Ⅰ.Correlation Research On The Expression Of P75NTR,Bax,Bcl-2,Caspase-3 And Apoptosis In Rat Cortical Neurons Following Mechanical Injury Ⅱ.Toxic Effect Of 1-trichloromethyl-1, 2, 3, 4-tetrahydro-carboline (TaClo) On Rat Dopaminergic Neuron

Ⅰ. Correlation research on the expression of P75NTR, Bax, Bcl-2, Caspase-3 and apoptosis in rat cortical neurons following mechanical injury1. Establishment and evaluation of rat neuron mechanical injury model Objective: To establish and evaluate the rat neuron mechanical injury model. Method: The cortical neuron cultures were prepared from the brain tissues of day 17 rat embryos. Briefly, cortex tissues were dissected from day 17 SD rat embryos. Cells were dissociated via gentle mechanical trituration in Hanks' Balanced Salt Solution (HBSS) containing newborn calf serum (3.5:1 v/v), the concentration of the cell suspension was 1.0 -1.2×10~7cells/ml before seeding, and the cells were seeded in poly d-lysine (50μg/ml) pre-coated 6-well plates (4×10~6/well) or in pre-coated 24-well plates for western blot (1×10~6 or 2×10~6cells/well). Cells were fed with Dulbecco's Modified Eagle Medium (DMEM/F12) containing 10% horse serum and 10% fetal bovine serum. Twenty four hours after seeding, 5μM Ara-C was added into the culture medium for 48 h, followed by replacement with fresh DMEM/F12 medium with 5% horse serum and fetal bovine serum. Cultures were exposed to treatment seven days after seeding. A traumatic neuron injury model was created by means of centrifugating culture plate which had different weight (3g, 6g, and 9g) steel plate on it for compression with vital neuron in vitro, and then the shapes of neurons and axons were observed with inverted phase contrast microscope. Meanwhile the activity of lactate dehydrogenase (LDH) was measured in culture medium at the different stages and different time-points (at 15mins, 30mins, 1h, 6h, 12h, 24h, 48h, and 72h) respectively. All the data of experiment groups are compared with them of control groups, then analysed stastically by SPSS 11.5, A value of P<0.05 was considered statistically significant.Results: The percent of neurons was more than 90% according to the morphous and the immunohistochemistry at the 7 day. Apoptosis were discovered after mechanical injury. Axonal swelling, axonal bulb, disruption of axon and neuron were observed with inverted phase contrast microscope in injury group. The cellular viability decreased and the LDH concentration increased after mechanical injury of neurons in vitro. LDH in injury groups shown significant decrease (P<0.05) compared with LDH in control groups, and there also shown significant difference within the injury groups (P<0.05).Conclusion: Cortical neurons were successfully cultured. It was confirmed that our injury model could actually produce neuronal insults.2. Test the apoptosis in neuron by flow cytometry analysis (FCM)Objective: To test the apoptosis in neuron by FCM.Method: The cortical neuron cultures were prepared from the brain tissues of day 17 rat embryos. Cultures were exposed to treatment seven days after seeding. A traumatic neuron injury model was created by means of centrifugating culture plate with vital neuron in vitro. After that, neurons were tested in several time-points (2h, 6h, 12h, 24h, 48h, 72h, and 96h). The cell suspension was prepared by the trypsinization method, stained with Annexin V-FITC/Propidium Iodide, and tested by FCM, and there were 10,000 test cells per sample.Results: Cellular relative apoptosis ratio in each group was shown after FCM analysis. The apoptosis ratio in severe group was higher significantly than the one in light and moderate groups (P<0.05).Conclusion: FCM showed the different apoptosis ratio after cell injury in each group. The apoptosis ratio in severe group was higher significantly than the one in light and moderate group.3. The correlation between the expression of p75NTR, Bax, Bcl-2, Caspase-3 and apoptosis in rat neuron mechanical injury modelObjective: To test the correlation between the expression of p75NTR, Bax, Bcl-2, Caspase-3 and apoptosis in rat neuron after mechanical