Male C57Bl/6J WT mice (n = 120), AT1aR-/- mice (B6.129P2-Agtr
/J) (Jackson Laboratories Bar Harbor) (n = 35), and BM chimeric mice (n = 81) were studied. All experimental procedures involving the use of animals were reviewed and approved by the Institutional Animal Care and Use Committee of LSU Health Sciences Center and performed according to the Guide for the Care and Use of Laboratory Animals outlined by the National Institutes of Health.
WT to WT (WT→WT, n = 41) and AT1aR-/- to WT (AT1aR-/-→WT, n = 40) BM chimeras were produced, as previously described , by transplanting BM from either WT or AT1aR-/- into WT mice. In brief, BM cells, collected from the femurs and tibias of donor mice were injected (2 × 106 BM cells) via the femoral vein into recipient mice (congenic WT with same phenotype as C57BL/6J mice; B6.SJLPtprcaPep3b/BoyJ), following total-body irradiation sufficient to eliminate the recipient's blood cells. The BM chimeras were housed in autoclaved cages, with 0.2% neomycin added to drinking water for the first two weeks. After six to eight weeks, reconstitution of BM cells was verified using flow cytometry by testing for the % blood leukocytes positive for CD45.1 (recipient isoform of CD45) vs CD45.2 (donor isoform of CD45). We used BM chimeras in which > 90% of recipient marrow was replaced by donor BM.
Angiotensin II infusion
Mice received a 14-day infusion of either saline or Ang II (Bachem Americas, Inc.) using micro-osmotic pumps (Durect Corporation), which were implanted subcutaneously in the intrascapular region under isofluorane anesthesia. Sterile procedures were used and a topical antibiotic Neosporin (Johnson & Johnson) was applied to prevent postoperative infection at the site of implantation. The pumps in the control group were loaded with only the saline vehicle, while the experimental groups received Ang II loaded pumps that delivered the peptide at a rate of 2000 ng/kg/min. In some experiments, a range of Ang II doses (400 - 2000 ng/kg/min) were used to determine the dose-dependent responses of leukocyte and platelet adhesion, and blood pressure to Ang II in the absence of I/R.
Measurements of blood pressure in conscious mice were obtained using a tapered femoral artery catheter implanted (under isofluorane anesthesia) three hours prior to data collection via a pressure transducer coupled to a computer system, as previously described . Mean arterial pressure was recorded over the 1-hour period preceding the experiment.
Middle cerebral artery occlusion and reperfusion
Transient (45 minutes) focal cerebral ischemia was induced in ketamine (100 mg/kg, i.p., Lloyd Laboratories) and xylazine (10 mg/kg, i.p., Hospira, Inc.) anesthetized mice by occluding the right middle cerebral artery with 7-0 silicone-coated nylon monofilament (Doccol Corp.). After the 45-minute occlusion period, the nylon fiber was gently removed and the common carotid artery was reopened [19, 20]. In sham mice, the arteries were visualized but not disturbed. Ischemia and reperfusion were verified using a laser Doppler flowmeter (ML191 Blood Flow Meter, ADInstruments) to monitor cerebral blood flow. We have demonstrated that middle cerebral artery occlusion is associated with a 90% reduction in brain perfusion . Core temperature was kept at 36 - 37°C.
Four hours following reperfusion after middle cerebral artery occlusion, mice were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) then prepared for intravital microscopic observation and evaluation of the cerebral microvasculature, as previously described [22, 23]. Briefly, cerebral venules (100 μm length, 25-50 μm diameter) were viewed through a cranial window using an upright fluorescent microscope using a 20× water immersion lens. Color images were captured with a 3 charge coupled device color video camera. Ex vivo carboxyfluorescein diacetate succinimidyl ester (CFDSE) (Molecular Probes, Invitrogen) labeled platelets (100 × 106 cells) derived from a matching donor mouse were administered for visualization and quantification of (green) adherent platelets. Rhodamine 6G (Sigma Chemical) was administered for detection of leukocytes (red). Adherent leukocytes and platelets were defined as cells bound to the venular wall for ≥ 30 and 2 seconds, respectively [6, 15]. Cell adhesion data are expressed as number of cells per millimeter squared of venular surface, calculated from venular diameter and length, assuming cylindrical geometry.
After 24 hours of reperfusion, mice were decapitated under deep anesthesia with ketamine (200 mg/kg, i.p.) and xylazine (20 mg/kg, i.p.) then 1-mm-thick coronal sections of the brain were immersed in 0.05% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich) solution for 30 minutes. The total areas of each brain section and the infarct region were quantified with the software program, NIH image. Infarct volume was corrected for edema as previously described .
Changes in the barrier properties of the cerebral microvasculature were monitored using the Evans blue (EB) extravasation method, as previously described [19, 20]. A 2% solution of EB (Sigma-Aldrich) was administered (4 ml/kg, i.v.) immediately following I/R or after sham operation. Twenty-four hours later, mice were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) then plasma sample was collected and brain tissue (right hemisphere with brain stem and dura mater removed) sample was collected after transcardial perfusion with PBS (100 mm Hg, 5 minutes). EB concentrations were determined using a fluorescence spectrophotometer (FLUOstar Optima; BMG LABTECH, Inc.). BBB permeability was estimated by dividing tissue EB concentration (ng/g brain weight) by the plasma concentration (ng/ml).
All of the values are reported as mean ± SE. One-way analysis of variance with Fisher's post-hoc test was used to determine statistical differences between groups. Statistical significance was set at P < 0.05.