Post-transcriptional inactivation of matrix metalloproteinase-12 after focal cerebral ischemia attenuates brain damage

Abstract

This study highlights the possible pathological role of MMP-12 in the context of ischemic stroke. Male rats were subjected to a two-hour middle cerebral artery occlusion (MCAO) procedure. MMP-12 shRNA expressing plasmid formulation was administered to these rats twenty-four hours after reperfusion. The results showed a predominant upregulation of MMP-12 (approximately 47, 58, 143, and 265 folds on days 1, 3, 5, 7 post-ischemia, respectively) in MCAO subjected rats. MMP-12 expression was localized to neurons, oligodendrocytes and microglia, but not astrocytes. Transcriptional inactivation of MMP-12 significantly reduced the infarct size. The percent infarct size was reduced from 62.87±4.13 to 34.67±5.39 after MMP-12 knockdown compared to untreated MCAO subjected rats. Expression of myelin basic protein was increased, and activity of MMP-9 was reduced in ischemic rat brains after MMP-12 knockdown. Furthermore, a significant reduction in the extent of apoptosis was noticed after MMP-12 knockdown. TNFα expression in the ipsilateral regions of MCAO-subjected rats was reduced after MMP-12 knockdown in addition to the reduced protein expression of apoptotic molecules that are downstream to TNFα signaling. Specific knockdown of MMP-12 after focal cerebral ischemia offers neuroprotection that could be mediated via reduced MMP-9 activation and myelin degradation as well as inhibition of apoptosis.

Figure 1. Regulation of MMPs in rats after focal cerebral ischemia.

Quantitative real-time PCR data of MMPs in the ischemic brains of rats subjected to a two-hour MCAO procedure followed by various reperfusion times. Error bars indicate SD. n = 6. Values on y-axis represent fold change compared to sham controls.

Figure 2. MMP-12 protein expression in rats subjected to focal cerebral ischemia and reperfusion.

(A) Immunoblot analysis depicting the protein expression of MMP-12 in the ipsilateral brain regions of rats subjected to ischemia and reperfusion. GAPDH was used as a loading control. Bar graph represents the quantification of MMP-12 protein expression. n = 6. R = reperfusion. *p < 0.05 vs. sham. (B) Immunofluorescence analysis depicting MMP-12 protein expression (green fluorescence) in the ipsilateral brain regions of sham controls and the rats subjected to a two-hour MCAO procedure followed by seven days or fourteen days reperfusion. Nuclei were stained with DAPI. n = 6.

Figure 3. Cellular localization of MMP-12 in neuronal cells of rats, which were subjected to focal cerebral ischemia and reperfusion.

Immunofluorescence analysis showing the protein expressions of MMP-12 (green fluorescence) and NeuN/GFAP/MOG/Iba1 (red fluorescence) in the ipsilateral brain regions of rats subjected a two-hour MCAO procedure followed by seven days reperfusion. Yellow fluorescence in the merged images indicates the cellular localization of MMP-12. Nuclei were stained with DAPI. Insets in the merged images show DAPI staining. n = 6.

Figure 4. In vitro and in vivo efficiency of plasmids expressing MMP-12 shRNA.

(A) RT-PCR and immunoblot analysis depicting the mRNA and protein expression, respectively, of MMP-12 after transfection of C6 rat glioma cell lines with plasmids containing an empty vector, a vector ligated with a scrambled sequence (SV-sh) and a vector ligated with MMP-12 shRNA (M-12-sh). βActin and GAPDH were used as loading controls. n = 3. Expression of MMP-12 in the ischemic brain regions of rats subjected a two-hour MCAO followed by seven days reperfusion and/or treated with SV-sh or M-12-sh. Bar graph represents the quantification of MMP-12 protein expression. n = 6. *p < 0.05 vs. sham; ^#p < 0.05 vs. untreated. (B) Immunofluorescence analysis depicting the protein expressions of MMP-12 (green fluorescence) in untreated and M-12-sh treated rats subjected to a two-hour MCAO procedure followed by seven days reperfusion. Nuclei were stained with DAPI. n = 6.

