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Caveolin-1 deletion reduces early brain injury after experimental intracerebral hemorrhage - PubMed

Caveolin-1 deletion reduces early brain injury after experimental intracerebral hemorrhage

Che-Feng Chang et al. Am J Pathol. 2011 Apr.

Abstract

Intracerebral hemorrhage (ICH) is a subtype of stroke with high rates of morbidity and mortality. Caveolin-1 (Cav-1) is the main structural protein of caveolae and is involved in regulating signal transduction and cholesterol trafficking in cells. Although a recent study suggests a protective role of Cav-1 in cerebral ischemia, its function in ICH remains unknown. In this study, we examined the role of Cav-1 and in a model of collagenase-induced ICH and in neuronal cultures. Our results indicate that Cav-1 was up-regulated in the perihematomal area predominantly in endothelial cells. Cav-1 knockout mice had smaller injury volumes, milder neurologic deficits, less brain edema, and neuronal death 1 day after ICH than wild-type mice. The protective mechanism in Cav-1 knockout mice was associated with marked reduction in leukocyte infiltration, decreased expression of inflammatory mediators, including macrophage inflammatory protein (MIP)-2 and cyclooxygenase (COX)-2, and reduced matrix metalloproteinase-9 activity. Deletion of Cav-1 also suppressed heme oxygenase-1 expression and attenuated reactive oxygen species production after ICH. Moreover, deletion or knockdown of Cav-1 decreased neuronal vulnerability to hemin-induced toxicity and reduced heme oxygenase (HO)-1 induction in vitro. These data suggest that Cav-1 plays a deleterious role in early brain injury after ICH. Inhibition of Cav-1 may provide a novel therapeutic approach for the treatment of hemorrhagic stroke.

Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1

Up-regulation of Cav-1 protein expression in WT brains subjected to ICH. A: Representative immunoblots of the Cav-1 protein in the ipsilateral (hemorrhagic) and contralateral hemispheres from ICH or sham-operated mice, 1 day after collagenase injection. Bar graph of densitometric analysis of bands showing a significant increase in Cav-1 protein level in the ipsilateral hemispheres of WT mice 1 day after ICH, compared with the contralateral hemispheres. B: Identification of Cav-1–positive cells 1 day after ICH in the perihematomal area by double immunofluorescence labeling. Cav-1 immunoreactivity is shown in red, and immunolabeling of neuronal nuclei (NeuN; neurons), CD31 (endothelial cells), GFAP (glial fibrillary acidic protein; astrocytes), or Iba1 (ionized calcium binding adaptor molecule 1; microglia) is shown in green. White arrows indicate co-localization. Cav-1 localized to neurons, endothelial cells, and microglia. Sections were stained with DAPI (blue) to show all nuclei. Scale bars: 50 μm (merged column); 50 μm (inset column).

Figure 2
Figure 2

Effect of Cav-1 deletion on brain injury volume, neurologic deficits, collagenous-induced bleeding, and brain edema in mice subjected to ICH. A: Representative sections from WT and Cav-1 KO mice 1 day after sham operation (top) or ICH (bottom) stained with Luxol fast blue/cresyl violet; areas of injury are represented by the lack of staining in the lower panels. Quantification shows significantly smaller brain injury volumes in Cav-1 KO mice, compared with WT mice, 1 day after ICH. Scale bar = 1 mm. *P < 0.05 versus WT mice. B: Neurologic deficits were significantly less severe in Cav-1 KO than in WT mice at day 1. *P < 0.05 versus WT mice. C: Hemoglobin levels in WT and Cav-1 KO mice were not significantly different 6 or 24 hours after ICH. D: Hemispheric enlargement was significantly smaller in Cav-1 KO mice than in WT mice at day 1. **P < 0.01 vs WT mice. E: Brain water content in the ipsilateral basal ganglion of WT and Cav-1 KO mice was significantly higher than in the contralateral basal ganglion 1 day after ICH. In the ipsilateral basal ganglion, brain water content of Cav-1 KO mice was significantly lower than in WT mice. Cerebel, cerebellum; Cont-BG, contralateral basal ganglia; Cont-CX, contralateral cortex; Ipsi-BG, ipsilateral basal ganglia; Ipsi-CX, ipsilateral cortex. *P < 0.05 vs WT mice, **P < 0.01 vs contralateral.

