Crotalus atrox disintegrin reduces hemorrhagic transformation by attenuating matrix metalloproteinase-9 activity after middle cerebral artery occlusion in hyperglycemic male rats

Abstract

Hemorrhagic transformation after ischemic stroke is an independent predictor for poor outcome and is characterized by blood vessel rupture leading to brain edema. To date, no therapies for preventing hemorrhagic transformation exist. Disintegrins from the venom of Crotalus atrox have targets within the coagulation cascade, including receptors on platelets. We hypothesized that disintegrins from C. atrox venom can attenuate hemorrhagic transformation by preventing activation of matrix metalloproteinase after middle cerebral artery occlusion (MCAO) in hyperglycemic rats. We subjected 48 male Sprague-Dawley rats weighing 240-260 g to MCAO and hyperglycemia to induce hemorrhagic transformation of the infarction. At reperfusion, we administered either saline (vehicle), whole C. atrox venom (two doses were used), or fractionated C. atrox venom (HPLC Fraction 2). Rats were euthanized 24 hr post-ictus for measurement of infarction and hemoglobin volume. Reversed-phase HPLC was performed to fractionate the whole venom and peaks were combined to form Fraction 2, which contained the disintegrin Crotatroxin. Fraction 2 protected against hemorrhagic transformation after MCAO, and attenuated activation of matrix metalloproteinase-9. Administering matrix metalloproteinase antagonists prevented the protection by Fraction 2. The results of this study indicate that disintegrins found in C. atrox venom may have therapeutic potential for reducing hemorrhagic transformation after ischemic stroke. Moreover, the RP-HPLC fractions retained sufficient protein activity to suggest that gentler and less efficient orthogonal chromatographic methods may be unnecessary to isolate proteins and explore their function.

Keywords: MCAO; crotatroxin; hemorrhagic transformation; hyperglycemia; snake venom disintegrin.

Figure 1

HPLC chromatograph of crude venom from C. atrox. The red box indicates the peaks combined to form Fraction 2 used in this study. These peaks contained isoforms of disintegrin Crotatroxin (Prot ID: VM2A_CROAT).

Figure 2

Dextrose Administered 2 Hours post-Reperfusion Induces Hyperglycemia. All animals had blood glucose (mg/dL) testing before MCAO (Baseline), 3 hours after reperfusion which is also 1 hour after dextrose injection (1 Hr Post-Dextrose Injection), and before sacrifice (24 Hrs Post-MCAO). One cohort of Sham and MCAO + Vehicle, and MCAO + C. atrox (LD) and MCAO + C. atrox (HD) was analyzed for statistical significance (n=5-6/group). Another cohort of Sham and MCAO + Vehicle, and MCAO + C. atrox Fraction 2 and MCAO + (C. atrox Fraction 2 + Batimastat + Prinomastat) was analyzed for statistical significance (n=4-6/group). All interventions were administered immediately after MCAO. Analyzed using two-way ANOVAs with Tukey post-hoc tests. * p<0.05 vs Baseline, # p<0.05 vs 1 Hr Post-Dextrose Injection.

Figure 3

Crotalus atrox Venom Reduces Hemoglobin Volume, but Not Infarction Volume 24 hours after MCAO. (a) Representative images of TTC-stained brains. (b) Infarction volume (%) is significantly larger in MCAO rats compared to sham animals. Neither low nor high dose C. atrox venom reduced infarct volume. (c) Brain hemoglobin volume was significantly higher in MCAO rats treated with the vehicle compared to sham rats. C. atrox venom treatment significantly attenuates hemoglobin volumes in the brain. All interventions were administered immediately after MCAO. n=5-6/group. Analyzed using one-way ANOVAs with Tukey post-hoc tests. * p<0.05 vs Sham, # p<0.05 vs MCAO + Vehicle.

Figure 4

Crotalus atrox Disintegrin Crotatroxin (Fraction 2) Attenuates Hemoglobin Volume, but Not Infarction Volume 24 hours after MCAO. (a) Representative images of TTC-stained brains. (b) MCAO causes significantly larger infarct volumes compared to sham surgery. C. atrox Fraction 2 has no effect on infarct volume compared to vehicle-treated animals. Co-administration of MMP inhibitors with C. atrox Fraction 2 similarly does not have any effect on infarction. (c) MCAO rats receiving vehicle treatment had statistically more brain hemoglobin volume compared to sham rats. C. atrox Fraction 2 significantly reduces hemoglobin volumes in the brain. Co-administration of MMP inhibitors reverses the protective effect of C. atrox Fraction 2 on hemoglobin volume. All interventions were administered immediately after MCAO. n=4-6/group. Analyzed using one-way ANOVAs with Tukey post-hoc tests. * p<0.05 vs Sham, # p<0.05 vs MCAO + Vehicle, & p<0.05 vs MCAO + C. atrox Fraction 2.

Figure 5

Crotalus atrox Disintegrin Crotatroxin (Fraction 2) Attenuates MMP-9 Activity 24 Hours Post-MCAO. MMP-9 zymography of rat brain shows that animals treated with the vehicle had significantly greater MMP-9 activity compared to sham rats. The MMP-9 activity was reduced back to sham levels in rats receiving C. atrox Fraction 2. The protective effects of C. atrox Fraction 2 on MMP-9 activity was reversed by administration of MMP inhibitors. All interventions were administered immediately after MCAO. n=4-6/group. Analyzed using one-way ANOVA with Tukey post-hoc test. * p<0.05 vs Sham, # p<0.05 vs MCAO + Vehicle, & p<0.05 vs MCAO + C. atrox Fraction 2.