Effects of minocycline plus tissue plasminogen activator combination therapy after focal embolic stroke in type 1 diabetic rats

Abstract

Background and purpose: Poststroke hyperglycemia is associated with resistance to tissue plasminogen activator (tPA) reperfusion, higher risk of intracerebral hemorrhage, and worse neurological outcomes. In this study, we asked whether minocycline combined with intravenous tPA may ameliorate inflammation and brain injury after focal embolic stroke in type 1 diabetic rats.

Methods: Type 1 diabetic rats were subjected to a focal embolic stroke. Three treatment groups were used: (1) saline at 1.5 hours after stroke; (2) tPA alone at 1.5 hours after stroke; (3) combined minocycline (intravenously) at 1 hour plus tPA at 1.5 hours, and second treatment of minocycline (intraperitoneally) at 12 hours after stroke. Acute brain tissue damages were assessed at 24 hours after stroke. Inflammatory biomarkers interleukin-1β and matrix metalloproteinases 2 and 9 were examined in plasma. Neutrophil infiltration, microglia activation, matrix metalloproteinase activation, and degradation of the tight junction protein claudin-5 were examined in the brain.

Results: Compared with saline or tPA alone treatments, minocycline plus tPA combination therapy significantly reduced brain infarction, intracerebral hemorrhage, and hemispheric swelling at 24 hours after stroke. The combination also significantly suppressed the elevated plasma levels of matrix metalloproteinase-9 and interleukin-1β up to 24 hours after stroke. At 16 hours after stroke, neutrophil infiltration, microglia activation, matrix metalloproteinase-9, and tight junction protein claudin-5 degradation in the peri-infarct brain tissues were also significantly attenuated by the combination therapy.

Conclusions: Combination therapy with minocycline plus tPA may be beneficial in ameliorating inflammation and reducing infarction, brain swelling, and hemorrhage after ischemic stroke with diabetes mellitus/hyperglycemia.

Figure 1

Measurements of acute brain tissue damages in a focal embolic stroke model of diabetic rats. A. Representative images for TTC stained ischemic brain infarctions. B. Ischemic infarct volumes were quantified at 24 hours after stroke. C. Hemispheric swelling were quantified on TTC-stained brain slices. D. Intracerebral hemorrhage volumes were quantified at 24 hours after stroke by hemoglobin assay. E. Blood glucose concentrations were measured at before, 1.5, 2.5, and 24 hours after stroke. Data were expressed as mean + s.d., * P<0.05, n=11-14 per group.

Figure 1

Measurements of acute brain tissue damages in a focal embolic stroke model of diabetic rats. A. Representative images for TTC stained ischemic brain infarctions. B. Ischemic infarct volumes were quantified at 24 hours after stroke. C. Hemispheric swelling were quantified on TTC-stained brain slices. D. Intracerebral hemorrhage volumes were quantified at 24 hours after stroke by hemoglobin assay. E. Blood glucose concentrations were measured at before, 1.5, 2.5, and 24 hours after stroke. Data were expressed as mean + s.d., * P<0.05, n=11-14 per group.

Figure 2

Measurements of MMP-9 activity in plasma and brain tissues of a focal embolic stroke model in diabetic rats. Plasma samples were collected before stroke (pre-stroke), 1.5 hours (pre-tPA), 2.5 hours (post-tPA) and 24 hours after stroke. A. Representative zymography comparing tPA vs minocycline plus tPA. B. Quantitative data of MMP-9 activity. Data were expressed as mean + s.d., *P<0.05 vs pre-stroke, # P<0.05 vs tPA treatment, n=6 per group. C. At 16 hours after stroke, representative MMP activity assessed by in situ zymography on rat brain sections treated with saline, tPA and minocycline combined with tPA, respectively. Bar=100 μm. D. Quantitative data analysis of MMP activity on brain sections. Data were expressed as mean + s.d., * P<0.05, n=3 per group.

Figure 2

Measurements of MMP-9 activity in plasma and brain tissues of a focal embolic stroke model in diabetic rats. Plasma samples were collected before stroke (pre-stroke), 1.5 hours (pre-tPA), 2.5 hours (post-tPA) and 24 hours after stroke. A. Representative zymography comparing tPA vs minocycline plus tPA. B. Quantitative data of MMP-9 activity. Data were expressed as mean + s.d., *P<0.05 vs pre-stroke, # P<0.05 vs tPA treatment, n=6 per group. C. At 16 hours after stroke, representative MMP activity assessed by in situ zymography on rat brain sections treated with saline, tPA and minocycline combined with tPA, respectively. Bar=100 μm. D. Quantitative data analysis of MMP activity on brain sections. Data were expressed as mean + s.d., * P<0.05, n=3 per group.

Figure 3

Measurements of inflammatory IL-1β concentration in plasma and microglia activation in ischemic brains of diabetic rats. A. Plasma samples were collected before stroke (Pre-stroke), 1.5 hours, 2.5 hours and 24 hours after stroke. IL-1β concentrations in plasma were examined by ELISA analysis. Data were expressed as mean + s.d., *P<0.05 vs pre-stroke, # P<0.05 vs saline and tPA treatment at 24 hours after stroke, n=6 per group. B. At 16 hours after stroke, representative iba-1 positive cells by immunostaining on rat brain sections treated with saline, tPA and minocycline combined with tPA, respectively. Bar=100 μm. C. Quantitative data of microglia/macrophages activation (iba-1 positive cells). Data were expressed as mean + s.d., * P<0.05, n=3 per group.

Figure 4

Measurements of inflammatory neutrophil infiltration in ischemic brains of diabetic rats. A. At 16 hours after stroke, representative MPO positive cells by immunostaining on rat brain sections treated with saline, tPA and minocycline combined with tPA, respectively. Bar=100 μm. B. Quantitative data of neutrophils infiltration (MPO positive cells). Data were expressed as mean + s.d., * P<0.05, n=3 per group.

Figure 5

Measurement of tight junction protein claudin-5 degradation in ischemic brains of diabetic rats. A. At 16 hours after stroke, representative claudin-5 expression by immunostaining on rat brain sections treated with saline, tPA and minocycline combined with tPA, respectively. Bar=100 μm. B. Quantitative data of claudin-5 expression. Data were expressed as mean + s.d., * P<0.05, n=3 per group.