| ObjectivesCerebral hemorrhage, with its high mortality rate and high morbidity rate, brings great harm to human health. In situation of globalization, particularly China's social phenomenon of the growing aging population, study in prevention and treatment for hemorrhagic stroke is urgently demanded. Methods of improving microcirculation, increasing blood flow, reducing the brain tissues from oxidative stress, are one of the efficient ways of treatment for cerebral hemorrhage. Acupuncture treatment, due to its advantages of safety, effectiveness, simple use, economic application and side-effect free outcomes, has been applied in the treatment of cerebral hemorrhage. However, the timing of acupuncture treatment for brain hemorrhage has long been contr over sially disputed and therefore became a troublesome problem to clinical treatment. So this experiment was conducted using a rat model of Collagenase VII induced caudop utamen cerebral hemorrhage, from the aspect of oxidative stress within the brain, we studied the effect of electro-acupuncture(EA) on cerebral Heme Oxygenase-1 (HO-1) and Heme Oxygenase-2 (HO-2) expression at different time points. This study aims at discussing the time dependent effectiveness of electro-acupuncture on cerebral hem orrhage, so as to provide a reliable experimental basis for the clinical application.Methods56 healthy Wistar male rats, weighing 200-250g were numbered according to weight and then applied the randomization of division into Normal Group, Model Group(further divided into Model Group 1-4), 3h EA Group and 72h EA Group, each group included 8 rats. The Model Groups 1 and 2 underwent sampling at 3h and 72h after modeling. For the Model Groups 3 and 4, sampling was performed at 5 days after respectively 3h and 72h post modeling. The cerebral hemorrhage was induced by injecting 0.5u Collagenase VII into caudate nucleus (1 mm behind the anterior fontanelle, 4 mm away from the midsagittal line at right with 5 mm deep). All modeled rats were raised at animal center of Jianghan University). 3h EA Group and 72h EA Group were performed at 3h and 72h after modeling at acupoints as Shuigou, bilateral Neiguan and bilateral Sanyinjiao. The EA trea tment was conducted once immediately after modeling, and rats received the treatment once a day. A pair of electrodes was connected to Shuigou point and the right ear, and another pair to ipsilateral Neiguan and Sanyinjiao, with the parameters of "Continuous Wave", 120 impulses per min frequency and 1mA intensity. EA was performed on rats for 5 times with 30 min for each.For the above 7 groups, rats were anaesthetized with 10% chloral hydrate via intraperitoneal injection. The ascending thoracic aorta were perfused with 100ml saline, 100ml 4℃precooled 4% paraformaldehyde contained PBS (pH=7.4) via thoracotomy. Brain tissue was sampled after perfusion and put into 4% paraformaldehyde overnight. Coronal Paraffin sections (10 segments, 5μm thick for each) were attained around injection site. Then samples were stained with HE and HO-1 and HO-2 were measured by immunohistochemical method. HO-1 and HO-2 positive cells were counted at×200 magnification for 4 non-overlapping visual area around hematoma tissue. The attained data were analyzed statistically.Results:(1) On HE staining, normal cerebral tissue showed no abnormality. Severe hemorrhage in brain tissue was seen in Model Group 1, along with neurofibrillary tangles and fracture observed and non-obvious microglia infiltration discovered. In Model Group 2, mild hemorrhage and microglia infiltration (foam appeared in some cells) were seen, along with very obvious liquefaction necrosis (cribriform formation); Model Group 3 appeared obvious microglia infiltration and necrosis along with mild hemorrhage; In Model Group 4, liquefaction lesion was seen reduced and microglia infiltration obvious. Foam cells infiltration and hemorrhage were discovered in 3h EA Group; and a reduced necrotic foci in cerebral tissue was seen in 72h EA Group, along with microglia infiltration and very slightly bleeding. (2) On immunohistochemistry: Cerebral HO-1 expression: Model Groups wereextremely significantly higher than Normal Group (P<0.01). Model Group 3 was higher than Model Group 1 with obviously statistical difference (P<0.01). And the same result was seen in comparison between Model Group 4 and Model Group 2 with the former group higher (P<0.01). Moreover, 3h EA Group was higher than Model Group 1 and 3 (P<0.01) and 72h EA Group higher than Model Group 2 and 4 (P<0.01, P<0.05), and 72h EA Group higher than 3h EA Group (P<0.01), with extremely significant difference. Cerebral HO-2 expression: no HO-2 positive neuron was visible in infarction focus of Model Group 1-3; it started to express in Model Group 4, which was higher than that in Model Group 2 (P<0.01); 3h EA Group was higher than Model Group 1 and 3 (P<0.01), 72h EA Group higher than Model Group 2 and 4 and moreover 3h EA Group (P<0.01), all differences were of extreme significance.Conclusions1. The Oxidative Stress may be an important factor for cerebral hemorrhage injury for its playing a role in cerebral edema and neuron injury post brain hemorrhage.2. Electro-acupuncture has certain effectiveness for repairing neurons post acute brain hemorrhage by elevating cerebral contents of HO-1 and HO-2 after the hemorrhage.3. The fact of 72h EA Group's better effect than 3h EA Group but inferior to Model Group 4 suggests a prudence of electro-acupuncture application in early stage of acute cerebral hemorrhage. |
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