Tissue plasminogen activator treatment of stroke in type-1 diabetes rats

Abstract

Background and purpose: Diabetes mellitus (DM) is a major stroke risk factor and is associated with poor recovery compared with nondiabetic stroke patients. In the present study, we investigated the effects of tissue plasminogen activator (tPA) treatment of stroke in diabetic and non-diabetic rats.

Methods: Type-1 diabetes (T1DM) was induced by injection of streptozotocin. Non-T1DM and T1DM rats were subjected to embolic middle cerebral artery occlusion (MCAo) and treated with or without tPA 2h after MCAo. Functional outcomes and immunostaining for advanced glycation endproducts receptor (RAGE), matrix metalloproteinase-9 (MMP-9) and toll-like receptor 4 (TLR4) and Western blotting were performed.

Results: tPA treatment of WT-MCAo rats significantly improved the functional outcome and reduced the lesion volume compared with non-treatment WT-MCAo rats (p<0.05). There was no significant difference between treatment with or without tPA in the WT-MCAo group in brain hemorrhage, BBB leakage and expression of inflammatory mediators, RAGE, MMP-9 and TLR4. However, tPA treatment in T1DM-MCAo rats (T1DM-MCAo+tPA) significantly enlarged brain hemorrhage, augmented BBB leakage, and failed to decrease lesion volume and improve functional outcome after stroke compared to T1DM-MCAo control. tPA treatment also significantly increased the expression of RAGE, MMP-9 and TLR4 in the ischemic brain in T1DM-MCAo rats compared with T1DM-MCAo control rats (p<0.05). Brain hemorrhage was significantly correlated with functional deficit and RAGE and TLR4 expression, respectively.

Conclusions: Treatment of stroke with tPA increased brain hemorrhage, BBB leakage and failed to improve functional outcome in T1DM rats. The increased inflammatory response may contribute to the failed neuroprotective effects of tPA treatment in T1DM rats.

Fig. 1

Functional tests and lesion volume measurements: tPA treatment of stroke in WT rats significantly improves functional outcome and reduces lesion volume compared to WT-MCAo rats. T1DM-MCAo rats exhibit significantly attenuated functional outcome and increased lesion volume compared to WT-MCAo rats. tPA treatment of stroke in T1DM rats does not improve functional outcome or reduce lesion volume. (A) mNSS test. (B) Adhesive removal test. (C) Foot-fault test. (D) Lesion volume measurement.

Fig. 2

BBB leakage, brain hemorrhage and RAGE, TLR4 and MMP-9 expression in the ischemic brain: T1DM-MCAo rats show increased BBB leakage, brain hemorrhage volume, and MMP-9, TLR4 and RAGE expression after stroke compared to WT-MCAo rats. tPA treatment increases BBB leakage, brain hemorrhage volume, and MMP-9, TLR4 and RAGE expression in T1DM-MCAo rats compared to non-treatment T1DM-MCAo rats. (A) Brain hemorrhage measurement. (B) Evans Blue dye assay for BBB leakage. (C–E) MMP-9, TLR4 and RAGE immunostaining and quantitative data in IBZ. Scale bar in C–E = 0.1 mm.

Fig. 3

Western blot assay: T1DM-MCAo rats exhibit increased brain tissue TLR4, RAGE and MMP-9 levels after stroke compared to WT-MCAo rats. tPA treatment significantly increases brain tissue TLR4, RAGE and MMP-9 levels in T1DM-MCAo rats compared to non-treatment T1DM-MCAo rats (p < 0.05).

Fig. 4

Correlation analysis: brain hemorrhage significantly correlated with RAGE and TLR expression in the ischemic brain. RAGE expression is strongly correlated with TLR4 expression in the ischemic brain. (A) Correlation analysis of brain hemorrhage with RAGE. (B) Correlation analysis of brain hemorrhage with TLR4. (C) Correlation analysis of RAGE with TLR4.