injury.Method: The cortical neuron cultures were prepared from the brain tissues of day 17 rat embryos. Cultures were exposed to treatment seven days after seeding. After the traumatic neuron injury model was created, neurons were tested in several time-points. Detect the change in expression of Bax, Bcl-2, and Caspase-3 in each group after injury by immunohistochemistry method. Test the expression of p75NTR by Western-blot and Realtime PCR. Combine with cellular apoptosis ratio in each group shown after FCM analysis, the correlation can be ananlysis.Results: p75NTR, Bax, Bcl-2, and Caspase-3 protein expressed in each injured group. There was significant difference between each group. The expression of p75NTR, Bax, Caspase-3 in severe group was larger than the one in light and moderate groups (P<0.05). Bcl-2 protein expressed in 2 hour, then decreased and reached valley bottom at 48 hour. The ratio of Bax/Bcl-2 reached peak at 48-72 hour. p75NTR, Bax, and Caspase-3 had a long duration time and arrived their peaks at 48-72 hour, and the apoptosis curve showed the same.Conclusion: p75NTR, Bax, Bcl-2, and Caspase-3 play a important role in the neuronal injury. They all participate in the neuronal apoptosis. Ⅱ. Toxic effect of 1-Trichloromethyl-1,2,3,4-Tetrahydro-Carboline (TaClo) on rat dopaminergic neuronObjective: To study the toxic effect and its mechanisms of TaClo on rat dopaminergic neuron.Method: The Primary neuron-enriched cultures were prepared from the mesencephalic tissues of day 14 rat embryos. Briefly, cortex tissues were dissected from day 14 SD rat embryos. Cells were dissociated via gentle mechanical trituration in Hanks' Balanced Salt Solution (HBSS) containing newborn calf serum (3.5:1 v/v), the concentration of the cell suspension was 1.2×10~7cells/ml before seeding, and the cells were seeded in poly d-lysine (50μg/ml) pre-coated 24-well plates (1×10~5/well) or in pre-coated 24-well plates for western blot (1×10~6 or 2×10~6cells/well). Cells were fed with Dulbecco's Modified Eagle Medium (DMEM/F12) containing 10% horse serum and 10% fetal bovine serum. Twenty four hours after seeding, change the culture medium into Ara-C, followed by replacement with it. Cultures were exposed to treatment seven days after seeding. Five different doses of Taclo (50μM, 100μM, 200μM, 500μM, and 1000μM) were used to test its potential neurotoxicity, in morphology, TH staining and depolarization of mitochondrial membrane potential. Two different doses of Taclo (50μM and 200μM) were used to test p38 MAPK and Caspase-3 (by Western-blot). All the data of experiment groups are analyzed statistically by SYSTAT 10, A value of P<0.05 was considered statistically significant.Results: Taclo induces dopaminergic neuronal loss. Taclo induced 40% neuronal loss at 50μM, and about 90% at 200μM (P<0.01 and P<0.001). Under the same experimental condition, Taclo induced dopaminergic neuronal loss in a dose-dependent manner from 50uM to 200uM (P<0.001). Taclo induces the depolarization of mitochondrial membrane potential. In neuron-enriched culture, hyperpolarized mitochondria (Red fluorescence) were prominently present in the cytoplasm of control group, in contrast, most of neuronal mitochondria was depolarized (green fluorescence) upon all three different doses (50μM,100μM, and 200μM) of Taclo (p <0.05), demonstrated by the decrease in the ratio of red to green fluorescence. In order to explore whether Taclo neurotoxicity is associated with proinflammatory pathway and whether it induces neuronal death via apoptotic pathway, both phosphorylated p38 MAPK and cleaved caspase-3 were immunoblotted. Within one hour challenged with Taclo both phosphorylated p38 and cleaved caspase-3 were increased in Taclo group (50μM and 200μM), and they can still be observed till twenty-four hours upon Taclo administration, and the activity of p38 and caspase-3 seems to be increased in a dose-dependent manner.Conclusion: Taclo induces dopaminergic neuronal loss and the depolarization of mitochondrial membrane potential. The activity of p38 MAPK and caspase-3 are activated upon Taclo stimuli in dopaminergic neuron.