The Mechanism Of Electroacupuncture Against Brain Ischemia

Cell survival is regulated by the balance between death and survival signals. Previous studies have shown that the N-methyl-d-aspartate receptors (NMDARs) are responsible for the glutamate-induced excitotoxicity in the postischemic brain. Meanwhile, nerve growth factor (NGF) is critically involved in cell survival and neuroprotective effects via the extracellular signal-related kinase (ERK) pathway or the phosphatidylinositol 3-kinase (PI3-K) pathway mediated by the high affinity NGF receptor, tropomyosin-related kinase A (TrkA). NMDARs and NGF are both involved in the processes of transient cerebral ischemia. Clinically, electroacupuncture (EA) has been shown to produce beneficial effects on stroke patients. Our previous studies have shown that EA not only promote the expression of TrkA, but also inhibit the excessive expression of NMDARs in the postischemic brain. The present study will focus on the effect of EA on the relation between NR1 subunit and TrkA in a rat model of cerebral ischemia-reperfusion in vivo, and the transcriptional regulation of NR1 gene in oxygen-glucose deprivation model in vitro.Part I Electroacupuncture regulates NMDA receptor NR1 subunit expression via PI3-K pathway in a rat model of cerebral ischemia—reperfusion1, Effect of EA treatment on NR1 mRNA levels in ischemic cortexTo determine whether the expression of NR1 subunit was altered after EA treatment compared to that in normal and ischemic groups, cortex tissues in five groups were subjected to semiquantitative RT-PCR analysis. In Ischemia group, NR1 mRNA levels were significantly increased at 24 hr after cerebral ischemia-reperfusion in cortex (P<0.05) compared to Normal group. Interestingly, while EA treatment did not affect the expression of NR1 mRNA in non-ischemia normal rats, EA treatment significantly decreased NR1 mRNA levels in ischemic cortical areas compared with those of Ischemia and Ischemia-sham EA group (P<0.05), respectively. These results suggest that EA treatment reverses the increase of NR1 mRNA levels in ischemic cortical cells. 2, Effect of EA treatment on expression of TrkA in ischemic cortexWe observed TrkA-immunoreactivity (TrkA-IR) in right cortical region in five groups. TrkA-IR was not present in the cortical neural cells of Normal and Normal-EA group. Even in Ischemia and Ischemia-sham EA group, there was no apparent change in TrkA staining at 24 hr after cerebral ischemia-reperfusion. However, we found that EA treatment obviously increased TrkA-IR in the cortical neural cells in ischemic area. TrkA-IR was present in cellular cytoplasm throughout all layers of the ischemic cortex. These results indicate that EA treatment's neuroprotective effects against ischemia may be mediated via the up-regulation of TrkA. 3, Coexpression of TrkA and NR1 in ischemic cortex after EA treatmentTo address the possible role of the TrkA activation in the regulation of the expression of NR1 subunit in ischemic cortex after EA treatment, we observed the coexpression of TrkA and NR1 in the ischemic cortex with confocal laser fluorescence microscope after animals were treated with EA. The coexpression of TrkA and NR1 were found in the cortical neurons of the ischemic brain region. It was also found thatTrkA staining was located mainly in the neuronal cytoplasm and NR1 in the neuronalmembrane. The coexpression of TrkA and NR1 in the ischemic cortex after EAtreatment indicates that it is possible that TrkA and NR1 can "cross-talk" in the samecell.4, Effect of protein kinase inhibitors on expression of NR1 subunit in ischemiccortex after EA treatmentTo investigate which signaling pathways are necessary for the effect of EA on the expression of NR1 subunit, we used specific PI3-K and ERK inhibitors, wortmannin and U0126 for our experiments. There was a high level of expression of NR1 subunit observed in Ischemia group treated with DMSO. Administration of wortmannin or U0126 did not result in significantly changes of NR1 mRNA levels compared to DMSO treatment. Interestingly, the expression of NR1 mRNA was significantly increased (P<0.05) in Ischemia-EA group after administration of wortmannin, but not with U0126.Moreover, we compared the staining density of NR1 positive neurons in the ischemic cortex treated with and without EA by a morphological measurement method. The strong NR1 staining could be seen in the neurons of cerebral ischemic area and no significant difference was found after administration of DMSO, wortmannin, or U0126, which is also confirmed by measuring the optical density of the NR1 staining. However, EA treatment resulted in a significant decrease of the strong NR1 staining and optical density and only wortmannin treatment abandoned the effect of EA.PART II Neuron-restrictive silencer factor regulates NR1 gene through NGF-PB-K pathway in OGD model in vitro 1, Effect of NGF signals on the neuronal death rate in OGD modelThe neuroprotective effects of NGF signals was determined by FDA-PI double staining. 2h OGD stimulus induced more neuronal death than normal group (P<0.05). If the neurons were exposed to NGF after 2h OGD, however, the neuronal death rate was reduced significantly. Moreover, while U0126 treatment did not affect the neuronal death rate in OGD+NGF+U0126 group, wortmannin treatment significantly increased the death rate in OGD+NGF+wortmannin group compared with those of OGD+NGF group. These results indicate the OGD is a good in vitro model to study brain injury caused by ischemia-referfusion, and NGF prevent the neuronal death by PI3-K pathway.2, Effect of NGF signals on the expression of NR1 mRNA in OGD model Cultured hippocampal neurons were subjected to semiquantitative RT-PCRanalysis. 2h OGD stimulus induced the significant increase of NR1 mRNA expression (P<0.05). NGF treatment after 2h OGD can significantly reduced the excessive expression of NR1 mRNA induced by OGD. After using inhibitor U0126, the levels of NR1 mRNA had no obvious changes, but after using wortmannin, NR1 mRNA expression were significant increased compared with those of OGD+NGF group (P<0.05).3, Effect of NGF signals on the binding activity of NRSF and NR1/NRSE in OGD modelUsing electrophoresis mobility shift assay (EMSA) with chemiluminescent technique, we detected the Effect of NGF signals on the binding activity of NRSF and NR1/NRSE in OGD model. The results demonstrated 2h OGD stimulus resulted in a weak retarded band. However, NGF treatment changed the retarded band more clear. After using wortmannin, but not U0126, the retarded band became weak again. These results indicates NGF can increase the binding avticity of NRSF and NR1/NRSE in OGD model in vitro. In summary, we have found in the present study that: ①EA treatment can up-regulate TrkA in ischemic cortex, and reverses the increase of NR1 mRNA levels induced by ischemia-reperfusion; ②EA treatment's neuroprotective effects against ischemia may be mediated via the TrkA-PI3-K pathway; ③the binding avticity of NRSF and NR1/NRSE can be increased through NGF-PI3-K pathway in OGD model in vitro.