Ischemic postconditioning diminishes matrix metalloproteinase 9 expression and attenuates loss of the extracellular matrix proteins in rats following middle cerebral artery occlusion and reperfusion

Abstract

Aims: Ischemic postconditioning (IPostC) has been proved to have neuroprotective effects for cerebral ischemia, but the underlying mechanism remains elusive. This study aimed at validating the neuroprotective effects of IPostC and investigating whether the neuroprotection of IPostC is associated with matrix metalloproteinase 9 (MMP9) and the extracellular matrix proteins, laminin and fibronectin, following cerebral ischemia/reperfusion in rats.

Methods: The rats in middle cerebral artery occlusion (MCAO) group underwent MCAO and reperfusion, and the animals in MCAO + IPostC group were treated by occluding bilateral common carotid arteries for 10 seconds and then reperfusing for 10 seconds for five episodes at the beginning of MCAO. Apoptosis was detected with terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The expression of MMP9, laminin, and fibronectin was measured with immunofluorescence and enzyme-linked immunosorbent assay.

Results: IPostC reduced brain edema and infarct volume and improved the neurological function. Furthermore, IPostC decreased cell apoptosis compared with the MCAO group. Compared to the MCAO group, IPostC treatment reduced MMP9 expression. Moreover, the results showed that the expression of laminin and fibronectin significantly increased in the MCAO + IPostC group compared to the MCAO group.

Conclusion: These findings indicated that diminishment of MMP9 expression and the attenuation of degradation of laminin and fibronectin may be involved in the protective mechanisms of postconditioning against cerebral ischemia/reperfusion injury.

Figure 1

IPostC rats have significantly reduced ischemic injury compared with middle cerebral artery occlusion (MCAO) rats. (A) Representative brain slices with infarcts stained by TTC in the MCAO group and MCAO + IPostC group at 24 h after reperfusion. (B) Statistical analysis of the percentage of infarct volume of contralateral hemisphere. (C) Statistical analysis of brain edema in the MCAO group and MCAO + IPostC group at 24 and 72 h after reperfusion. (D) The neurological functional outcome of the MCAO and MCAO + IPostC groups at 24 and 72 h after reperfusion. n = 6. *P < 0.05 versus MCAO.

Figure 2

IPostC decreases the number of TUNEL‐positive cells. (A) The three black ovals indicate the regions selected for quantifying TUNEL‐staining cells. The mean of apoptosis rate of the three black ovals was calculated. (B) Representative sections stained for TUNEL at different times after reperfusion in sham‐operated, IPostC‐alone, middle cerebral artery occlusion (MCAO), and MCAO + IPostC groups. Bar = 50 μm. (C) The percentage of TUNEL‐positive cells in the selected brain regions in different groups at different times after reperfusion. IPostC significantly reduced the number of TUNEL‐positive cells at 4, 24, and 72 h of reperfusion. n = 5. *P < 0.01 versus sham or IPostC‐alone; ^♦ P < 0.01 versus sham or IPostC‐alone; ^# P < 0.05 versus MCAO.

Figure 3

IPostC decreases MMP9 expression in brain after middle cerebral artery occlusion (MCAO). (A) MMP9 stained by immunofluorescence (red) in the ischemic penumbra of the MCAO group and MCAO + IPostC group at different time points. (B) Quantitative evaluation of fluorescence intensity of MMP9 in the penumbra for different groups. Bar = 50 μm. (C) The expression of MMP9 assessed by ELISA method. n = 5. *P < 0.05 versus sham; **P < 0.01 versus sham; ^# P < 0.05 versus MCAO.

Figure 4

The effects of IPostC on laminin expression in brain after middle cerebral artery occlusion (MCAO). (A) Immunofluorescence staining for laminin (red) in the penumbra of the sham‐operated group, MCAO group and MCAO + IPostC group at different time points. (B) Quantitative evaluation of fluorescence intensity of laminin in the penumbra for different groups. Bar = 100 μm. (C) The expression of laminin assessed by ELISA method. n = 5. *P < 0.05 versus sham; ^# P < 0.05 versus MCAO; ^## P < 0.01 versus MCAO.

Figure 5

IPostC attenuates loss of fibronectin in brain after middle cerebral artery occlusion (MCAO). (A) Representative immunofluorescence images of fibronectin (red) in the ischemic penumbra at 4, 24, and 72 h after MCAO. (B) Quantitative evaluation of fluorescence intensity of fibronectin in the penumbra at different time points for different groups. Bar = 100 μm. (C) The expression of fibronectin assessed by ELISA method. n = 5. *P < 0.05 versus sham; ^# P < 0.05 versus MCAO.