Study Of Claudin-5and Occludin Expression Changes And The Intervention Effects Of Gelatinases In Cerebral Ischemia-reperfusion

Thrombolytic therapy is considered to be the most effective treatment for acute cerebralinfarction. However, the emergence of reperfusion injury can cause blood-brain barrier (BBB)disruption, directly contributes to cerebral vasogenic edema and hemorrhagic transformation.The tight junctions-at the endothelial cells between blood and brain cells are importantstructural components of the BBB. Tight junction proteins play a critical role for paracellularpermeability, with the most important two proteins being claudin-5and occludin. Thegelatinases, matrix metalloproteinase-2and-9(MMP-2and-9) are members of the matrixmetalloproteinase (MMPs) family. MMP-2and-9can promote breakdown of the BBB bydirect degradation of components of the extracellular matrix, and are implicated in ischemicinjury. MMP-2and-9have been shown to mediate claudin-5and/or occludin degradation invarious pathological conditions, which further led to the destruction of the BBB. Whatremains to be determined, however, is the effect of these gelatinases on claudin-5. Becausecontroversial results have been reported showing that gelatinases can target occludin, but notclaudin-5.Thus, understanding expression characteristics of claudin-5and occludin is crucial forreducing thrombolysis-associated cerebral vasogenic edema and hemorrhagic transformation.In addition, there are few studies specifically designed to look at reperfusion-associated tightjunction proteins alteration at ischemic core regions, such as at the striatum, by using animalstroke models at different time points of reperfusion. In addition, the molecular eventsmediating this tight junction proteins degradation remain virtually unknown. Therefore, in this study, we are interested in whether the activated MMP-2and-9are involved in thedegradation of claudin-5and occludin proteins, which would mediate BBB permeability inthe stage of reperfusion.First, to test this possibility, we quantified BBB permeability in the brain of ratssubjected to2h of middle cerebral artery occlusion followed by15min,3h,6h and22h ofreperfusion. We examined the expression of claudin-5and occludin proteins in the striataltissue, which was referred to as “ischemic core”. The ultrastructure of neurovascular unit andtight junction were examined using transmission electron microscope. To assess the anatomicdistribution of gelatinolytic activity, we have schematically represented the outline of theenhanced gelatinolytic activity in the brain regions, processed for in situ zymography.Moreover, we compared with the area of energy metabolism (mitochondrial) dysfunction byTTC-staining of coronal brain tissue sections from rats subjected to the same insult in rats.Then, we future choose two reperfusion time points of3h and24h to investigate therole of MMP-2and-9activation in the degradation process of claudin-5and occludin proteins.To determine whether enhanced MMPs activity was co-localized with Evan’s blue leakage inthe ischemic brain, we performed in situ zymography on cryosections obtained from braintissue injected with Evan’s blue at3h reperfusion. By gelatin zymography, we quantified theactivities of MMP-2and-9. We measured mRNA for MMP-2, MMP-9, claudin-5, andoccludin at2h and22h of reperfusion. Furthermore, we co-localized the fluorescence oncryosections from MMP-2, claudin-5or occludin immunostaining. Since the interactionsbetween tight junction and astrocytes are required for induction of BBB properties, themorphologic relationship of claudin-5and occludin with astrocytes was analyzed. Finally,using MMPs inhibitor GM6001, we further study and verify the effect of gelatinases on thedegradation of claudin-5and occludin proteins after3h of reperfusion.The results show BBB increased significantly with time of reperfusion for15minute,3h,6h and22h compared with the sham operated group, where no visible staining was found. The reperfusion resulted in a significant reduction of22-kDa claudin-5in the ischemic sidewith expanding reperfusion time. Similarly, Western blot showed a significant decrease in65-kDa claudin-5after15min,3h and6h of reperfusion in the ischemic side. The leak at theneurovascular unit and tight junction were represented at the level of transmission electronmicroscopy. By in situ zymography we have observed that MMPs become activated15minand3h after the beginning of reperfusion in the ischemic core and penumbra, respectively.TTC staining results showed increased infarct size with time after cerebral ischemiareperfusion.At3h, Evan’s blue leakage was mainly seen in the subcortical regions including striatumin the brain slices, and increased gelatinolytic activity of MMPs in the ischemic hemisphere,where Evan’s blue leakage concurrently occurred. No Evan’s blue leakage and weakgelatinolytic activity were seen in the corresponding region of the nonischemic striatal tissue.Gelatin zymography and real-time quantitative PCR showed greater increases in MMP-2thanin MMP-9. MMP-2was the main enzymatic source at3h reperfusion. Immunohistochemistrydouble staining reveals that astrocyte GFAP are co-localized with MMP-2, claudin-5oroccludin in the region of the striatal vessels. Claudin-5and occludin mRNA expressiondecreased at2h and22h in both hemispheres, with fragments of both proteins seen in theblots at3h on the ischemic side. By24h, gel zymography showed elevated levels of MMP-9.Western blot of claudin-5at24h showed a reduction in both hemispheres compared withsham operated group, but occludin showed no significant reductions. GM6001-treated ratsshowed significant reduction in Evan’s blue extravasation in the ischemic hemispherecompared with sham operated animal. MMPs inhibitor GM6001completely blocked gelatincleavage by the improved activities of MMP-2and-9, and prevent the degradation ofclaudin-5and occludin proteins during the early stage of cerebral ischemia reperfusion,respectively.In summary, we have demonstrates the timing of tight junction proteins claudin-5, occludin in light of BBB permeability associated with cerebral ischemia reperfusion. Ourstudy suggests that MMP-2and-9mediated degradation of the claudin-5and occludinproteins may represent an important mechanism for reperfusion-associated BBB disruption.And also this study provides a microscopic explanation for the beneficial effects of early useof the MMPs inhibitors, which would reduce the risk of brain edema and hemorrhage inthrombolytic therapy.