Limb Ischemic Perconditioning Attenuates Blood-Brain Barrier Disruption by Inhibiting Activity of MMP-9 and Occludin Degradation after Focal Cerebral Ischemia
Ren Changhong1,2,6,7, Li Ning1,6, Wang Brian2, Yang Yong3, Gao Jinhuan1, Li Sijie1,6, Ding Yuchuan4, Jin Kunlin1,2, Ji Xunming1,5,*()
1Institute of Hypoxia Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China 2Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107, USA 3Department of Herbal Medicine, School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China 4Department of Neurosurgery, Wayne State University School of Medicine, Detroit, MI 48201, USA 5Cerebrovascular Diseases Research Institute, Xuanwu Hospital, Capital Medical University, Beijing, China 6Center of Stroke, Beijing Institute for Brain Disorder, Beijing, China 7Beijing Key Laboratory of Hypoxia Translational Medicine. Beijing, China.
Remote ischemic perconditioning (PerC) has been proved to have neuroprotective effects on cerebral ischemia, however, the effect of PerC on the BBB disruption and underlying mechanisms remains largely unknown. To address these issues, total 90 adult male Sprague Dawley (SD) rats were used. The rats underwent 90-min middle cerebral artery occlusion (MCAO), and the limb remote ischemic PerC was immediately applied after the onset of MCAO. We found that limb remote PerC protected BBB breakdown and brain edema, in parallel with reduced infarct volume and improved neurological deficits, after MCAO. Immunofluorescence studies revealed that MCAO resulted in disrupted continuity of claudin-5 staining in the cerebral endothelial cells with significant gap formation, which was significantly improved after PerC. Western blot analysis demonstrated that expression of tight junction (TJ) protein occludin was significantly increased, but other elements of TJ proteins, claudin-5 and ZO-1, in the BBB endothelial cells were not altered at 48 h after PerC, compared to MCAO group. The expression of matrix metalloproteinase (MMP-9), which was involved in TJ protein degradation, was decreased after PerC. Interestingly, phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), an upstream of MMP-9 signaling, was significantly reduced in the PerC group. Our data suggest that PerC inhibits MMP-9-mediated occludin degradation, which could lead to decreased BBB disruption and brain edema after ischemic stroke.
Figure 1. Limb remote ischemic PerC signiﬁcantly reduced ischemic injury. (A) Neurological deficits were determined using neurobehavioral scoring system (higher scores correspond with more severe deficits) after MCAO. (B) Motor function was determined by ladder rung walking test (higher scores correspond with more severe deficits). *P<0.05 and **P<0.01, versus MCAO group. N=10 per group. Error bars indicate SD. (C) Representative image of TTC-stained coronal brain slices at 48 h of reperfusion in MCAO group and MCAO + PerC group. (D) Quantitative analysis of infarct volume in MCAO group and MCAO + PerC group. **P<0.01, versus MCAO group. N=5 per group.
Figure 2. Limb remote ischemic PerC reduced brain edema and BBB leakage. (A) PerC treatment attenuated stroke-induced brain edema. **P<0.01, versus ipsilateral control groups; #P<0.001, versus corresponding ipsilateral ischemic hemisphere. N=5 per group. (B) PerC reduced BBB leakage. *P<0.05, versus ipsilateral MCAO groups; #P<0.001, versus corresponding ipsilateral ischemic hemisphere. §P<0.01, versus ipsilateral sham group. N=5 per group. Error bars indicate SD.
Figure 3. Remote ischemic PerC reduced TJ gap formation. (A) Representative images of claudin-5 positive vessels (green) in the penumbra of the rat brain. Vessels in the penumbra of sham-operated brain showed a continuous, linear labeling of claudin-5. In the MCAO group, a discontinuous, less regular distribution of claudin-5 in the vessels was noted (arrows), which was barely detectable in the PerC-treated group. (B) Quantification of gap formation in sham group, MCAO group and MCAO + PerC group. Values are mean ± SD. **P<0.01, versus MCAO group; §P<0.01, versus sham group. N=5 per group. Scale bar = 25 μm.
Figure 4. Remote ischemic PerC upregulated occludin expression. (A) Representative Western blots showing occludin, claudin-5 and ZO-1 expressions in MCAO group and MCAO + PerC group. (B) Quantification of occludin, claudin-5 and ZO-1 in sham group, MCAO group and MCAO + PerC group. Values are mean ± SD. **P<0.01, versus MCAO group; §P<0.01, versus sham group. N=5 per group.
Figure 5. Remote ischemic PerC downregulated MMP-9 activity. (A) Representative Western blots showing MMP-9 and MMP-2 protein expressions after 48 h of reperfusion in MCAO group and MCAO + PerC group. (B) Densitometric quantification of MMP-9 expression in MCAO group and MCAO + PerC group. Values are mean±SD. **P<0.01, versus MCAO group; N=5 per group. (C) Representative gelatin zymography image of MMP-9 pro-form (proMMP-9) and pro-form of MMP-2 (proMMP-2) activities in each group. (D) Densitometric quantification of proMMP-9 (left panel) and proMMP-2 (right panel) in each group. Values are mean±SD. **P<0.01, versus MCAO group; §P<0.01, versus sham group. N=5 per group.
Figure 6. Remote ischemic PerC attenuated phosphorylated-ERK1/2 activity. (A) Representative Western blots of pERK1/2 and ERK1/2 protein levels in MCAO group and MCAO + PerC group. (B) Densitometric quantification of pERK1/2 (top panel) and ERK1/2 (bottom panel) in MCAO group and MCAO + PerC group. Values are mean±SD. **P<0.01, ***P<0.001, versus MCAO group; §P<0.01, versus sham group. N=5 per group.
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