatments. RT-PCR and western blot was applied respectively to detecting the expression of TNFa mRNA and protein; Elisa was performed to testing the content of TNFa in microglial conditioned medium. The results of OX-42 immunohistochemistry showed that after being treated with KA for 2 hours, MG showed amoeba like shape with expanded cell bodies and retracted neurites, it's OX-42 immunoreaction was obviously enhanced than those of the control group and the MC+KA group. The results of RT-PCR and western blot showed that the content of TNFa mRNA and protein in MG in KA group were obviously higher than those of the control group, and which was lower in MC+KA group than in KA group. The result of Elisa showed that TNFa levels in microglial conditioned media of KA group was significantly higher than those of the control group, whereas it was lower in MC+KA group than in KA group. The results indicate that KA significantly activated MG, and TNFa is over synthesized and secreted by activated MG. Pre-treatment with minocycline can inhibit MG activation and further antagonize above effects of KA.Because of the features of auto-discharge and low excitation threshold, hippocampal neurons have a close relationship with pathogenesis of epilepsy. A variety of cytokines expressed in the brain during the process of CNS diseases have an obvious effect on the excitability of hippocampal neurons, which may be rely on a mechanism of activation of their respective receptors and ion channels on neurons. In the first part of the study, we have confirmed that the synthesis and secretion of TNFa are increased in activated MG. To further explore the excitatory effects of KA-MCM on hippocampal neurons as well as the important role that KA-MCM contained TNFa plays in this event. Through in vitro and in vivo studies, we used patch clamp and evoked potential electrophysiological technology,RT-PCR and Western blot methods to study the effects of KA-MCM on hippocampal neuronal calcium current, population spike and the expression of related excitatory protein (NMDAR1 and iNOS). The antagonisms effects of TNFa antibody was observed when it was added into KA-MCM. The records of Patch clamp and population spike showed that KA-MCM significantly increased calcium current density in hippocampal neurons, and enhanced population spike on hippocampal CA3 region of rats in vivo. RT-PCR and Western blot showed that KA-MCM significantly enhanced the expression of mRNA and protein of NMDAR1 and iNOS in hippocampal neurons and tissues, whereas addition of anti-TNFa to KA-MCM significantly eliminated such effects. The results indicated that activated MG derived TNFa have strong excitatory effects on hippocampal neurons in vivo and in vitro and which can increase the body susceptibility to seizure and neuronal injury.Studies showed that pathological changes of brain include neuron loss and gliocyte hyperplasy in chemical or electrical kindling model of epilepsy as well as patients with temporal lobe epilepsy, and apoptosis is the major way of neuronal loss. Based on above two part of the studies, here we further explore the pro-apoptosis effects of KA-MCM in hippocampal neurons and the antagonistic effects of anti-TNFa. Through in vitro and in vivo studies, by using TUNEL detection, immunohistochemistry and RT-PCR method, we observe the effects of KA-MCM and anti-TNFα-KA-MCM (adding anti-TNFa into KA-MCM) on the expressions of TUNEL positive cells as well as caspase-3 mRNA and protein. The result of TUNEL detection showed that there are few TUNEL positive cells and no significant difference among them at 24,48 hr time period after incubated with three different kinds of MCMs, but they are obviously increased at 72 hr after incubated with KA-MCM. Apoptotic cells in anti-TNFa-KA-MCM group are less than those of KA-MCM group. The result of RT-PCR showed that the expression of caspase-3 mRNA is increased at 48 hr and increased obviously in 72 hr after intra-cerebroventricular injection of KA-MCM. At same time point they are less expressed in anti-TNFa-KA-MCM than in KA-MCM group. The result of caspase-3 immunohistochemistry has the same tendency with above result of RT-PCR. The results indicate that the TNFa derived from KA activated microglia can significantly promote apoptosis of hippocampal neurons. The activation of the caspase-3 is involved in this process.Summary:KA activated MG highly expressed and secreted TNFa. KA-MCM can significantly enhance hippocampal population spike in vivo, and increase calcium current,the expression of mRNA and protein of NMDAR1, iNOS,and caspase-3 of hippocampal neurons or hippocampal tissue in vitro. Apoptosis cells are increased significantly after being incubated with KA-MCM. However, the above effects of KA-MCM can be inhibited by adding anti-TNFa antibody into KA-MCM. The above results provided by present research indicate that TNFa derived from KA activated microglia is neuroexcitoxic to hippocampal neurons in vitro and in vivo, which can induce apoptosis on hippocampal neurons. On the basis of the results, We consider that the excito-toxic effect of TNFa on hippocampal neuron may be related to the activation of Glu receptors and voltage-gated calcium channels which result in calcium overload, nitric oxide (NO) production, and activation of caspase-3 (a key enzyme of apoptotic death), el al. The mechanism of neural-immune regulation is a hot spot of concern in therapy and study areas in recently years. This study provides new experimental materials and theory supports for the prevention and therapy of epilepsy, as well as the rational design of neuroprotective drugs.