| Objective:Our research was based on mentor preliminary research results. We setted up MCAO of rats by improved suture method. According to principles of treatment which is "Yiqihuoxue and Quyushengxin", we electro-acupuncture (EA) the piont "Baihui""Shuigou","Zusanli". Then we observed the affect of EA to MCAO rats neurobehavioral score and TTC staining, HE staining, and the expression of HIF-1α, MVD (CD34+) in brain tissure and the level of serum albumin, S-100P (blood-brain barrier permeability index).By doing this, we wanted to investigate the mechanism for the regulation of angiogenesis after focal cerebral ischemia and reperfusion. At the same time, we tried to explain the relationship between the role of angiogenesis and blood-brain barrier permeability by EA.Methods:According to the number table, we divided84SD rats (180g-220g, male)of clean grade into four groups randomly including normal control group, sham operation group, model group and EA group. The normal control group haved12rats, which not be processed, the normal feeding. The sham operation group haved12rats, which haved only separation of the CCA and IPA to the PPA, do not plug wire tied. The model group haved30MCAO rats, which is divided into3subgroups and each subgroup of10according to ischemia/reperfusion24h,48h,72h. EA group also haved30MCAO rats, which is divided into3subgroups and each subgroup of10according to ischemia/reperfusion24h,48h,72h, but coupled with electro-acupuncture treatment based on of the model group. We use the HANS-200A type acupoint nerve stimulation instrument, the use of density wave (frequency2Hz/15Hz), about1mA current intensity, a little quiver in order to stimulate local muscle for the degree, to stimulate "Baihui","Shuigou" and "Zusanli" of the EA group rats, each time to stimulate30mins. The1st electro-acupuncture stimulation happened at2h after ischemia and reperfusion. And then, the rats in EA group accepted an electro-acupuncture stimulation every12h.According to the literature method, we have done the following research. We observed changes in the experimental rat neural behavior Zea-longa score. We observed the changes of the brain in general structural by ordinary optical microscope through HE staining way. We calculated the ratio of cerebral infarct volume by Weidner’s methods through TTC staining way. We determined the expression of HIF-1a, MVD (CD34+) in brain tissure by immunohistochemistry SP way. We determined the level of serum albumin by BCG and the level of serum S-100β by EILSA way. We measured the brain water content of wet/dry weight method.Result: (1) HE staining observed by optical microscope:Normal control group, sham operation group have no abnormal result. The HE staining of model group showed that pathological change to the neuronal damage with the extension of the time points from small to big after ischemia. Loose ischemia necrosis structure of the EA group compared with the sham group, but interstitial edema was not obvious, the number of necrotic cells were reduced compared with the model group, arranged compared with the model group rules.(2) Zea-longa score:Normal group and sham-operated rats did not appear the symptoms of neurological deficit. Neurological deficit scores of the model group were most obvious in the I/R24hours, at the peak, and then decreased gradually to I/R72h, but neurological deficit scores were still higher than normal control group and sham-operated group (P<0.01).The neurological deficit scores of the EA group were most obvious in the I/R24h, but it is still difference from model group (P<0.05). The neurological deficit score of EA group and model group compared with I/R24h showed a significant difference (P<0.05); In I/R48h and72h, the neurological deficit score still showed a particularly significant difference (P<0.01) compared with the model group. The neurological deficit scores of EA group did not fully recover to normal levels. Compared with normal control group and sham operation, it haved a special significant difference (P<0.01)(3) The expression of HIF-1a:Micro-expression of HIF-1α appeared in the rat brain of normal group and sham-operated group. The expression of HIF-1α in the rat brain of model group increased with I/R time. It arrived at the highest point in I/R72h.It constituted a particularly significant difference with the normal group and sham-operated group (P<0.01). The stronger expression of HIF-1a in the rat brain of EA group increased with I/R time compared with model group (P<0.05).It also arrived at the highest point in I/R72h.(4) The ratio of cerebral infarct volume:Normal group and sham-operated rats did not appear to cerebral infarction. In our observation time window, the volume of cerebral infarction in rats brain of model group and EA group presented a basic normal distribution. The two peak appeared at I/R48h both. In the I/R24h time window, the ratio of cerebral infarct volume between the EA group and model group showed significant difference (P<0.05) Special significant difference (P<0.01) between the EA group and model group appeared in the I/R48h,72h time window. The reducing of ratio of cerebral infarct volume in rats of EA group was bigger than that of model group with I/R time increasing. The peak appeared at I/R72h.(5) The MVD in ischemia brain regions:The MVD in the rats brain of normal control group and sham group were almost zero. The limited growth of MVD in rats ischemia brain regions of model group constituted a particularly significant difference with the normal control group and sham group (P<0.01). The MVD in rats ischemia brain regions of EA group increased with time especially in the I/R72h time point. Compared with the model group, it constituted a particularly significant difference (P<0.01) (6) The level of serum albumin:The level of serum albumin of normal control group keeped stability.Compared with the normal control group, the level of serum albumin of sham group rat was almost unchangde except the slight decrease in I/R24h which was no significant difference(P>0.05). The level of serum albumin of model group and EA group showed a clear downward trend with particularly significant difference (P<0.01), in particular the model group, compared with normal control group and sham group during the entire observation period. The level of serum albumin of EA group rat compared with model group showed significant difference (P<0.05). But the level of serum albumin of of EA group rat compared with normal control group and sham-operation group showed no difference (P>0.05).(7) The level of serum S100β:We could not detected any S-100β protein in normal control group and sham-operated rats serum at each time point. The S-100β protein could be detected in the serum of model group and EA group rats at each time piont, which haved a very significant difference from the normal control group and sham-operation group (P<0.01). Both model group and EA group haved the similar expression pattern of the level of serum S100β.The level of serum S100β increased from I/R24h began, reached the peak at I/R48h and then decreased. It still maintained a high level at I/R72h during the observation period we setted. The level of serum S100β of EA group indicaed significant differences from the model group at each time point (P<0.05)(8) Brain water content:Thebrain water content in bothnormal control group and sham-operation group rats keeped normal levels.Compared with normal control group and sham-operation group, the brain water content in both model group and EA group rats showed significantly increased after ischemia and reperfusion. The difference was obvious. Both model group and EA group haved the similar expression pattern of the brain water content.The brain water content increased from I/R24h began, reached the peak at I/R48h and then decreased. It still maintained a high level at I/R72h during the observation period we setted. The brain water content of EA group indicated significant difference from the model group at each time point (P<0.05)Conclusion:Early EA intervention can play as a positive and effective treatment against brain ischemia and reperfusion injury by improving the symptoms of neurological deficit, improving cerebral hypoxia tolerance, reducing the volume of cerebral infarction, promoting angiogenesis, increasing serum albumin levels to a certain extent endogenous, protecting the blood-brain barrier and reducing the severity of cerebral edema. |
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