Ethics statement and Mice.
C57BL/6 WT mice and B6.IL-21-VFP knock in reporter mice (B6.Cg-Il21tm1.1Hm/DcrJ) were obtained from Jackson Laboratory. All mice underwent SHAM or 1-hour occlusion and 4 hour or 24-hour reperfusion. All animal procedures used in this study were conducted in strict compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of Wisconsin Center for Health Sciences Research Animal Care Committee. All mice (∼25 g) were anesthetized with 5% halothane for induction and 1.0% halothane for maintenance vaporized in N2O and O2 (3:2), and all efforts were made to minimize suffering. Male and Female mice between 10 weeks and 14 weeks were used for all studies.
Regional cerebral blood flow (rCBF) measurement.
Changes in rCBF at the surface of the left cortex were recorded using a blood perfusion monitor (Laserflo BPM2; Vasamedics) with a fiber optic probe (0.7 mm diam). The tip of the probe was fixed with glue on the skull over the core area supplied by the MCA (2 mm posterior and 6 mm lateral from bregma). Changes in rCBF after MCAO were recorded as a percentage of the baseline value. Mice included in these investigations had >80% relative decrease in rCBF during MCAO.
Investigation of intracranial vasculature.
Mice were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). After thoracotomy was performed, a cannula was introduced into the ascending aorta through the left ventricle. Transcardial perfusion fixation was performed with 2 ml saline and 2 ml of 3.7% formaldehyde. Carbon lampblack (C198-500; Thermo Fisher Scientific) in an equal volume of 20% gelatin in ddH2O (1 ml) was injected through the cannula. The brains were removed and fixed in 4% PFA overnight at 4°C. Posterior communicating arteries (PComA) connect vertebrobasilar arterial system to the Circle of Willis and internal carotid arteries, and its development affects brain sensitivity to ischemia among different mouse strains [46]. Development of PComA in both hemispheres was examined and graded on a scale of 0–3, as reported previously[47]: 0, no connection between anterior and posterior circulation; 1, anastomosis in capillary phase (present but poorly developed); 2, small truncal PComA; 3, truncal PComA.
Focal ischemia model.
Focal cerebral ischemia in mice was induced by occlusion of the left MCA, as described previously [48]. Operators performing surgeries were masked to experimental groups. In brief, the left common carotid artery was exposed, and the occipital artery branches of the external carotid artery (ECA) were isolated and coagulated. After coagulation of the superior thyroid artery, the ECA was dissected distally and coagulated along with the terminal lingual and maxillary artery branches. The internal carotid artery (ICA) was isolated, and the extracranial branch of the ICA was then dissected and ligated. A standardized polyamide resin glue-coated 6.0 nylon monofilament (3021910; Doccol Corp) was introduced into the ECA lumen, and then advanced ∼9–9.5 mm in the ICA lumen to block MCA blood flow. During the entire procedure, mouse body temperature was kept between 37 and 38°C with a heating pad. The suture was withdrawn 60 min after occlusion. The incision was closed, and the mice underwent recovery.
Infarct Measurement and Motor-Function Determination
Postischemic motor function was evaluated by the Rotarod test (4 min on a cylinder rotating at 8 rpm) as described previously[49]. On day 7, animals were killed by transcardiac 4% phosphate-buffered paraformaldehyde (PFA) perfusion. Each brain was postfixed, cryoprotected, and sectioned (coronal; 30 μm thickness at an interval of 320 μm). Serial sections were stained with cresyl violet and scanned using the National Institutes of Health ImageJ software. Ischemic infarct volume was estimated by numeric integration of data from five serial coronal sections in respect to the sectional interval as described previously [50, 51]. Each infarct volume was corrected to account for edema and differential shrinkage during tissue processing using the Swanson formula [52].
Lymphocyte isolation, and cytofluorometry (FACS).
