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. 2010 Nov;226(1):183-90.
doi: 10.1016/j.expneurol.2010.08.023. Epub 2010 Sep 15.

Progesterone and Allopregnanolone Attenuate Blood-Brain Barrier Dysfunction Following Permanent Focal Ischemia by Regulating the Expression of Matrix Metalloproteinases

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Free PMC article

Progesterone and Allopregnanolone Attenuate Blood-Brain Barrier Dysfunction Following Permanent Focal Ischemia by Regulating the Expression of Matrix Metalloproteinases

Tauheed Ishrat et al. Exp Neurol. .
Free PMC article

Abstract

Blood-brain barrier (BBB) breakdown after stroke is linked to the up-regulation of metalloproteinases (MMPs) and inflammation. This study examines the effects of progesterone (PROG) and its neuroactive metabolite allopregnanolone (ALLO) on BBB integrity following permanent middle cerebral artery occlusion (pMCAO). Rats underwent pMCAO by electro-coagulation and received intraperitoneal injections of PROG (8 mg/kg), ALLO (8 mg/kg) or vehicle at 1 h post-occlusion and then subcutaneous injections (8 mg/kg) at 6, 24, and 48 h. MMP activation and expression were analyzed by Western blot, immunohistochemistry and gelatin zymography 72 h post-pMCAO. Occludin1, claudin5, tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) were analyzed at 72 h post-pMCAO with Western blots. BBB permeability was measured by Evans blue extravasation and infarct size was evaluated by cresyl violet at 72 h after pMCAO. Ischemic injury significantly (p<0.05) increased the expression of MMP-9, MMP-2, TNF-α and IL-6, and reduced the levels of occludin1 and claudin5. These changes were followed by increased infarct size (% contralateral hemisphere) and Evans blue extravasation into the brain indicating compromise of the BBB. PROG and ALLO attenuated BBB disruption and infarct size following pMCAO by reducing MMPs and the inflammatory response and by preventing the degradation of occludin1 and claudin5. We conclude that PROG and ALLO can help to protect BBB disruption following pMCAO.

Figures

Figure 1
Figure 1
Damage to the left cortical region (unstained core area) at 72 h after pMCAO is shown by 2, 3, 5-triphenyltetrazolium chloride (TTC)-staining. The box shows the penumbral region (peri-infarct cortical tissue) selected for Western blotting and immunostaining.
Figure 2
Figure 2
Evans blue leakage is significantly increased at 72 h after ischemic injury. PROG and ALLO treatments significantly attenuate Evans blue leakage in ischemic cortex. *Lesion (L) vs. Sham (S); L vs. #Lesion + Progesterone (LP) and #Lesion + Allopregnanolone (LA); p<0.05; n = 6.
Figure 3
Figure 3
PROG and ALLO reduce infarct size in a rat model of pMCAO. (A) CV-stained coronal sections from representative rats given vehicle, PROG or ALLO, brains harvested at 72 h post-occlusion. Infarcts are shown as unstained regions. The infarct area in PROG- and ALLO-treated rats is substantially reduced. (B) Bar diagram shows the percent area distribution of infarction compared to the area of the contralateral side in pMCAO, pMCAO plus PROG, and pMCAO plus ALLO groups.
Figure 4
Figure 4
Figure 4.1. (A) Representative Western blots of MMP-9 and MMP-2 in peri-infarct cortex at 72 h after pMCAO. (B) Densitometric analysis shows that expression of MMP-9 and MMP-2 is significantly up-regulated after pMCAO, and decreased by PROG and ALLO treatments (n = 6). 4.2 Immunohistochemical representation of secondary antibody staining for endogenous peroxidase activity as a negative control for the expression of MMP-9 and MMP-2 in the peri-infarct cortex 72 h after pMCAO (n = 3). Compared to shams, MMP-9 and MMP-2 signals increased at 72 h after pMCAO, and were decreased by PROG and ALLO treatments. 4.3. (A) Representative zymography shows effects of PROG and ALLO on MMP-9 and MMP-2. (B) Densitometric analysis shows that MMP-9 and MMP-2 levels are enhanced at 72 h after pMCAO, and decreased by PROG and ALLO treatments. *L vs. S; L vs. #LP and #LA; p<0.05; n = 6.
Figure 4
Figure 4
Figure 4.1. (A) Representative Western blots of MMP-9 and MMP-2 in peri-infarct cortex at 72 h after pMCAO. (B) Densitometric analysis shows that expression of MMP-9 and MMP-2 is significantly up-regulated after pMCAO, and decreased by PROG and ALLO treatments (n = 6). 4.2 Immunohistochemical representation of secondary antibody staining for endogenous peroxidase activity as a negative control for the expression of MMP-9 and MMP-2 in the peri-infarct cortex 72 h after pMCAO (n = 3). Compared to shams, MMP-9 and MMP-2 signals increased at 72 h after pMCAO, and were decreased by PROG and ALLO treatments. 4.3. (A) Representative zymography shows effects of PROG and ALLO on MMP-9 and MMP-2. (B) Densitometric analysis shows that MMP-9 and MMP-2 levels are enhanced at 72 h after pMCAO, and decreased by PROG and ALLO treatments. *L vs. S; L vs. #LP and #LA; p<0.05; n = 6.
Figure 4
Figure 4
Figure 4.1. (A) Representative Western blots of MMP-9 and MMP-2 in peri-infarct cortex at 72 h after pMCAO. (B) Densitometric analysis shows that expression of MMP-9 and MMP-2 is significantly up-regulated after pMCAO, and decreased by PROG and ALLO treatments (n = 6). 4.2 Immunohistochemical representation of secondary antibody staining for endogenous peroxidase activity as a negative control for the expression of MMP-9 and MMP-2 in the peri-infarct cortex 72 h after pMCAO (n = 3). Compared to shams, MMP-9 and MMP-2 signals increased at 72 h after pMCAO, and were decreased by PROG and ALLO treatments. 4.3. (A) Representative zymography shows effects of PROG and ALLO on MMP-9 and MMP-2. (B) Densitometric analysis shows that MMP-9 and MMP-2 levels are enhanced at 72 h after pMCAO, and decreased by PROG and ALLO treatments. *L vs. S; L vs. #LP and #LA; p<0.05; n = 6.
Figure 5
Figure 5
(A) Representative Western blots showing that junction protein levels were decreased in peri-infarct cortex after pMCAO compared to Shams, and increased after treatment with PROG and ALLO. (B) Quantitative analysis showing significantly lower levels of junction proteins following stroke compared to Shams. The level of claudin5 and occludin1 is significantly increased after treatment with PROG and ALLO following stroke. *L vs. S; L vs. #LP and #LA; p<0.05; n = 6.
Figure 6
Figure 6
(A) Representative Western blot showing that TNF-α and IL-6 are highly expressed in injured brain compared to Shams, and are reduced by treatment with PROG and ALLO. (B) Quantitative analysis with significant expression of TNF-α and IL-6 following stroke compared to Shams. Expression of TNF-α and IL-6 is significantly reduced after treatment with PROG and ALLO following stroke. *L vs. S; L vs. #LP and #LA; p<0.05; n = 6.

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