| ObjectiveIn cerebrovascular disease, ischemic cerebrovascular disease accounted for about80percent, and the most effective treatment is promptly restore blood perfusion of the ischemic brain tissue area. However, the patient that came to the hospital within thrombolytic therapy time window (4.5hours after the onset) is still less than5%, and intracranial and extracranial organ bleeding after thrombolysis and other side effects also limit the application of ultra-early thrombolytic. It is crucial to take effective measures to protect the brain and reduce ischemia-reperfusion injury. Ischemic preconditioning and postconditioning can inspire potential protective mechanism in the body, and reduce brain tissue damage after stroke. Ischemic preconditioning is proposed first in the experimental of myocardial ischemic injury, that is a brief, mild and nonfatal ischemic preconditioning before serious fatal ischemic events can protect subsequent severe ischemic events. Such preconditioning phenomenon can also protect brain ischemic injury, called brain ischemic tolerance(BIT). Because ischemic events may occur at any time and with the unpredictability of features, there are a lot of limitations in clinical application of ischemic preconditioning. Ischemic postconditioning (IP) is proposed first in a myocardial ischemia model of rat, that is slight, transient and non-lethal ischemic in the early stages of ischemia and reperfusion can make the body adapt to ischemia-reperfusion and tolerate long-term ischemia. Experiments confirmed lately IP also exist in cerebral ischemia.Although IP is applied after ischemic events, brain tissue and other organs are very sensitive to ischemia and hypoxia and the degree of ischemia is difficult to control. So the clinical application of IP is difficult. Later research turned to other organs taken after ischemia treatment. In mice coronary ischemia-reperfusion model, ischemia of renal artery can reduce significantly myocardial ischemia-reperfusion injury. Subsequently remote ischemic postconditioning (RIP) is proposed. That is an organ can be reduced ischemia-reperfusion injury by transient and non-lethal ischemia and reperfusion on distant organs after ischemic events. The kidney is the first to be studied to take RIP, but the operation in clinical practice is difficult. Later studies comfirmed that lower limb arteries ischemia can be implement in RIP, which has been demonstrated in cardiac and cerebral ischemia reperfusion injury in animal models. RIP can stimulate endogenous protective mechanism, enhance ischemic tolerance. As the organ of RIP implemention, lower limbs are distant from brain and can endure ischemia and hypoxia. Away from the ischemia organs of brain, operation, observation and assession is easy and simple. RIP is more easily applied to studies of cerebral ischemia protective mechanism, and has a great prospect of clinical application.The mechanisms of cerebral ischemia reperfusion injury (CIRI) include the role of oxygen free radicals, the toxic effects of excitatory amino acids, intracellular calcium overload, inflammatory response and cell apoptosis. These mechanisms is interactive. Inflammatory response is an important mechanism for CIRI, including a large number of inflammatory cytokines. Interleukin (IL) is an important inflammatory cytokines in CIRI, including facilitative and inhibitory inflammatory factors. IL-1β is the most important facilitative pro-inflammatory cytokines. IL-1Ra is unique and selective competitive antagonist produced in vivo, and is a specific inhibitor of IL-1, belonging to the suppression of inflammatory cytokines. It may be alleviated by CIRI against inflammatory response. In acute phase of CIRI, apoptosis and necrosis coexist. In delayed neuronal death (DND) occuring in a few hours to several days, neuronal apoptosis is main. Bcl-2family plays an important role in neuronal apoptosis process. Bcl-2family contain anti-apoptotic genes and pro-apoptotic genes. Bcl-2is the most important anti-apoptotic gene, by inhibiting free radicals, intracellular calcium overload, the activation of Caspases mechanisms and the release of cytochrome C and enhancing the stability of the membrane and reducing. Bax is the most important pro-apoptotic gene and play a reverse biological role of Bcl-2.The study was to research the neuroprotective effect of RIP with bilateral femoral artery occlusion on neurological function, infarct volume, brain water content, pathological changes, apoptosis, and the expression of inflammatory cytokines IL-1β mRNA and IL-1Ra mRNA following focal cerebral ischemia and reperfusion by suture method in rats, to investigate the protective mechanism of RIP, and lay the foundation for further clinical application.Methods147healthy10-week-old male SD rats were randomly divided into three groups by non-surgery:sham-operated group, model group and RIP group. The model of middle cerebral artery occlusion and reperfusion(MCAO/R) was set up. Embolization thread was inserted into all rats except those in the sham-operated group for the establishment of model of middle cerebral artery occlusion for2hours and then thrown out to reperfusion for24hours. In RIP group, bilateral femoral arteries were exposed before