The Study Of Presynaptic Membrane Protein (Munc18) Antibody Chronically Kindling Rat And Its Mechanism

Antibody against GluR3 (in 1994) and antibody against Munc18 (in 2000) were found in patients with Rasmussen's Encephalitis (childhood epilepsy), whose titer of both antibodies correlated with clinical improvement. There were some reports about GluR3 antibody, whereas no seldom involved in the role of Muncl8 antibody. In this work, we investigated the role of Muncl8 antibody on epilepsy in vitro and in vivo experiment on Sprague Dawley (SD) rat, and combined clinical samples for demonstrating the results of animal experiment. GluR3 antibody was chose as a positive control.Part I (vitro experiment): Muncl8 antibody and GluR3 antibody induced cytotoxicity and apoptosis of hippocampal neuronsPrimary neurons were cultured from hippocampal tissue of neonatal SD rat. Antibodies were carried out at two different time window of 20 hours and 40 hours and at four different dilutions. MTT cell proliferation assay and lactate dehydrogenase (LDH) release were used to evaluate antibody-induced cytotoxicity of nerve cells. TUNEL staining was applied to measure apoptosis and apoptotic index (AI) of nerve cells.As target cell, hippocampal nerve cells were designed to four groups: A treated with Muncl8 antibody, B did with GluR3 antibody, C did with both Muncl8 and GluR3 antibodies, D no treatment as negative control.The results showed both Muncl8 antibody and GluR3 antibody could induce cytotoxicity and apoptosis on nerve cells (P<0.05 or P<0.01), which were dose-dependent and time-dependent. The cytotoxicity produced by Muncl8 antibody was less intense than that by GluR3 (P <0.05). In contrast, AI induced by Munc18 antibody was significantly stronger than that by GluR3 (P<0.05). No synergistic effect was indicated upon treatment with both Muncl8 antibody and GluR3 antibody. Part II (animal experiment): To established Muncl8 antibody animal epilepsy models on SD rat by chronic kindling wayThe model was made by unplaced a cannulas in the hippocampal CA1 area of SD rat (8-weeks-old). After the 4th day of model, the antibody was micro-injected throughthe cannulas according to 1 u, 1/ every other day for five times, followed by every other week for additional four times. Both' electroencephalogram (EEG) and epileptiform behaviors of rats were monitored or evaluated.Rats were divided into 6 groups: A injected with Muncl8 antibody, B did GluR3 antibody, C did 0.9%NaCL solution, D only implaced a cannulas, E was sham surgical group and F normal rat.The results indicated Muncl8 antibody could induce epilepsy by chronic kindling way. It had been observed that 83% rats had epileptiform EEG and 75% rat did epileptiform behaviors. Most of rats started seizure after the 3rd ~ 5th injection. Most of epileptiform behaviors were the 2nd ~ 4th grade according to Racine's criterion, but seldom the 5th epileptiform behaviors .There were five rats which epileptiform behaviors was above the 3rd grade, so the successful ratio of this model was 42 %( 5/12). After the 8th week, 44% rats kept epileptiform EEG and 50% rats maintained epileptiform behaviors. No marked epileptiform EEG and behaviors, however, were detected in GluR3 antibody-injected group and control groups. Part III (vivo experiment): Muncl8 antibody induced apoptosis, expression of c-FOS and c-JUN of hippocampal neurons on ratAnimals were injected antibodies and grouped as described above. Batch-slaughter rats and taken brain section were in order to dynamic study. The HE Staining, Nissl Staining and TUNEL Staining were applied to observe the degeneration, necrosis and apoptosis of neurons. Cell counting was used to evaluate loss of neurons. Apoptosis index (AI) detected the apoptosis induced by antibodies in the whole hippocampus area. The expression of c-FOS and c-JUN (immunohistochemistry method) responded degree and scope of damage induced by antibody on cortex's neurons.The results showed Muncl8 antibody not only can result in damage in local implanted area, but also induce the degeneration and apoptosis of neurons in hippocampus area, such as: dentate gyrus area,...