To Perform Double Immunofluorescence Staining

All experimental protocols in this study were approved by the Institutional Animal Care and Use Committee (nn) of Loma Linda University, were in accordance with the ARRIVE (Animal Research: Reporting in vivo Experiments) guidelines, and comply with the National Institutes of Health guidelines for the Use of Animals in Neuroscience Research. The adult male Sprague-Dawley rats (280-340 g, Harlan, IN, United States) used in this study were housed in a light and temperature-controlled room with unlimited access to food and water. The endovascular perforation model has been described in detail in previous manuscripts (Duris et al., 2011). Briefly, rats were maintained in anesthesia with 3% isoflurane in 70/30% medical air/oxygen by a rodent ventilator (Harvard Apparatus, Holliston, MA, United States) after intubation. Devascularization of the left external carotid artery was performed to prepare a stump for further puncture. A sharpened 4-0 nylon suture was inserted into the left internal carotid artery through a small incision next to the stump. The suture was advanced until resistance was detected at the bifurcation of the anterior and middle cerebral artery.



Then, an extra puncture was performed to perforate with an immediate withdrawal. The sham-operated group underwent the same procedure without endovascular puncture. Basic characteristics of the study. Experimental design and animal groups. Animal usage and mortality rates of all groups at 24 h after SAH. Western blots. There is no significant difference in SAH grading scores and body MedicGLP Weight Support loss among all SAH groups. GLP-1R, a glucagon-like peptide 1 receptor; SAH, subarachnoid hemorrhage; WB, western blot; IF, immunofluorescence; h, h; Ex-4, Exendin-4; Vehicle, 0.9% sterile NaCl; DMSO, dimethyl sulfoxide; Dorsomorphin, a specific inhibitor MedicGLP Online of AMPK; siRNA, small interfering ribonucleic acid; Scr siRNA, scrambled siRNA; BWC, brain water content; AMPK, adenosine monophosphate-activated protein kinase. To determine the endogenous GLP-1R expression in endothelial cells after SAH, 3 rats underwent SAH and double immunofluorescence staining of GLP-1R with von Willebrand factor (vWF), an endothelial cell marker, MedicGLP Weight Support at 24 h after SAH. 6 per group). Vehicle or Ex-4 was administered intraperitoneally (i.p.) at 1 h after SAH.



SAH grading score, modified Garcia, and beam balance tests were assessed 24 h after SAH. According to these results, the 3 μg dose of Ex-4 was selected for the following experiments. Plasma glucose level was measured and recorded at 8-time points during the first 24 h after SAH. 12 per group). Evans blue dye (2% in sterile 0.9% of NaCl; 4 ml/kg of body weight) was injected i.p. 6 per group). SAH grading and neurological scores were evaluated 24 h after SAH in animals used for Western blots. 6 per group). GLP-1R siRNA and scrambled siRNA were injected intracerebroventricularly (i.c.v) 48 h before SAH (Salahpour et al., 2007), while DMSO and Dorsomorphin were injected i.c.v. 6 per group) were performed on the ipsilateral hemisphere samples of the brains collected in Experiments 3 and 4 at 24 h after SAH. Plasma glucose levels of animals in Experiment 2 were measured before SAH, at 0 (immediately), 30, 60, 90, 120, 180 min, and 24 h after delivering the vehicle or drug (Kuroki et al., 2016). At each time point, the blood samples were taken from the tail vein and tested with a blood glucose meter (Ascensia Diabetes Care US Inc., NJ, United States).



Photographs of the basal surface of the brain sample were captured immediately after euthanasia and evaluated blindly, following a previously described procedure (Sugawara et al., 2008). Briefly, the scores of six predetermined segments (scored from 0 to 3 in each segment) were assessed according to the amount of clotted subarachnoid blood. The total SAH grading score was calculated as the sum of six segments. Modified Garcia and the beam balance tests were performed to assess neurological function 24 h after SAH, as previously described (Garcia et al., 1995; Suzuki et al., 2010). Six subtests of the modified Garcia test (spontaneous activity, spontaneous movement of four limbs, forelimb outstretching, vibrissa touch, body proprioception, and climbing capacity) were evaluated and recorded by an independent observer. To evaluate beam balance score, animals were put on a narrow wooden beam for a 1-min observation and scored according to their beam walking ability. The mean score from three consecutive trials (scored from 0 to 4) represented the final beam walking score of the targeted animal.