Influence of hyperglycemia on oxidative stress and matrix metalloproteinase-9 activation after focal cerebral ischemia/reperfusion in rats: relation to blood-brain barrier dysfunction

Abstract

Background and purpose: Hyperglycemia is linked to a worse outcome after ischemic stroke. Among the manifestations of brain damage caused by ischemia are blood-brain barrier (BBB) disruption and edema formation. Oxidative stress and matrix metalloproteinase-9 (MMP-9) activation are implicated in BBB dysfunction after ischemia/reperfusion injury. Our present study was designed to clarify the relation among hyperglycemia, oxidative stress, and MMP-9 activation associated with BBB dysfunction after transient focal cerebral ischemia (tFCI).

Methods: We used a model of 60 minutes of middle cerebral artery occlusion on the following animals: normoglycemic wild-type rats, wild-type rats with hyperglycemia induced by streptozotocin, and human copper/zinc superoxide dismutase (SOD1) transgenic rats with streptozotocin-induced hyperglycemia. We evaluated edema volume, Evans blue leakage, and oxidative stress, such as the carbonyl groups and oxidized hydroethidine (HEt), SOD activity, and gelatinolytic activity, including MMP-9.

Results: Hyperglycemia significantly increased edema volume and Evans blue leakage. Moreover, it enhanced the levels of the carbonyl groups, the oxidized HEt signals, and MMP-9 activity after tFCI without alteration in SOD activity. Gelatinolytic activity and oxidized HEt signals had a clear spatial relation in the hyperglycemic rats. SOD1 overexpression reduced the hyperglycemia-enhanced Evans blue leakage and MMP-9 activation after tFCI.

Conclusions: Hyperglycemia increases oxidative stress and MMP-9 activity, exacerbating BBB dysfunction after ischemia/reperfusion injury. Superoxide overproduction may be a causal link among hyperglycemia, MMP-9 activation, and BBB dysfunction.

Figure 1

A, Serum glucose level in normoglycemic Wt rats (NG), hyperglycemic Wt rats (HG), and hyperglycemic SOD1 Tg rats (HGT) before, during, or after MCAO (^*P<0.05). B, Neurological scores 24 hours after tFCI in NG and HG rats. Each symbol depicts the individual score of a single animal (^*P<0.05). C, Representative photographs of 2,3,5-triphenyltetrazolium chloride staining 24 hours after tFCI in NG and HG rats. Bar = 1 cm. D, Infarct volume 24 hours after tFCI in NG and HG rats (^*P<0.05). E, Edema volume 24 hours after tFCI in NG and HG rats (^*P<0.05). F, Representative photographs of Evans blue extravasation in the brains and coronal sections (bregma +0.70 mm) of NG and HG rats 24 hours after tFCI. Bars = 5 mm. G, Quantitative assay of Evans blue leakage in NG and HG rats 24 hours after tFCI (^*P<0.05).

Figure 2

A, Western blot analysis of the carbonyl groups in normoglycemic Wt rats (NG) and hyperglycemic Wt rats (HG). Hyperglycemia markedly raised the levels of the carbonyl groups 24 hours after tFCI (^**P<0.01). C indicates control. B, In situ detection of oxidized HEt in the ischemic area of NG and HG rats 24 hours after tFCI. The oxidized HEt signals were significantly increased in the HG rats compared with the NG rats (^*P<0.05). Bar = 50 μm. C, Western blot analysis of Cu/Zn-SOD and MnSOD in NG and HG rats. There was no significant difference in Cu/Zn-SOD or MnSOD immunoreactivity between the NG and HG rats. β-actin and COX were used as internal controls. D, Assay of total SOD activity in NG and HG rats. Hyperglycemia had no significant effect on SOD activity in the rat brains. Note: Duplicate samples were run at 7 and 24 hours (A and C).

Figure 3

A, Zymographic analysis in normoglycemic Wt rats (NG) and hyperglycemic Wt rats (HG). Hyperglycemia significantly increased MMP-9 activity 24 hours after tFCI (^**P<0.01), but did not change MMP-2 activity in any group. MMP-9 bands (92 kDa and 88 kDa) and MMP-2 bands (68 kDa) were detected. C indicates control; hSt, human gelatinase standard. B, Representative photomicrographs of gelatinase activity in the sham controls and the ischemic areas of NG and HG rats 24 hours after tFCI. In the sham control brains, there was no difference in gelatinase activity between the NG and HG rats. After 24 hours of reperfusion, gelatinase activity in vessels (arrows) and cells (arrowheads) was prominent in the HG rats compared with the NG rats. Bar = 100 μm. C, Colocalization of gelatinase activity and oxidized HEt in vessels (arrows) and cells (arrowheads) of the HG rats 24 hours after tFCI. Bar = 100 μm.

Figure 4

A, Assay of SOD activity in hyperglycemic Wt rats (HG) and hyperglycemic SOD1 Tg rats (HGT) (^**P<0.01). B, Zymographic analysis in HG and HGT rats 24 hours after tFCI. MMP-9 activity was significantly reduced in the HGT rats compared with the HG rats (^*P<0.05). C, Quantitative analysis of Evans blue leakage in the HG and HGT rats 24 hours after tFCI. The level of Evans blue leakage was remarkably decreased in the HGT rats compared with the HG rats (^*P<0.05).