Matrix Metalloprotease 3 Exacerbates Hemorrhagic Transformation and Worsens Functional Outcomes in Hyperglycemic Stroke

Abstract

Background and purpose: Acute hyperglycemia worsens the clinical outcomes and exacerbates cerebral hemorrhage after stroke. The mediators of hemorrhagic transformation (HT) in hyperglycemic stroke are not fully understood. Matrix metalloproteinase 3 (MMP3) plays a critical role in the tissue-type plasminogen activator-induced HT. However, the role of MMP3 in exacerbating the HT and worsening the functional outcomes in hyperglycemic stroke remains unknown.

Methods: Control/normoglycemic and hyperglycemic (blood glucose, 140-200 mg/dL) male Wistar rats were subjected to middle cerebral artery occlusion for 90 minutes and either 24 hours or 7 days reperfusion. MMP3 was inhibited pharmacologically (UK 356618, 15 mg/kg IV at reperfusion) or knocked down in the brain by shRNA lentiviral particles (injected intracerebroventricular). Neurovascular injury was assessed at 24 hours, and functional outcomes were assessed at 24 hours, day 3, and day 7. MMP3 activity was measured in brain homogenate and cerebral macrovessels. Localization of MMP3 within the neurovascular unit after hyperglycemic stroke was demonstrated by immunohistochemistry.

Results: Hyperglycemia significantly increased MMP3 activity in the brain after stroke, and this was associated with exacerbated HT and worsened functional outcomes. MMP3 inhibition significantly reduced HT and improved functional outcomes.

Conclusions: MMP3 plays a critical role in mediating cerebrovascular injury in hyperglycemic stroke. Our findings point out MMP3 as a potential therapeutic target in hyperglycemic stroke.

Keywords: blood glucose; brain ischemia; hyperglycemia; intracranial hemorrhages; stroke.

Figure 1

Blood glucose and MMP3 activity. A, Administration of 30% glucose solution achieved and maintained blood glucose levels of 140–200 mg/dl through 90 minutes of ischemia. Acute mild HG significantly increased MMP3 activity in B, brain homogenates and C, cerebral vasculature after stroke and the use of MMP3 inhibitor significantly blunted this effect at 24 h. *p<0.05 vs control, #p<0.05 vs HG, ^αp<0.05 vs sham, n=6–8 per group.

Figure 2

Localization of MMP3 in the neurovascular unit in hyperglycemic stroke at 24 h. A, MMP3 co-localizes with NeuN positive neurons and B, isolectin positive cerebral vessels and C, NG2 positive pericytes in the peri-infarct region in both control and hyperglycemic groups. However, MMP3 was not seen in D, GFAP positive astrocytes. n=6 per group.

Figure 2

Localization of MMP3 in the neurovascular unit in hyperglycemic stroke at 24 h. A, MMP3 co-localizes with NeuN positive neurons and B, isolectin positive cerebral vessels and C, NG2 positive pericytes in the peri-infarct region in both control and hyperglycemic groups. However, MMP3 was not seen in D, GFAP positive astrocytes. n=6 per group.

Figure 3

MMP3 pharmacological inhibition and neurovascular injury at 24 h. Acute HG significantly exacerbated the vascular injury and bleeding in the brain. MMP3 inhibition in hyperglycemic animals significantly reduced the HG induced B, brain edema, C, HT (The HT was seen in the cortex and striatum. The figure explains the score and regional localization of hemorrhage, with the empty boxes indicating striatum and solid boxes indicating the cortex, and position of the boxes indicates the score) and D, Hb content in ischemic hemispheres without affecting the A, infarct size. *p<0.05 vs control, #p<0.05 vs HG, n=6–8 per group.

Figure 4

MMP3 pharmacological inhibition and functional outcomes. Acute mild HG significantly worsened the functional outcomes. Inhibition of MMP3 activity and reduction in vascular injury was associated with significant increase in A, neurobehavioral composite score and B, grip strength in hyperglycemic animals at 24 h. C) Early inhibition of MMP3 significantly increased composite scores and improved recovery at day 3 and day 7 after stroke. *p<0.05 vs control, #p<0.05 vs HG, ^αp<0.05 vs pre-stroke, n=6–7 per group.

Figure 5

MMP3 knockdown in the brain. A, Stereotaxic injection of MMP3 shRNA lentiviral particles significantly knocked down the expression of MMP3 in the brain compared to sham or animals injected with empty vector (^αp<0.05 vs sham and EV). MMP3 knockdown significantly reduced the HG induced B, brain swelling without affecting A, infarct size at 24 h. *p<0.05 vs control EV and control shRNA, #p<0.05 vs HG EV, n=6–7 per group.

Figure 6

MMP3 focal knockdown in rat brain and stroke outcomes. Knocking down MMP3 in rats’ brains significantly reduced the HG induced increase in A, HT and B, Hb content in ischemic hemispheres and improved the HG induced neurological deficits as indicated by a significant increase in C, neurobehavioral composite score and D, grip strength at 24 h. *p<0.05 vs control EV and control shRNA, #p<0.05 vs HG EV, n=6–7 per group.