middle cerebral artery occlusion and were clamped for10minutes then opened10minutes for a total of three cycles after reperfusion2h and before reperfusion immediately. In sham-operated group and model group, femoral arteries were isolated without occlusion. After reperfusion for24hours, the neurological function was evaluated by granding on a scale of0-5, infarct volume was measured by TTC staining, brain water content was measured by wet and dry weight ratio method, pathological structural changes in ischemic rat hippocampal CA1region were observed by HE staining under light microscope and electron microscope, apoptosis in CA1was assay by TUNEL, Bcl-2and Bax protein were detected by SABC immunohistochemical staining method, and the expression of IL-1β mRNA and IL-1Ra mRNA were compared by RT-PCR. All data were expressed as mean±standard deviation indicated. Different groups were compared by analysis of variance and q test. Data were analyzed using SPSS19.0. P<0.05indicates a statistically significant difference.Results1, Compared with sham-operated group, in model group neurological dysfunction is apparent and neurological score was also significantly increased (P<0.05); compared with the model group, neurological function scores in RIP group were lower, but not statistically significant.2, RIP reduce infarct volume after MCAO/R. Observed by TTC staining, brain tissue in sham-operated group was red, and no infarction was be detected. In model group, the brain tissue swelled obviously with white ischemic area and larger infarct size involving the cortex, basal ganglia and hippocampus. While compared with model group, infarct volume in RIP group decreased (P<0.05). 3, RIP reduce brain edema after MCAO/R. Compared with sham-operated group, the brain water content in the model group was significantly higher (P<0.05), while compared with model group, brain water content in RIP group was significantly lower (P<0.05).4,Pathological changes in ischemic hippocampal CA1region was observed by HE staining. In sham-operated group, no edema and necrosis was detected and nerve cells arranged in neat rows with normal morphology and pale staining. In model group, obvious pathological changes can be seen with loose interstitial cells, interstitial edema, peripheral widened gap, disorder and shrinkage. In RIP group, shrinkage and interstitial edema significantly decreased with more regular arrangement.5, Pathological changes of brain tissue were observed by electron microscopy. In sham-operated group neurons was normal with evenly distributed chromatin nucleus, and cell membrane, nuclear membrane and organelles is complete, and mitochondrial membrane is integrity with more mitochondria. In model group, serious pathological changes occurred in nuclei and mitochondria. In RIP group, mitochondrial structure was roughly normal.6, RIP reduce apoptosis in ischemic hippocampal CA1region. TUNEL-positive cells in sham-operated group were less. Compared with sham-operated group, TUNEL-positive cells in model group were significantly increased (P<0.05), with brown irregular cellular nucleus concentrated. Compared with model group, TUNEL-positive cells were significantly decreased (P<0.05), and apoptosis rate decreased.7, RIP impact differently on protein expression of Bcl-2and Bax in ischemic hippocampal CA1region. In sham-operated group, positive cells of Bcl-2and Bax scattered in the brain and were small in hippocampal CA1region. In model group, positive cells of Bcl-2and Bax were significantly more than those in sham-operated group (P<0.05), and ratio of Bcl-2/Bax was decreased compared with sham-operated group (P<0.05). In RIP group, Bcl-2positive cells were significantly more than those in sham-operated group and in model group (P<0.05), and Bax positive cells were more than those in sham-operated group (P<0.05) while less than those in model group (P<0.05), and ratio of Bcl-2/Bax was the most in the three groups (P<0.05).8, The expression of IL-1β mRNA in ischemic hippocampal CA1region was detected by RT-PCR. In sham-operated group, IL-1β mRNA level is lower. In model group, IL-1β mRNA level increased significantly compared with sham-operated group (P<0.05). In RIP group, IL-1β mRNA level was lower than that in model group, but still more than that in sham-operated group (P<0.05).9, The expression of IL-1Ra mRNA in ischemic hippocampal CA1region in sham-operated group by RT-PCR was lower, while that in model group and RIP group were higher (P<0.05), and among which the expression of IL-1Ra mRNA in RIP group was the highest (P<0.05). Conclusions1, RIP can reduce neurological deficits, infarct volume, cerebral edema and neuronal apoptosis following MCAO/R, that resulted in cerebral protection.2, IL-1β is a kind of pro-inflammatory cytokine, and IL-1Ra is a kind of inhibiting inflammatory cytokine. They played an important role in cerebral ischemia-reperfusion injury.3, RIP can reduce inflammation and apoptosis by down-regulating IL-1β mRNA and up-regulating IL-1Ra mRNA and reduce ischemia-reperfusion injury.4, As an organ implemented in RIP, lower extremity is safe and easy on clinical operability, that has a great prospect of clinical application. |
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