| Object:To study endocrine hormone changes of the hippocampus after cerebral ischemia-reperfusion injury and explore the effect of electro-acupuncture at meridian points on cerebral ischemia-reperfusion injury in rat on the expression of hippocampal corticotrophin releasing factor (CRF) and cortisol (CORT) gene. By comparing the meridian point electro-acupuncture and model group rat’s hippocampal CRF and CORT mRNA expression levels that electro-acupuncture at meridian points on cerebral ischemia-reperfusion injury to study the curative therapy, possible mechanisms of cerebral ischemia reperfusion injury in rats.Method:Modified line plug prepared the middle cerebral artery occlusion and reperfusion models. 240 rats were brought in five times, each has 48 rats. The rats in each time was randomly allocated to four groups, meridian point electro-acupuncture, non-meridian point electro-acupuncture, model and sham operation group, each group of 12. meridian point electro-acupuncture group acupuncture"LI11"(radial joints at the bottom of the lateral front sag) on both sides, bilateral "ST36"(knee posterolateral fibular head down about 5 mm), "GV20"(the center of the parietal bone), "GV16"(after the occipital crest pillow atlas joints back cavity). Electro-acupuncture parameters:2/30 Hz frequency, the density wave, current strength 1 mA, in the degree of the micro movement of rats’tentacles and ears,30 minutes a time. Non-meridian point electro-acupuncture group rat’s abdomen after disinfection, select six meridian points on both sides to acupuncture. The method is same with meridian point electro-acupuncture group, sham operation group and model group rats only fixed. Respectively to proceed the neural function defect scale after cerebral ischemia reperfusion 6h, 12h, Id,3d,7d, and then put them to death before eviscerating the hippocampus. Anesthesia the rats with the method of intra-peritoneal injection of 10% chloral hydrate, beheaded kill, remove the brain tissue, rapid separate of hippocampus on ice dish, The hippocampus were temporarily stored in the liquid nitrogen, to -80℃ after refrigerator under test. PCR detection:ultrapure RNA extraction kit was used to extract the rat hippocampal tissue, total RNA in each sample 2μg total RNA as a template, a random primer 0.5μg as primer, reverse transcription reaction 2 h,42℃ 70℃ for 5 minutes for c-DNA reverse transcription termination reaction. Purpose gene sequences were obtained from the gene pool, primers using Primer Premier 5.0 software design. Finally the 2-delta delta Ct method of real-time fluorescence PCR results were used to analysis relative quantitative of the data, with each group of beta actin expression for internal samples. Results: 1)After cerebral ischemia reperfusion, the expression level of rats hippocampal CRF mRNA and CORT mRNA increased obviously.2) In the model group and the curative group, the supersession of hippocampal CRF mRNA expression in the initial peak continued increases in twelve hours, from 1d all the groups except sham, the CRF mRNA expression in the hippocampus began to fall. Electric acupuncture intervention can significantly reduce the hippocampus after 1 d CRF mRNA expression levels.3)All the group except sham group, in cerebral ischemia reperfusion injury in rats after 6 h, CORT mRNA is a high expression in the hippocampus. Comparing to model group and non-meridian point group, CORT mRNA expression level of meridian point group decreased obviously in 6th hour. Meridian and non-meridian point group can significantly reduce rat hippocampus’s CORT mRNA expression level after one day. From 1st day to 3rd day electric acupuncture meridian point group decreased more significantly than electric acupuncture non-meridian point group. But there was no statistically significant difference of the two groups after 7th day. Both acupuncture meridian and non-meridian groups can play to reduce the hippocampus CORT mRNA expression levels, but earlier than in those with electric acupuncture non-meridian point can more prominent role.4) Comparing to the electric acupuncture non-meridian point group, electric acupuncture meridian point group can significantly improve the neurological score. Conclusion:Hippocampus after cerebral ischemia-reperfusion injury related endocrine hormone (CRF and CORT) high expression may aggravate the brain tissue and injure hippocampal neurons. In the experiment, the electric acupuncture meridian point can decrease the CRF mRNA and CORT mRNA expression to recover the neural function and have a protective effect on hippocampal neurons. So we believe that the mechanism of electro-acupuncture’s protection on cerebral ischemia-reperfusion injury may relate to lower the expression of hippocampal CRF and CORT mRNA. |
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