Mice were deeply anesthetized with isoflurane and then transcardially perfused with cold PBS. Single cell suspensions were made from cervical lymph nodes (CLNs) and spleens by grinding the tissues between the frosted ends of glass slides[53]. Red blood cells were lysed using ACK lysis buffer, and cells were washed with HBSS. Brain and spinal cord tissues were minced with razor blades and pushed through 70 μm nylon cell strainers. Cells were washed, resuspended in 70% Percoll and overlaid with 30% Percoll. The gradient was centrifuged at 2400 rpm for 30 min at 4°C without brake. The interface was removed and washed before plating. All collected organs were weighed, and live cells were counted using a hemocytometer. Data were acquired on a BD LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).
Fluorescent microscopy.
For frozen sections, mice were first perfused with cold PBS, followed by perfusion with 4% PFA/PBS. Harvested tissues were left in 25% sucrose/PBS overnight at 4°C. 10-40 μm-thick tissue cryosections were cut and stored at −80°C until staining. Floating sections were incubated in PBS 2 times for 10 minutes at room temperature before applying primary conjugated antibodies in FACS buffer (PBS/ with 2% BSA/ and 0.1% Sodium Azide) with 0.1% Triton X-100 (1:1000) overnight at 37°C. Sections were then washed 2 times for 10 min each time with PBS and secondary antibodies were applied in PBS (1:500) for 2 hours if necessary. Lastly, sections were washed 3 times for 10 min each time with PBS and mounted with ProLong Gold antifade reagent containing DAPI (Invitrogen). All images were acquired with a camera (Optronics Inc., Goleta, CA) mounted on a fluorescence microscope (Olympus BX41, Leeds Precision Instruments). The brightness/contrast of the acquired digital images was applied equally across the entire image and equally to control images and analyzed using Adobe Photoshop CS4 software (Adobe Systems Inc., San Jose, CA).
Antibodies The following fluorophore-conjugated antibodies were purchased from BD Biosciences (Franklin Lakes, NJ): anti-CD4 (RM4-5), anti-ICOS-1 (D10.G4.1), anti-CXCR5 (2G8). All isotype controls were purchased from BD Biosciences. Anti-Fcγ-R (2.4G2) was produced from a hybridoma. IBA-1(019-19741) was purchased from WAKO (Richmond, VA). Anti-MAP2 (MAB8304) and anti-CXCL13 (AF470) were purchased from R and D systems (Minneapolis, MN). Anti-GFAP (AB5541), Anti-NeuN (MAB377B) and Anti-IL-21 (06-1074) was purchased from EDM Millipore (Hayward, CA). Anti-IL-21R (PA5-19982) was purchased from Invitrogen (Carlsbad, CA). JAK/STAT Phospho Antibody Array was purchased from Full Moon BioSystems (Sunnyvale, CA).
Oxygen glucose deprivation.
Primary neuronal cultures derived from embryonic day 14–18 mouse cortices were grown to 80% confluency in neural basal media supplemented with B27 (2%) and penicillin/streptomycin (1%), as previously described [54]. Astrocytic and microglial contamination was excluded based on the absence of GFAP+ and CD11b+ cells when stained by immunocytochemistry. For OGD, media was replaced with neural basal media with or without glucose and placed in a hypoxic chamber or under Normoxic conditions for 90 min at 37°C.
Human tissue samples.
Unidentified human brain samples were received from the Department of Pathology tissue bank and were exempt by IRB. Pathologists Dr. Krisztian Kovacs and Dr. Shahriar Salamat evaluated all acute human stroke tissue samples.
Statistical analyses and quality standards.
All surgeries were performed in a blinded manner and measurements masked where possible. Infarct volume measurements from Cresyl Violet stained sections were averaged from multiple independent blinded observers. Based on power calculations, n = 3–8 sex- and age-matched mice were used for each experiment and group assignment was randomized. Among animals receiving tMCAO procedure, 83% of WT mice were included in analysis as 17% displayed no injury. Mice were excluded due to premature death or vessel variation. Results are given as means ± s.e.m. Multiple comparisons were made using one-way ANOVA. Where appropriate, two-tailed nonparametric Mann-Whitney U test analysis was used for comparing measures made between two groups. P-values <0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s. = not significant.