C3H/HeOuJ mice were obtained from Jackson Laboratories (Bar Harbor, ME). Male mice aged 14–18 weeks and weighing 28–35 g were used for all experiments. All procedures were carried out with the approval of the Institutional Animal Use and Care Committee of the University of Pennsylvania.
Intracerebral hemorrhage surgery
Mice were anesthetized with inhaled 70% N2O, 30% O2, and 1–5% Isoflurane, and given buprenorphine 0.1 mg/kg subcutaneously for analgesia. Autologous blood from the ventral tail artery was injected at 0.5 μl/min for a total of 15 μl by microinfusion pump (World Precision Instruments, Sarasota, FL) with a 5-min pause midway through the infusion, at coordinates 2.5 mm right of the bregma, angled 5° medial and 3 mm deep. The needle was left in place for 30 additional minutes to allow blood clotting and then withdrawn at 1 mm/min. Incisions were closed using Vetbond (3 M, St. Paul, MN). Body core temperature was maintained at 37 ± 0.5°C by rectal thermometer using a thermistor-controlled heating pad throughout the procedure. Sham surgeries were also performed that included all procedures (including needle insertion) except blood injection.
Immediately after sacrifice, the brain was inspected for ICH success based on gross inspection of a coronal section at the needle insertion site, blinded to mouse treatment. Hemorrhages that tracked down to the base of the brain, up the needle track past the corpus callosum, or into the ventricles were deemed unsuccessful and that mouse was eliminated from all analyses. Three mice were eliminated after ICH resulting in a success rate 81%.
Mice were injected intraperitoneally with rat anti-mouse Ly6G monoclonal antibody (clone 1A8, BD Biosciences, San Jose, CA), 5 mg/kg, n = 6, to deplete circulating neutrophils or control rat IgG (5 mg/kg, n = 6) 12 h prior to ICH surgery and then every 36–48 h until sacrifice on post-ICH day 3.
Quantification of neurobehavioral deficit
Cylinder testing was performed postoperatively each morning, blinding to treatment, and videotaped for review of the scoring. Each mouse was placed in a 12-cm-diameter clear glass cylinder and observed for 20 rears. The initial placement of the forelimbs on the wall of the cylinder was scored per rear. Subsequent movements (such as lateral exploration) were not scored until the mouse returned to the ground; the next rear was then scored. The laterality index was calculated as (number of right forelimb placements on the side of the cylinder – number of left forelimb placements)/(number of right + number of left + number of both), where 0 indicates no forelimb preference, and 1 indicates only the right forelimb was used.
Mice were euthanized at 72 ± 2 h after ICH; their brains were removed and immediately frozen in Tissue-tek O.C.T. (Andwin Scientific, Addison, IL), and stored at −80°C until analysis. Then 6-μm sections were fixed with 75% acetone/25% ethanol and blocked with 2% normal goat serum. Slides were incubated with rat anti-mouse Ly6G (5 μg/ml) or rat anti-mouse CD11b (2.5 μg/ml) (eBioscience, San Diego, CA) followed by secondary antibody [Cy3 Affinipure goat anti-rat IgG (Jackson Immunoresearch, West Grove, PA)] at 1:500. DAPI was used at 0.5 μg/ml (Roche Diagnostics, Mannheim, Germany).
Images were acquired using a Nikon E600 fluorescence microscope equipped with a CoolSNAP CCD camera (Photometrics, Tucson, AZ) and processed with NIS Elements software (Nikon, Melville, NY). Neutrophil infiltration was quantified by summing the number of perihematomal neutrophils in five perihematomal 40× fields per mouse to yield the neutrophil count for each mouse. CD11b-positive cells were quantified by summing the number of positive cells in five 20× fields.
Tissue preparation for flow cytometry
Immediately following sacrifice, 1 ml of venous blood was withdrawn and mixed with heparin 200 U/ml. Mice were then perfused with 50 mL of ice cold PBS, and the brains and spleens removed. The two cerebral hemispheres were divided along the inter-hemispheric fissure so that the ipsilateral and contralateral hemispheres could be analyzed separately. Each hemisphere was placed in 4 ml of complete RPMI 1640 (Life Technologies, Gaithersburg, MD) medium supplemented with 10% fetal calf serum, 1% sodium pyruvate, 1% non-essential amino acids, 0.1% β-mercaptoethanol, 100 U penicillin/mL, and 100 μg/ml streptomycin (all Gibco, Invitrogen Incorporation, Grand Island, NY). Tissues were mechanically dissociated and incubated with 100 μl of collagenase/dispase (10 mg/ml, Roche Diagnostics, Indianapolis, IN) and 300 μl DNase (10 mg/ml, Sigma) for 45 min at 37°C. The suspension was then passed through a 70-μm cell strainer, pelleted at 2,000 × g for 10 min, and resuspended in 60% isotonic Percoll (GE Healthcare, Pittsburgh, PA) solution, overlaid with 30%, and centrifuged at 1,000 × g for 25 min. Brain mononuclear cells were harvested at the 60% and 30% inter-phase layer.
Peripheral blood leukocytes were overlaid on 4 ml Lympholyte-M and centrifuged at 800 ×g for 20 min. Leukocytes at the interface were harvested and washed with complete RPMI.
Cells were washed in PBS and then blocked with 50 μl Fc block [10% CD16/CD32 10 μg/ml, BD Biosciences, 0.5% normal rat IgG in FACS buffer (1× PBS, 0.2% BSA, and 2 mM EDTA)] for 15 min prior to staining with CD45-APC, CD11b-PerCp Cy5.5, Ly6G-Pacific Blue, CD11c-PECy7, CD3-FITC, CD19-FITC, NK1.1-FITC, and Gr-1-PE (eBioscience) for 15 min. Data were acquired on a BD Canto II using FACsDIVA 6.0 software (BD Biosciences). Analysis was performed using FlowJo software (Treestar Inc., Ashland, OR). Microglia were identified as CD45intCD11b+Gr-1- cells. Neutrophils were identified as CD45hiCD3-CD19-NK1.1-CD11b+Ly6G+ F4/80- cells. Monocytes were identified as CD45hiCD3-CD19-NK1.1-CD11b+Ly6G-CD11c-F4/80int cells. Dendritic cells were identified as CD45hiCD3-CD19-NK1.1-CD11b+Ly6G-CD11c+ cells.
Cell counts by immunohistochemistry and flow cytometry were tested for normality, and differences between treatment groups were compared by two-sided t-test or Wilcoxon rank sum, as appropriate. Cells counts in peripheral blood were compared by t-test. The ratios of ipsilateral/contralateral neutrophils, monocytes, and dendritic cells in the brain were calculated to account for differences in the success of perfusion in each mouse and compared by t-test. Cylinder test results on each day were compared by ANOVA followed by t-tests for each pair of cohorts since the comparisons were pre-specified and distribution was normal. Analysis was performed with Stata IC/10 (College Station, TX).