Protective Effects And Mechanisms Of Anfibatide On Focal Cerebral Ischemia-reperfusion Injury In Mice

Ischemic cerebrovascular disease is a kind of common and frequently encountered diseases of the central nervous system, which is characterized by suspension of cerebral blood flow, local brain tissue ischemia anoxic necrosis and neurological deficit, which is resulted from various causes. In clinical treatment, thrombolytic therapy and mechanical recanalization could restore the blood supply without delay, but inevitably, ischemia-reperfusion injury is a critical reason leading to exacerbations. Anfibatide(ANF), a polypeptide purified from agkistrodon acutus venom, is a specific platelet glycoprotein(GP) Ib receptor antagonist. Previous researches showed that treatment with ANF could decrease infarct volumes and improve neurobehavioral function in MCAO mice. Our research focuses on exploring the protective effects of ANF and its potential mechanisms on MCAO mice.Aim To study the protective effects and mechanisms of anfibatide(ANF) on focal cerebral ischemia-reperfusion injury in mice.Methods The focal cerebral ischemia-reperfusion model was built by the suture method for the right middle cerebral artery occlusion(MCAO) in mice. Male Kunming mice were randomly assigned to six groups including sham group, model group, ANF(1, 2, 4 ug/kg) and positive drug tirofiban(TRF, 0.5 mg/kg) treatment groups. Respectively drugs were administrated by intravenous injection after 90 min cerebral ischemia followed by 1 h reperfusion. 24 h after reperfusion, the indexes of MCAO mice were observed using the following methods: infarct volumes were determined by MRI T2-weighted imaging(T2WI); neurological deficit scores were evaluated using modified neurological severity score(m NSS); balance and motor functions were observed by inclined board test; the number of intact neuronal cells were counted by nissl staining; cerebral microthrombi were detected by hematoxylin and eosin(HE) staining; evans blue staining was performed to assess the permeability and integrity of blood brain barrier(BBB); abundance of GPIbα and v WF were observed by immunofluorescence(IF); expressions of P-selectin and macrophage-1 antigen(MAC-1) were detected by immunohistochemistry(IHC); expression of the Fibrin(ogen) was examined by IHC and western blotting(WB); tail bleeding time was measured to evaluate the affect of hemostasis; a hemoglobin spectrophotometry method using darkbin’s reagent was used to quantify cerebral hemorrhage; platelet counts were determinated to assay whether ANF could cause thrombocytopenia.Results ANF could significantly reduce infarct volumes, improve neurobehavioral function and motor function. ANF could increase the number of intact neuronal cells and decrease the number of microthrombi. ANF could decrease the permeability and integrity of BBB, the abundance of GPIbα and v WF and suppress the expressions of P-selectin, MAC-1 and Fibrin(ogen). Compared with TRF, ANF could not only shorten the bleeding time, but also reduce hemorrhage volumes. ANF could not change the platelet counts.Conclusion These results suggest that ANF exerts protective effect on cerebral ischemia and reduces bleeding complications, which may be associated with inhibition of the interactions between GPIbα with v WF, P-selectin and MAC-1, inhibition of platelet adhesion and aggregation, decrease of Fibrin(ogen) deposition and finally reduction of thrombus formation.