Figure 5. In vivo efficacy of plasmids expressing MMP-12 shRNA.

(A) Representative TTC staining images of the rat coronal brain sections of sham-operated, untreated MCAO-subjected and MMP-12 shRNA (M-12sh) treated MCAO-subjected rats sacrificed seven days after reperfusion. n = 6. The white-colored areas represent the infarct regions in these sections, and the red-colored areas represent normal areas. (B) Quantification of infarct volume from TTC stained sections using image analysis software. The possible influence of edema on infarct volume was corrected by standard methods (volume of contralateral hemisphere − volume of non-ischemic ipsilateral hemisphere), with infarcted volume expressed as a percentage of the contralateral hemisphere. n = 6. Values are expressed as mean ± SEM; *p < 0.05 vs. untreated, MCAO-subjected animals. (C) Immunohistochemical analysis of ipsilateral rat coronal brain sections depicting DAB staining (brown), representative of neurons. The missing NeuN-DAB staining in injured brain sections due to neuronal loss is restored after MMP-12 knockdown. Brain sections are counterstained with hematoxylin for nuclear localization. Results are from six independent sections obtained from six different rats. I-ischemia; R-reperfusion. (D) Quantification of NeN positive cells in the cortex of ipsilateral brain. n = 6. *p < 0.05 vs. sham; ^#p < 0.05 vs. untreated MCAO-subjected animals.

Figure 6. Effect of MMP-12 knockdown on other possible MMP-12 substrates.

(A) Gelatin zymogram showing MMP levels in sham control and ischemic brains of rats subjected to a two-hour MCAO followed by seven days reperfusion without treatment and treatment with plasmids containing a vector ligated with a scrambled sequence (SV-sh) and a vector ligated with MMP-12 shRNA (M-12-sh). Quantification of MMP-9 gelatinolytic activity. n = 6. *p < 0.05 vs. sham; ^#p < 0.05 vs. untreated. R = reperfusion (B) Immunoblot showing the protein expression of MBP in the ischemic brains of rats subjected to a two-hour MCAO followed by seven days reperfusion with and without treatments. GAPDH was used as a loading control. Quantification of MBP protein expression. n = 6. *p < 0.05 vs. sham; ^#p < 0.05 vs. untreated. (C) DAB immuno-staining depicting the protein expression of MBP in the contralateral and ipsilateral rat coronal brain sections obtained from various groups of rats. Arrows demonstrate the marked loss of MBP-immunostained axonal processes with clear structural abnormalities of rarefaction and fragmentation. Brain sections are counterstained with hematoxylin for nuclear localization. n = 6. Scale bar = 200 μm.

Figure 7. Effect of MMP-12 knockdown after focal cerebral ischemia on TNFα-mediated apoptosis.

(A) Immunofluorescence analysis showing the protein expressions of MMP-12 (green fluorescence) and TNFα (red fluorescence) in the ischemic core and the penumbra of rats subjected a two-hour MCAO procedure followed by seven days reperfusion with and without treatments. Nuclei were stained with DAPI (blue fluorescence). Yellow fluorescence in the merged images indicates the colocalization of MMP-12 and TNFα. n = 6. Scale bar = 100 μm. (B) TUNEL assay on the paraffin-embedded coronal brain sections of rats subjected to a two-hour MCAO procedure followed by seven days reperfusion with and without treatments. TUNEL-positive cells were quantified in the ipsilateral regions of rat brain that consisted of the ischemic core and the penumbra. n = 6. Values are expressed as mean ± SEM. *p < 0.05 compared to untreated animals subjected to a two-hour MCAO followed by seven days reperfusion. (C) Immunoblots showing the protein expression of various apoptotic molecules in the ischemic brains of rats subjected to a two-hour MCAO followed by seven days reperfusion with and without treatments. SV-sh: treated with plasmid expressing a scrambled sequence (vehicle control). M-12sh: treated with plasmid expressing MMP-12 shRNA. n = 6. GAPDH was used as a loading control. (D) Quantification of immunoblots. n = 6. *p < 0.05 vs. sham; ^#p < 0.05 vs. untreated.