Figure 3
Figure 3

Cav-1 deletion reduced neuronal degeneration and apoptotic cell death in mice subjected to ICH, as evaluated by FJB and TUNEL staining. A: Brain atlas coronal brain section of a core hemorrhagic region at 0.24 mm from the bregma. Quantification analysis indicates that Cav-1 KO mice had significantly fewer degenerating neurons than WT mice, 1 day after ICH. The total number of FJB-positive cells is expressed as the mean number per field of view (0.63 mm2). B: Representative TUNEL (top) and DAPI-stained (bottom) brain sections of a WT and a Cav-1 KO mouse 1 day after ICH. The inset is a representative TUNEL-positive cell at higher magnification. Quantification analysis shows that the percentage of TUNEL-positive cells was significantly lower in the Cav-1 KO than in the WT hemorrhagic brain, 1 day after ICH. The percentage of TUNEL-positive cells is expressed as the percentage of nuclei that were stained by the TUNEL method/the total number of DAPI-stained nuclei. Sections were stained with DAPI (blue) to show all nuclei. Scale bars: 50 μm (A and B); 10 μm (A and B insets). *P < 0.05, ***P < 0.001 vs WT mice.

Figure 4
Figure 4

Cav-1 deletion reduced HO-1 expression and reactive oxygen species production in mice subjected to ICH. A: Representative immunoblots of the HO-1 protein in the ipsilateral and contralateral hemispheres from WT and Cav-1 KO mice, 1 day after ICH or sham operation. Bar graph of densitometric analysis of bands showing a significant decrease in HO-1 protein level in the ipsilateral hemispheres of Cav-1 KO mice compared with WT mice, 1 day after ICH. B: Bar graphs show HO-1 mRNA expression in WT and Cav-1 KO mice in the ipsilateral hemispheres 6 and 24 hours after injury. Cav-1 deletion significantly reduced injury-induced HO-1 mRNA expression in the ipsilateral hemispheres 6 and 24 hours after ICH, compared with WT mice. C: Double immunofluorescence labeling of Cav-1 and HO-1 in the perihematomal area of WT mice, 1 day after ICH. Cav-1 immunoreactivity is shown in green, and HO-1 is shown in red. Co-localization of HO-1 and Cav-1 were observed in both microglia/macrophage-like cells (arrowhead) and vascular-like structures (arrow). Sections were stained with DAPI (blue) to show all nuclei. D: Representative NT-stained brain sections of a WT and a Cav-1 KO mouse 1 day after ICH. The inset is a representative NT-positive cell at higher magnification. Cell count analysis indicates that Cav-1 KO mice had significantly fewer NT-positive cells than WT mice, 1 day after ICH. The total number of NT-positive cells is expressed as the mean number per field of view (0.63 mm2). Scale bars: 20 μm (C); 100 μm (D); 10 μm (D inset). Contra, contralateral; Ipsi, ipsilateral. *P < 0.05, ***P < 0.001 vs WT mice.

