Abstract 2611: Inhibition Of Rho-kinase By Hydroxyfasudil Attenuates Early Brain Injury After Subarachnoid Hemorrhage In Rats

Abstract

Background: Early brain injury comprising of blood-brain barrier (BBB) disruption and brain edema is an important part of subarachnoid hemorrhage (SAH) pathophysiology. Although several reports have shown that inhibition of Rho-kinase (ROCK) plays an important role in suppressing barrier permeability, the effect of ROCK on the BBB in SAH is not well understood. Therefore, we assessed the hypothesis that (A) ROCK inhibitors, hydroxyfasudil (HF) and Y27632 attenuate early brain injury after SAH, and (B) HF decreases brain edema via preservation of the BBB. Methods: Adult male rats were randomly assigned to five groups; sham-operated, SAH with saline, SAH with HF (10mg/kg, i.p.) treatment at 0.5 hrs, SAH with HF (10mg/kg, i.p.) treatment at 0.5 and 6 hrs each , and SAH with Y27632 (10mg/kg, i.p.) treatment at 0.5 hrs. The endovascular perforation method was used to produce SAH. Neurological scores were evaluated before sacrifice at 24 and 72 hours after injury. Brain water content, Evans blue dye extravasation assay, Rho-kinase activity assay, and Western blotting analyses were performed. Results: Among 175 surgeries performed, 53 animals were excluded. Of those excluded, 26 died from severe SAH and 27 had only mild SAH. There were no differences between the SAH with saline group (Vehicle group), SAH with HF group, and SAH with Y27632 group in SAH grading and mortality. HF but not Y27632 significantly improved neurological outcomes. Both groups significantly attenuated brain water content (BWC) in the ipsilateral hemisphere compared with the Vehicle group at 24 hrs after SAH for single treatment. The BWC of the HF group was also significantly lower than that of the Vehicle group in the contralateral hemisphere and cerebellum. There was no difference in neurological score between the Vehicle and HF group with two treatments, however, HF significantly ameliorated BWC in the ipsilateral hemisphere compared with vehicle at 24 hrs after SAH. There was no difference in neurological score and BWC between the Vehicle and HF groups with single treatment at 72 hrs after SAH. Evans blue extravasation in the ipsilateral hemisphere in the Vehicle group was significantly higher than in the HF group for single treatment at 24 hours after SAH. Supernatants of the ipsilateral hemisphere of single treatment groups sacrificed at 24 hours showed significantly higher ROCK activity in the Vehicle group than the sham group, and the HF group showed significantly lower activity than the Vehicle group. As the tight junction (TJ) is one of the components of the endothelial junctional complex in the BBB, we evaluated TJ proteins, occludin and Zona occludens-1 (ZO-1) in Western blotting analyses. Occludin and ZO-1 levels were significantly lower in the Vehicle group than the HF group. Conclusion: The ROCK inhibitor, HF, attenuates early brain injury by decreasing brain edema after SAH via protection of tight junction proteins.