| Purpose:To establish the rat model of cerebral ischemic-reperfusion (CIRI),and cured by eye acupuncture therapy, studying the relationship between the injury by cerebral ischemia-reperfusion and TLR4/NF-κB signal transduction pathways, exploring the underlying mechanism and roles of eye acupuncture therapy in the protective effect on the injure caused by cerebral ischemic-reperfusion.Materials and Methods:Healthy male SPF Wistar rats were randomly divided into normal group, sham-operative group(sham group), focal cerebral ischemic-reperfusion group(model group), eye acupuncture treated group and body acupuncture treated group.In addition to the normal group and the sham group, the middle cerebral artery occlusion model(MCAO) was established by suture method on the others. Which neurological deficit score was 1 to 3 points were considered successful model, and randomly divided into model group, eye acupuncture treated group and body acupuncture treated group. Eye acupuncture treated group and body acupuncture treated group were acupunctured immediately after ischemia and reperfusion and 12 h after ischemia and reperfusion.24 h after MCAO, the neurological function was evaluated by neurological deficit scoring, the moisture of brain tissue was detected by dry-wet weight assay, morphological changes in ischemic cortex were observed by HE staining, the TNFa, IL-1βlevels were determined by Elisa method, the TLR4, MyD88 and IKKβmRNA expression in rat ischemic cortex tissue were determined by RT-PCR; immunohistochemistry and Western blot were used to measure TLR4, MyD88, IKKβand NF-κB (p65) protein expression in ischemic cortex tissue.Results:1. In normal and sham group,there were normal and no symptoms of neurological deficit. Compared with the normal group and sham group, the model group has obvious signs of neurological impairment (p<0.01);Compared with the model group, the symptoms of neurological deficit were improved significantly (p< 0.05) in the eye acupuncture group and body acupuncture group; and the eye acupuncture group improved more significant than the body acupuncture group, but no significant difference (p> 0.05); Compared with the normal group and sham group,the model group was significantly higher brain water content (p<0.01); Compared with the model group, brain water content of the eye acupuncture group and body acupuncture group significantly decreased (p<0.05); and the brain tissue water content in eye acupuncture group and body acupuncture group was no significant difference (p> 0.05). By HE staining, in normal and sham group,there have the right structural integrity of cerebral cortex nerve cells, and hierarchy, no edema, rich cytoplasm in neuron, nuclear membrane clear; Compared with the normal group and sham group,the model group on the right cerebral cortex has typical changes in the performance of cerebral infarction:mesh-like necrosis, the edge of necrosis is unclear, neurons in the core area of infarction were disorder, nuclear condensation or fragmentation, dissolution or disappearance, cytoplasmic osteoporosis, edema, shallow staining, cells interstitial edema, inflammatory cell infiltration and can see the expansion of the capillaries. Compared with the model group, the mesh-like necrosis of eye acupuncture group and body acupuncture group on the right cerebral cortex reduced significantly, the edge of the necrosis was more clearly than the model group, but also see the expansion of Inflammatory cell infiltration and capillaries. The pathological changes also improved; sham group compared with the normal group, the serum TNFa, IL-1βlevels did not differ significantly (p> 0.05).The serum TNFa of model group was significantly increased (p<0.01); and the serum TNFa of eye acupuncture group and body acupuncture group decreased significantly (p<0.01), there was no obviously difference between the two groups.2. By RT-PCR and gel electrophoresis, in the normal and sham group, the expresstion of TLR4, MyD88 mRNA were less and there was no significant difference between the two groups(P> 0.05); Compared with the above two groups, in the model group, the expresstion of TLR4, MyD88 showed significantly higher (P<0.01); the expresstion of TLR4, MyD88 in the eye acupuncture group and body acupuncture group was significantly decreased compared with the model expression (P<0.05). Bands of gel electrophoresis showed that the TLR4 expression in the eye acupuncture group and body acupuncture group has no statistically significant (P> 0.05). Immunohistochemical staining also showed that in the normal group and sham group there were no TLR4, MyD88-positive cells in rat brain cortex. In model group, there were more TLR4, MyD88-positive cells than above two groups, cytoplasmic colored, and the different was significantly (P<0.01); Compared with the model group, the number of positive cells in eye acupuncture group and body acupuncture group reduced significantly (P<0.05), and there was no significant difference between eye acupuncture group and body acupuncture group (P> 0.05).The results of Western blot analysis indicated the same results.3. By RT-PCR and gel electrophoresis, in the normal and sham group, the expresstion of IKKβwas less and there was no significant difference between the two groups(P> 0.05); Compared with the above two groups, in the model group, the expresstion of IKKβshowed significantly higher (P<0.01); the expresstion of IKK(3 in the eye acupuncture group and body acupuncture group was significantly decreased compared with the model expression (P <0.05). Immunohistochemical staining also showed that in the normal group and sham group there were no NF-κB (p65)-positive cells and less IKKβ-positive cells in cerebral cortex. In model group, there were more IKKβ, NF-κB (p65)-positive cells than above two groups, cytoplasmic colored, and the different was significantly (P<0.01); Compared with the model group, the number of positive cells in eye acupuncture group and body acupuncture group reduced significantly (P<0.05), and there was no significant difference between eye acupuncture group and body acupuncture group (P> 0.05).The results of Western blot analysis also indicated the same results.Conclusion:1. Model group has obvious symptoms of neurological impairment, and brain edema. Compared with the model group, the eye acupuncture and the body acupuncture can significantly improve the neurological function and brain edema symptoms, and no significant differences between the two groups.2. Model group which has obvious pathological changes in the right cerebral cortex, showing that a large number of inflammatory cell infiltration. Compared with the model group, the eye acupuncture and the body acupuncture can significantly improve the pathological changes in varying degrees, and from the morphological point of view the eye acupuncture improed more obviously than body acupuncture, but there was not statistically significant between the two groups, which indicating that eye acupuncture can be effective in preventing the injure caused by cerebral ischemic-reperfusion.3.In model group, the levels of serum TNFa, IL-1βwas significantly higher; Compared with the model group, the eye acupuncture and the body acupuncture can significantly reduce serum TNFa, IL-1βlevels, reduce inflammation, that showing the eye acupuncture and the body acupuncture have a protective role in brain tissue.4. In model group, the expression of TLR4, MyD88, IKKβmRNA and their protein were increased; Compared with the model group, the eye acupuncture and the body acupuncture group can significantly reduce the levels of TLR4, MyD88, IKKβmRNA and the protein expression. And there was not statistically significant between the eye acupuncture group and the body acupuncture group.5. In model group, the expression and activity of NF-κB was increased, Compared with the model group, the eye acupuncture and the body acupuncture group can significantly reduce the levels of NF-κB expression,and inhibited activation of NF-κB. And there was not statistically significant between the eye acupuncture group and the body acupuncture group.6. In model group,the TLR4/NF-κB MyD88 dependent signaling pathway was activated, but in the eye acupuncture group this signaling pathway was inhibited, that showed eye acupuncture can reduce inflammation, achieving the purpose of cerebral protection.
|
PDF Full Text Request