The Roles Of EphrinB In The Rat Hippocampus After Transient Forebrain Ischemia And Reperfusion

Objective: Hippocampal CA1 pyramidal neurons are particularly sensitive to ischemic injury, and showed Delayed neuronal death (DND) and CA3 and dentate gyrus neurons were not affected. Occurred in the course of ischemia reperfusion process, including the occurrence of excitatory neurotoxicity, oxidative stress, inflammation and other kinds of biochemical reactions. In a very short period of time after ischemia, due to sharp decline in energy supply, such as changes in ionic gradients, and extracellular glutamate and other neurotransmitter ATP-dependent increase of such failures have been caused by a variety of functions, these factors start The cell death process. However, if the ischemic process was rather short, the cell death mainly as delayed neuronal death. However, cerebral ischemia on their participation in delayed neuronal death mechanisms has not been elucidated. ephrinB2 is an important member of the Eph-ephrin receptor ligand system. In physiological conditions, ephrinB2 in the hippocampus CA1 pyramidal neurons are abundant, and in the CA3 region neurons content is less about content of CA1 pyramidal neurons in the 1/10 [53,29]. ephrinB2 and NMDA glutamate receptors in the functional activity of receptors has a very close relationship. The excessive activation of NMDA receptor activation in the regulation or not stroke, brain trauma, the middle temporal lobe epilepsy, Parkinson's, Huntington's disease and Alzheimer's disease and many acute and chronic central nervous system disorders through Excitotoxicity mediated effects leading to neuronal death. However, the ephrinB2 is involved in cerebral ischemia-reperfusion injury and its mechanism is not been reported.MAPK signal transduction pathway in neurons after cerebral ischemia plays an important role in the survival of which is relatively clear of ERK1/2, its activation by the Raf-MEK-ERK kinase via regulation, has been reported ERK1/2 activation in Cerebral ischemic injury, the phosphorylation increased after ischemia in the brain, but there are also reports p-ERK1/2 in cerebral ischemia and reperfusion play a neuroprotective role. On ERK1/2 activation in the ischemic injury is controversial.The subject of cerebral ischemia by four vessel occlusion and reperfusion model, Western blot, caspase-3, the original end of the tunel apoptosis detection and in vivo administration, we observed after transient forebrain ischemia in adult rat hippocampal pyramidal Neurons ephrinB2, p-ERK1/2, NMDARs and other protein changes. To determine the cerebral ischemia, reperfusion CA1 pyramidal neurons in the mechanism of delayed death of hippocampal CA1 area and the generation of neurons and the CA3 injury reasons for the differences.Methods:1. Transient forebrain ischemia modelAfter transient forebrain ischemia-reperfusion model using the modified method of Pulsinelli four-vessel occlusion transient forebrain ischemia (15min). Fasted overnight before surgery with a constant blood sugar levels. Rats were anesthetized with chloral hydrate (40 mg/100g, ip), the neck incision, isolated bilateral common carotid artery. The rats were then fixed on the positioning system on the two-flank holes in coagulation of the vertebral artery, the night fasting. Rats prepared in this way no apparent brain damage, behavior intact. The next day, awake with the artery clip occlusion of bilateral common carotid arteries of rats 15min after transient forebrain ischemia severe. Rats in 30 ~ 60s in a coma, loss of righting reflex, bilateral dilated pupils, and the observation of significantly lighter look for the red eye, pain reflex, spontaneous breathing can. 15min after the lifting of arterial ischemia folder to restore cerebral blood flow. Rats were treated with the survival of these symptoms 6h, 24h or 48h after the experiments. Seizures in rats after ischemia be ruled out, the experiment to maintain rat body temperature 37.0℃.In the treatment group before burning off the vertebral artery fixed in rat stereotaxic apparatus, the head incision, separated from the skull to determine the points before the skull to skull, according to the former point back 0.8mm, moved next to 1.3mm, from the skull 4.05mm for positioning under the lateral ventricle, giving U0126 1ug (5ul), then with the simple method of surgery and ischemia reperfusion group.2. The experimental group(1) Control group (n = 6);(2) 6 h, 24 h, 48 h after ischemia-reperfusion group (n = 6);(3) U0126 + 6 h, 24 h, 48 h after ischemia-reperfusion group (n = 6);3. The detection method(1) The end of the TUNEL apoptosis detection control group, ischemia-reperfusion group and advance to give U0126 group after ischemia apoptosis observed.(2) Caspase-3 activity detected in normal group, ischemia-reperfusion group and advance to give U0126 in the ischemic reperfusion group the level of Caspase-3 activity. (3) Western-blot control group, ischemia-reperfusion group and advance to give U0126 in the ischemic reperfusion group ephrinB2, p-ERK1/2, and NMDARs detect such phosphorylation.4. StatisticalAnalyze data using statistical software SPSS15.0 Data presented as mean±standard deviation ( x±s), said multiple samples of group design was used to compare the mean single factor analysis of variance (ANOVA), homogeneity of variance between groups were tested with LSD, the variance between the two groups missing persons With Games-Howell test, test levelα= 0.05, p <0.05 was considered statistically significant.Results:TUNEL staining showed that, with the cerebral ischemia time, the hippocampal CA1 pyramidal neurons in a large number of apoptosis and apoptosis rates in the 48 h 80%; Caspase-3 activity at 24 h reperfusion peak, 48 h appeared slightly decreased, while CA3 neurons in areas badly affected. Western blot results showed that, after ischemia, the p-ERK1/2, ephrinB2 and NMDA receptor phosphorylation showed rapid increasing trend, indicating that these proteins are involved in the ischemic hippocampus Neurons after reperfusion injury.2, In the cerebral ischemia reperfusion given before the earlier p-ERK1/2 inhibitor U0126 results show that: given early after U0126 cerebral ischemia survival of hippocampal neurons has a very good improvement, antagonized the ischemia-reperfusion induced hippocampal neuronal death. Western results suggest that: U0126 antagonized the advance use of p-ERK1/2 phosphorylation, while inhibition of the ephrinB2 expression and the activation of NMDARs, and the high correlation between each other. In addition, U0126 has given in advance, the better the inhibition of the Caspase-3 activity.In advance of cerebral ischemia to give p-ERK1/2 inhibitor U0126 antagonized the ischemia-reperfusion after induced death of hippocampal neurons, TUNEL staining and Caspase-3 showed that the survival of hippocampal neurons markedly Improvement. Western blot results suggest that, given advance U0126 antagonized p-ERK1/2 phosphorylation also inhibited the expression of ephrinB2 and NMDA receptor activation, in a sharp decline in the expression of 48 h, and has some time dependence. In addition, the pre-grant U0126 inhibited the activity of Caspase-3.Conclusions:1. In cerebral ischemia reperfusion, ephrinB2 involved in ischemia-reperfusion induced neuronal damage; 2. In cerebral ischemia reperfusion, ephrinB2 involved in hippocampal CA1 pyramidal neuronal damage by regulating the activation of NMDA receptors;3. In cerebral ischemia reperfusion, ephrinB2 is activated by ERK1/2 regulation and the p-Erk1/2 involved in hippocampal neurons in ischemia-reperfusion injury by regulating the expression of ephrinB2;4. In cerebral ischemia, the lack of ephrinB2 in the CA3 neurons has no significant damage. This may be very little hippocampal CA3 neurons in one of the factors of death.