A total score was calculated by adding the scores from six segments (0-18 points). mediated by Frizzled 4 receptor activation which promoted -catenin nuclear translocation, which then enhanced Occludin, VE-Cadherin and ZO-1 expression. Norrin might have potential to safeguard FRAX1036 BBB after SAH. Keywords: Subarachnoid hemorrhage, Norrin, Blood-brain barrier, Frizzled-4, -catenin == Introduction == The pathophysiology of subarachnoid hemorrhage (SAH) and other stroke events involve the cerebral vascular neural network which includes arterial and venous systems as well as neuronal cells and other support cells and matrix1. Within this vascular neural network, blood-brain barrier (BBB) damage contributes to brain edema after SAH2. Therefore , to improve clinical outcomes of SAH patients, it may be crucial to develop new therapies against BBB disruption3. Norrin, encoded by Norrie Disease Protein gene, is a secreted small molecule protein which highly expressed in embryo development to regulate angiogenesis4. Recent studies demonstrated that Norrin is also essential to BBB formation5. Norrin activates the Frizzled FRAX1036 4 receptor, which is also the receptor intended for canonical Wnt/-catenin pathway6. The canonical Wnt/-catenin pathway is reported to exhibit neuroprotective and angiogenesis actions7. However , it is unclear whether endogenous Norrin is induced and whether ID2 Norrin/Frizzled 4 will be protective against BBB disruption after SAH. This study investigated a potential role and mechanisms of Norrin and its receptor in the protection of BBB in a rat model of SAH (Supplemental Determine I). == Materials and Methods == == Animals == One hundred and seventy-eight male adult Sprague-Dawley rats (Harlan, Indianapolis, IN) weighting 300-350 g were used in the present study. All experimental protocols were approved by the Institutional Animal Treatment and Use Committee of Loma Linda University. == Experimental Design == The experiment was designed as follows. == Experiment I (Supplemental Determine II) == To determine the time course of Norrin and TSPAN12 after SAH, twenty rats were randomly assigned into five groups: Sham (n = 4), SAH 6 hours (n = 4), SAH 12 hours (n = 4), SAH 24 hours (n = 4), and SAH 72 hours (n=4). Western blots were used to detect the protein expression of Norrin and TSPAN12 in ipsilateral/left hemisphere of each group. Double immunohistochemistry staining of Norrin and GFAP were also performed in at 12 hours after SAH(n = 1). == Experiment II (Supplemental Figure II) == Intended for outcome evaluation, Sixty-four rats were randomly divided into six groups: Sham (n = 13), SAH (n = 7), SAH + Vehicle (5 ul sterile saline8) (n = 15), SAH + 5 ng/ul9r-Norrin (25 ng in 5 ul sterile saline) (n = 8), SAH + 25 ng/ul r-Norrin (125 ng in 5 ul sterile saline) (n = 7) and SAH + 50 ng/ul r-Norrin (250 ng in 5 ul sterile saline)(n = 14). Vehicle or r-Norrin was injected intracerebroventricularly a few hours after SAH onset. Neurologic Scores, including Modified Garcia Test and Beam Balance Test, SAH Grading Scores and Brain Water Content were assessed at 24 and 72 hours after SAH FRAX1036 in all groups (n = 6). Evans Blue Extravasation was evaluated at 24 hours after SAH in Sham, SAH + Vehicle and SAH + 50 ng/ul r-Norrin groups (n = 5). Double immunohistochemistry staining of ZO-1 and FRAX1036 vWF were also performed in Sham, SAH, and SAH + 50 ng/ul r-Norrin groups (n = 2). == Experiment III (Supplemental Figure II) == Nine rats were randomly assigned into three groups: Sham (n = 3), SAH + 500 pmol Scrambled siRNA (in 5.