Intracerebroventricular treatment of Norrin promotes angiogenesis and repair of blood brain barrier in mouse models of ischemic stroke

Abstract

Objective To identify the effects of intracerebroventricular treatment of Norrin on vascular density in peri-infarct area and blood brain barrier (BBB), and explore the mechanisms underlying these changes. Methods (1) Twenty-four mice were randomly divided into sham-operated group, ischemia for 6, 24 and 72 h groups (n=6); middle cerebral artery occlusion (MCAO) models in the later 3 groups were induced by suture-occluded method; Western blotting was employed to detect the Norrin expression in the brain tissues. (2) Male C57BL/6 mice were randomly divided into sham-operated group, vehicle group and Norrin treatment group (n=30); MCAO models in the later two groups were induced by suture-occluded method; intracerebroventricular (i.c.v) injection of 250 ng Norrin was performed immediately after MCAO; the changes of neurological function were assessed by modified neurological deficits scale (mNSS); infarct volume was examined by triphenyl tetrazolium chloride (TTC) staining; the changes of brain water content were identified by dry-wet method; immunofluorescence staining was performed to detect the vascular density; Evens blue (EB) extravasation was assessed by means of EB fluorescent quantitation; Western blotting was used to detect the expressions of angiopoietin-2 (Ang-2), zonula occludens protein-1 (ZO-1) and nucleus β-catenin. Results (1) Western blotting results demonstrated that as compared with that in the sham-operated group, the Norrin expression in the ischemia for 6 h group was significantly increased (P<0.05); further increased trend was noted in the ischemia for 24 and 72 h groups. (2) One and 3 d after MCAO, the mNSS scores in the Norrin treatment group was significantly decreased as compared with those in the vehicle group (P<0.05); 3 d after MCAO, Norrin treatment group had significantly reduced infarct volume, dramatically increased vascular density in peri-infarct area, significantly decreased water edema and EB extravasation, and remarkably up-regualted expressions of Ang-2 and Zo-1 and accumulation of β-catenin in nucleus as compared with the vehicle group, with significant differences (P<0.05). Conclusion Ischemic stroke induces the up-regulation of Norrin; exogenous Norrin enhances angiogenesis and promotes BBB repair, the underlying mechanism involves in the up-regulation of Ang-2 and Zo-1 mediating by Norrin/β-catenin signaling. Key words: Norrin; Ischemic stroke; Angiogenesis; Blood brain barrier