| ObjectiveStroke is a kind of health-threatening disease especially for the older people. The mechanism of stroke has been studied deeply, but is not fully understood and the treatment measures are not well satisfactory.Preconditioning with brief, nonlethal ischemia (ischemic preconditioning, IPC) can reduce the injury of subsequent severe ischemia. This phenomenon is called ischemia tolerance. The precise mechanism of brain ischemia tolerance is unclear and a lot of substances may have been involved in the process. Studies revealed there was relationship between brain ischemia tolerance and the injury of cerebral ischemia. The triggering mechanism of brain IPC is similar to that of cerebral ischemia injury, except that different extent of stimuli results in different effects. Studies have illustrated that many substances deteriorating brain damage in ischemia can be protective in the process of brain IPC.Bradykinin (BK) is considered an important mediator of the inflammatory response in both peripheral and central nervous system, and it has attracted recent interest as a potential mediator of brain injury following stroke. An increase in plasma bradykinin levels was observed during cerebral ischemia. Early treatment after cerebral ischemia with bradykinin B2 receptor antagonist was reported to improve outcome in ischemic rats. During the late stage of ischemic stroke, kallikrein gene transfer provided neuroprotection against cerebral ischemia injury. These results suggest that during the different period of cerebral ischemia bradykinin acts differently. Studies in heart IPC implicated that not only endogenous bradykinin mediated IPC but also exogenously administrated bradykinin mimicked IPC and protected ischemic myocardium. In the study of rat mesenteric microcirculation, exogenous bradykinin preconditioning resisted inflammatory effects after ischemia. Therefore, we tested bradykinin's ability in a rat middle cerebral artery occlusion model to induce ischemia tolerance against ischemic neuronal injury. It will provide theoretical basis for the studies of brain ischemic tolerance mechanism and drug preconditioning.Blood-brain barrier (BBB) is composed of brain microvascular endothelial cells, basal membrane and glial cell processes, and which is important to hold the balance between brain tissue and blood. During brain ischemia, the disruption of BBB results in the increase of brain vascular permeability and it could further promote ischemic brain injury and brain edema. Brain microvascular endothelial cells are characterized by the presence of tight junctions and the absence of fenestrations and vesicles. After brain ischemia, the tight junctions of BBB are disrupted, transcytosis vesicles are increased, and leukocytes adherence and migration rate is enhanced. Therefore, we primary culture the rat brain microvascular endothelial cells as BBB model in vitro, take oxygen glucose deprivation (OGD) to mimic ischemia in vivo, and then tested the changes of BBB construction and function. In addition, studies showed that ischemic preconditioning not only could protect neurons but also protect endothelial cells. At last, we tested the influence of preconditioning on BBB construction and function in vitro. It will further provide theoretical basis for the studies of brain ischemic tolerance mechanism.Materials and MethodsPartâ… : Effects of bradykinin preconditioning on focal cerebral ischemia rats.1. Materials(1) Animals: female SD rats weighing 250-300g.(2) Experimental reagents: bradykinin, 2,3,5-triphenyltetrazolium, Evans blue, rabbit anti-basic fibroblast growth factor antibody, SABC kit, goat anti-rabbit IgG antibody, rabbit anti-β-actin antibody, enhanced chemiluminescence kit.(3) Experimental instruments: micro-perfusion pump, spectrophotometer, electrophoresis apparatus, transmark apparatus, freeze microtome. 2. Methods(1) Rats middle cerebral artery ischemia-reperfusion model by intraluminal suture.(2) Measurement of infarct volume by 2,3,5-triphenyltetrazolium staining and image analysis to test the effects of bradykinin preconditioning.(3) Measurement of brain hemispheric water content to demonstrate the mechanism of bradykinin preconditioning.(4) The blood-brain barrier permeability was quantitatively evaluated by extravasation of Evans blue to demonstrate the mechanism of bradykinin preconditioning.(5) Immunohistochemical assessment the expression levels of basic fibroblast growth factor surrounding the infarct area to demonstrate the mechanism of bradykinin preconditioning.(6) Western-blot assessment the expression levels of basic fibroblast growth factor surrounding the infarct area to demonstrate the mechanism of bradykinin preconditioning.Partâ…¡: Effects of preconditioning on brain endothelial cells tight junctions and cell adherence.1. Materials(1) Animals: 3-5d SD rats.(2) Experimental reagents: DMEM, fetal calf serum, ECGS, trypsinase, typeâ…¡collagenase, dextran, rabbit anti-â…§related antigen antibody, rabbit anti-occludin antibody, rat anti-ZO-1 antibody, TRITC-phalloidin, rabbit anti-ICAM-1 antibody, rabbit anti-VCAM-1 antibody, SABC kit.(3) Experimental instruments: CO2 incubator, inverted microscope, low-oxygen incubator, fluorescence microscope, electro microscope.2. Methods(1) Rat brain microvascular endothelial cells culture and identification.(2) Analyze the cell culture fluid by blood-gas analyzer to ensure the deoxygenated culture.(3) Measurements of cell ultrastructure changes by electro microscope to test the effects of OGD preconditioning.(4) Immunofluorescence and fluorescence-labeled assessment the expression of tight junction proteins ZO-1 and F-actin, to demonstrate the effects of OGD preconditioning on BBB tight junctions.(5) Immunohistochemical assessment the expression levels of ICAM-1 and VCAM-1 to demonstrate the mechanism of OGD preconditioning.ResultsPartâ… : Effects of bradykinin preconditioning on focal cerebral ischemia rats.1. TTC staining results showed that bradykinin preconditioning could reduce infarct volume significantly. And the infarct volume decreased as the time interval between bradykinin administration and ischemic insults shortened.2. Brain water content in the ischemic hemisphere was significantly reduced and the brain edema was alleviated by bradykinin preconditioning.3. EB extravasation content and scope of blue-staining were significantly decreased and blood-brain barrier function was protected by bradykinin preconditioning.4. Both immunohistochemical and Western-blot assessment showed that bradykinin preconditioning significantly increased bFGF-like immunoreactivity surrounding the infarct area and promoted the neuronal survival.Partâ…¡: Effects of preconditioning on brain endothelial cells tight junctions and cell adherence.1.OGD preconditioning could alleviate the injury of brain microvascular endothelial cells, decrease the opening of tight junctions, protect the brain microvascular endothelial cells.2. OGD preconditioning could lighten the changes of tight junction proteins ZO-1 and F-actin location.3. OGD preconditioning could decrease the expression of inter-cellular adherence molecules ICAM-1 and VCAM-1.Conclusion1. Bradykinin preconditioning provided neuroprotection against ischemic injury induced by transient middle cerebral artery occlusion in rats. Bradykinin preconditioning decreased infarct volume, brain edema and the permeability of blood-brain barrier and increased the expression of basic fibroblast growth factor surrounding infarct area. The ischemia tolerance in the brain induced by bradykinin preconditioning may be due to the protection of cerebral vasculature and the promotion of neuronal survival.2. OGD and re-oxygenation/glucose destroyed the ultrastructure such as tight junctions of rat brain microvascular endothelial cells, changed the expression location of cellular tight junctions proteins and cellular framework proteins, increased the expression of inter-cellular adherence molecules. OGD preconditioning induced ischemic tolerance and provided protective effects on rat brain microvascular endothelial cells injury.
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