The Coagulation Factor XIIa Inhibitor rHA-Infestin-4 Improves Outcome after Cerebral Ischemia/Reperfusion Injury in Rats

Abstract

Background and purpose: Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach.

Methods: For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation.

Results: Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis.

Conclusions: With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.

Fig 1. Prophylactic treatment with rHA-Infestin-4 reduces infarct area and brain edema formation after tMCAO in rats.

(A) Upper panel shows representative 2,3,5-triphenyltetrazolium chloride stainings of 3 coronal rat brain sections 24 h after transient middle cerebral artery occlusion (tMCAO) in rats. Rats were treated with 100 mg/kg rHA-Infestin-4 or saline (control) 15 min prior to tMCAO. tMCAO was held up for 90 min followed by reperfusion until 24 h. Corresponding individual infarct areas, analyzed by planimetry, are shown in the lower panel. Prophylactic rHA-Infestin-4 treatment significantly reduced infarct areas compared to saline treated rats (n = 29-31/group). **p<0.01, Mann-Whitney test compared to saline treated animals. Dots above the dashed horizontal line indicate dead animals per group. Mortality was included in statistical analysis. Horizontal solid lines indicate mean values (+SEM) of surviving animals. (B) In addition, brain edema formation 24 h after tMCAO was reduced in rHA-Infestin-4 treated rats compared to saline treated control rats (n = 29-31/group). **p<0.01, Mann-Whitney test compared to saline treated animals. Dots above the dashed horizontal line indicate dead animals per group. Mortality was included in statistical analysis. Horizontal solid lines indicate mean values (+SEM) of surviving animals.

Fig 2. Prophylactic treatment with rHA-Infestin-4 ameliorates neurological outcome after tMCAO in rats.

(A) Zea Longa score 24 h after transient middle cerebral artery occlusion (tMCAO) in rats. Rats were treated with 100 mg/kg rHA-Infestin-4 or saline (control) 15 min prior to tMCAO. tMCAO was held up for 90 min followed by reperfusion until 24 h. Prophylactic rHA-Infestin-4 treatment significantly reduced the Zea Longa score compared to saline treated rats (n = 29-31/group). *p<0.05, Mann-Whitney test compared to saline treated animals. Horizontal solid lines indicate mean values (+SEM). (B) In addition, the Neuroscore was reduced in rHA-Infestin-4 treated rats compared to saline treated rats 24 h after tMCAO (n = 29-31/group). **p<0.01, Mann-Whitney test compared to saline treated animals. Dots above the dashed horizontal line indicate dead animals per group. Mortality was included in statistical analysis. Horizontal solid lines indicate mean values (+SEM) of surviving animals. (C) Functional performance on RotaRod and (D) grip strength in percent compared to the individual baseline of saline treated and rHA-Infestin-4 treated animals 24 h after tMCAO. rHA-Infestin-4 significantly improved functional performance in these two assays compared to saline treated controls (n = 28-30/ group). **p<0.01 between rHA-Infestin-4 treated and saline treated animals, Mann-Whitney test. Dots above the dashed horizontal line indicate dead animals per group. Mortality was included in statistical analysis. Horizontal solid lines indicate mean values (+SEM) of surviving animals.

Fig 3. Therapeutic treatment with rHA-Infestin-4 tends to reduce infarct area and brain edema formation after tMCAO in rats.

(A) Upper panel shows representative 2,3,5-triphenyltetrazolium chloride stainings of 3 coronal rat brain sections 24 h after transient middle cerebral artery occlusion (tMCAO) in rats. Rats were subjected to 90 min of tMCAO followed by reperfusion until 24 h. Rats were treated with 100 mg/kg rHA-Infestin-4 or saline (control) directly after start of reperfusion. Corresponding individual infarct areas, analyzed by planimetry, are shown in the lower panel. A trend towards reduced infarct areas could be detected after therapeutic rHA-Infestin-4 treatment compared to saline treated rats (n = 28-30/group). p<0.1 and >0.05, Mann-Whitney test compared to saline treated animals. Dots above the dashed horizontal line indicate dead animals per group. Mortality was included in statistical analysis. Horizontal solid lines indicate mean values (+SEM) of surviving animals. (B) In addition, a trend towards reduced brain edema formation 24 h after tMCAO was visible in rHA-Infestin-4 treated rats compared to saline treated control rats (n = 28-30/group). p<0.1 and >0.05, Mann-Whitney test compared to saline treated animals. Dots above the dashed horizontal line indicate dead animals per group. Mortality was included in statistical analysis. Horizontal solid lines indicate mean values (+SEM) of surviving animals.

Fig 4. Therapeutic treatment with rHA-Infestin-4 improves neurological performance after tMCAO in rats.

(A) Zea Longa score 24 h after transient middle cerebral artery occlusion (tMCAO) in rats. Rats were subjected to 90 min of tMCAO followed by reperfusion until 24 h. Rats were treated with 100 mg/kg rHA-Infestin-4 or saline (control) directly after start of reperfusion. Therapeutic rHA-Infestin-4 treatment significantly reduced the Zea Longa score compared to saline treated rats (n = 28-30/group). *p<0.05, Mann-Whitney test compared to saline treated animals. Horizontal solid lines indicate mean values (+SEM). (B) In addition, the Neuroscore was reduced in rHA-Infestin-4 treated rats compared to saline treated rats 24 h after tMCAO (n = 28-30/group). *p<0.05, Mann-Whitney test compared to saline treated animals. Dots above the dashed horizontal line indicate dead animals per group. Mortality was included in statistical analysis. Horizontal solid lines indicate mean values (+SEM) of surviving animals. (C) Functional performance on RotaRod and (D) grip strength in percent compared to the individual baseline of saline treated and rHA-Infestin-4 treated animals 24 h after tMCAO. rHA-Infestin-4 had no effect in these two assays compared to saline treated controls (n = 18-20/ group). p>0.05 between rHA-Infestin-4 treated and saline treated animals, Mann-Whitney test. Dots above the dashed horizontal line indicate dead animals per group. Mortality was included in statistical analysis. Horizontal solid lines indicate mean values (+SEM) of surviving animals.