Figure 5
Figure 5

Cav-1 deletion reduced neutrophil infiltration and MIP-2 expression but had no effect on MCP-1 expression in mice subjected to ICH. A: Representative MPO-stained brain sections of a WT and a Cav-1 KO mouse 1 day after ICH. The inset is a representative MPO-positive cell at higher magnification. Cell count analysis indicates that Cav-1 KO mice had significantly fewer infiltrating neutrophils than WT mice, 1 day after ICH. The total number of MPO-positive cells is expressed as the mean number per field of view (0.63 mm2). ***P < 0.001 vs WT mice. Scale bars: 100 μm (A); 10 μm (A insert). B and C: Bar graphs showing MIP-2 and MCP-1 protein concentrations in WT and Cav-1 KO 1 day after ICH. ICH significantly increased MIP-2 and MCP-1 protein levels in both WT and Cav-1 KO mice. The Cav-1 KO hemorrhagic hemispheres exhibited significantly reduced MIP-2 protein level, compared with the WT hemorrhagic hemispheres. However, no significant difference was observed in MCP-1 protein level between the two groups. *P < 0.05 vs WT mice, ***P < 0.001 vs contralateral. D and E: Bar graphs showing MIP-2 and MCP-1 mRNA expression in WT and Cav-1 KO mice 6 and 24 hours after ICH. Cav-1 deletion significantly reduced injury-induced MIP-2 mRNA expression in the ipsilateral hemisphere compared with WT mice 6 and 24 hours after ICH (*P < 0.05 vs WT mice). No significant difference was observed in MCP-1 mRNA expression between the two groups.

Figure 6
Figure 6

Cav-1 deletion reduced COX-2 expression and MMP-9 enzymatic activity in mice subjected to ICH. A: Representative immunoblots of the COX-2 protein in the ipsilateral and contralateral hemispheres from WT and Cav-1 KO mice 1 day after ICH or sham-operation. Bar graph of densitometric analysis of bands showing a significant decrease of COX-2 protein level in the ipsilateral hemispheres of Cav-1 KO mice 1 day after ICH, compared with WT mice. B: Bar graphs showing that Cav-1 deletion significantly reduced COX-2 expression in the ipsilateral hemispheres compared with WT mice 1 hour after ICH. (C) Representative zymography of the MMP-9 and MMP-2 activity from WT and Cav-1 KO mice 1 day after ICH. The gelatinase activity of MMP-9 was increased in both WT and Cav-1 KO hemorrhagic brains. MMT-9 activity was decreased in Cav-1 KO mice compared with WT mice. Weak MMP-2 gelatinase activity was also observed. Contra, contralateral; Ipsi, ipsilateral. *P < 0.05 vs WT mice.

Figure 7
Figure 7

Cav-1 deletion or knockdown increased neuronal survival and reduced HO-1 expression after stimulation with hemin in neuronal culture. A: Bar graphs showing the cell viability of primary neurons after stimulation with various concentrations of hemin, as assessed by MTT assay. Hemin (10, 15, and 20 μmol/L) stimulation significantly induced cell viability loss in both WT and Cav-1 KO neuronal cultures. Cav-1 KO neurons had a significant decrease in cell death compared with WT neurons. *P < 0.05 vs WT neuron. B: Representative immunoblots of the HO-1 protein in the WT and Cav-1 KO neurons after stimulation with 15 μmol/L hemin. Bar graph of densitometric analysis of bands showing a significant decrease of HO-1 protein level in the Cav-1 KO neurons, compared with WT neurons. *P < 0.05 vs WT neuron. C: Representative immunoblots of the HO-1 protein in human neuroblastoma SK-N-MC cells after treatment with Cav-1 siRNA. Bar graph showing a significant decrease of hemin-induced HO-1 protein expression in SK-N-MC cells compared with the control siRNA group. *P < 0.05 vs Con siRNA. D: Bar graph showing that lentiviral–Cav-1 shRNA (661) and (663) effectively suppressed Cav-1 protein expression compared with the lentiviral-control shRNA. *P < 0.05 vs control shRNA, ***P < 0.001 vs control shRNA. E: Representative immunoblots of the HO-1 protein in primary cortical neuron cultures after lentiviral infections. Bar graph showing that the hemin-induced HO-1 expression was significantly attenuated in lentiviral–Cav-1 shRNA (661, 663)–infected neurons. ***P < 0.001 vs control shRNA. F: Bar graphs showing the cell viability of primary neurons after hemin stimulation in the MTT assay. Primary neurons infected with lentiviral–Cav-1 shRNA (661) had a decrease in cell death compared with primary neurons infected with lentiviral-control shRNA. Consi, control siRNA; CAV-1si, Cav-1 siRNA. *P < 0.05 vs control shRNA.

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