Title: Evidence that Meningeal Mast Cells Can Worsen Stroke Pathology in Mice
Abstract: Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine (C-C motif) ligand 7, can contribute to stroke pathology. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke. Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine (C-C motif) ligand 7, can contribute to stroke pathology. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke. Stroke, the leading cause of adult disability and the fourth most common cause of death in the Unites States,1Go A.S. Mozaffarian D. Roger V.L. Benjamin E.J. Berry J.D. 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Systemic augmentation of αB-crystallin provides therapeutic benefit twelve hours post-stroke onset via immune modulation.Proc Natl Acad Sci U S A. 2011; 108: 13287-13292Crossref PubMed Scopus (122) Google Scholar, 7Becker K.J. Modulation of the postischemic immune response to improve stroke outcome.Stroke. 2010; 41: S75-S78Crossref PubMed Scopus (36) Google Scholar However, successfully exploiting this therapeutic potential requires a detailed understanding of the interplay between the immune system and the brain after stroke.4Iadecola C. Anrather J. The immunology of stroke: from mechanisms to translation.Nat Med. 2011; 17: 796-808Crossref PubMed Scopus (1808) Google Scholar An understudied but important aspect of this interplay is the role of meningeal-located immune cells in modulating brain pathology. The meninges have long been recognized as an anatomical barrier that protects the central nervous system (CNS). However, accumulating evidence suggests that the meninges are important for communication between the CNS and immune system during health and disease.8Shechter R. London A. Schwartz M. Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates.Nat Rev Immunol. 2013; 13: 206-218Crossref PubMed Scopus (280) Google Scholar, 9Androdias G. Reynolds R. Chanal M. Ritleng C. Confavreux C. Nataf S. Meningeal T cells associate with diffuse axonal loss in multiple sclerosis spinal cords.Ann Neurol. 2010; 68: 465-476Crossref PubMed Scopus (98) Google Scholar, 10Derecki N.C. Cardani A.N. Yang C.H. Quinnies K.M. Crihfield A. Lynch K.R. Kipnis J. Regulation of learning and memory by meningeal immunity: a key role for IL-4.J Exp Med. 2010; 207: 1067-1080Crossref PubMed Scopus (580) Google Scholar All blood vessels pass through the meningeal subarachnoid space before entering the brain, and this vascular connection and the close proximity of the meninges to the underlying parenchymal nervous tissue make them ideally located to act as a gatekeeper to modulate immune cell trafficking to the CNS. To support this gatekeeper function is evidence that the meninges modulate brain infiltration of T cells, neutrophils, and monocytes during meningitis and autoimmune conditions,11Christy A.L. Walker M.E. Hessner M.J. Brown M.A. Mast cell activation and neutrophil recruitment promotes early and robust inflammation in the meninges in EAE.J Autoimmun. 2012; 42: 50-61Crossref PubMed Scopus (127) Google Scholar, 12Kivisakk P. Imitola J. Rasmussen S. Elyaman W. Zhu B. Ransohoff R.M. Khoury S.J. 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Brown M.A. Mast cell activation and neutrophil recruitment promotes early and robust inflammation in the meninges in EAE.J Autoimmun. 2012; 42: 50-61Crossref PubMed Scopus (127) Google Scholar, 13Kim J.V. Kang S.S. Dustin M.L. McGavern D.B. Myelomonocytic cell recruitment causes fatal CNS vascular injury during acute viral meningitis.Nature. 2009; 457: 191-195Crossref PubMed Scopus (277) Google Scholar Emerging evidence suggests that the actions of immune cells resident in the meninges are important for this gatekeeper function.11Christy A.L. Walker M.E. Hessner M.J. Brown M.A. Mast cell activation and neutrophil recruitment promotes early and robust inflammation in the meninges in EAE.J Autoimmun. 2012; 42: 50-61Crossref PubMed Scopus (127) Google Scholar, 12Kivisakk P. Imitola J. Rasmussen S. Elyaman W. Zhu B. Ransohoff R.M. Khoury S.J. Localizing central nervous system immune surveillance: meningeal antigen-presenting cells activate T cells during experimental autoimmune encephalomyelitis.Ann Neurol. 2009; 65: 457-469Crossref PubMed Scopus (211) Google Scholar, 15Sayed B.A. Christy A.L. Walker M.E. Brown M.A. Meningeal mast cells affect early T cell central nervous system infiltration and blood-brain barrier integrity through TNF: a role for neutrophil recruitment?.J Immunol. 2010; 184: 6891-6900Crossref PubMed Scopus (134) Google Scholar Mast cells (MCs), best known as proinflammatory effector cells, can play critical roles in the development of inflammation in many disease settings.16Abraham S.N. St John A.L. Mast cell-orchestrated immunity to pathogens.Nat Rev Immunol. 2010; 10: 440-452Crossref PubMed Scopus (738) Google Scholar, 17Galli S.J. Grimbaldeston M. Tsai M. Immunomodulatory mast cells: negative, as well as positive, regulators of immunity.Nat Rev Immunol. 2008; 8: 478-486Crossref PubMed Scopus (647) Google Scholar, 18Rao K.N. Brown M.A. Mast cells: multifaceted immune cells with diverse roles in health and disease.Ann N Y Acad Sci. 2008; 1143: 83-104Crossref PubMed Scopus (214) Google Scholar MCs reside in high numbers within the meninges, but their function in this site has not been fully investigated in stroke pathology. Unlike most immune cells, mature MCs do not circulate in the blood but are long-term residents of tissues, often in perivascular locations, and can rapidly perform their functions in situ. CNS MCs are found in the brain parenchyma and the meninges of rodents and humans.18Rao K.N. Brown M.A. Mast cells: multifaceted immune cells with diverse roles in health and disease.Ann N Y Acad Sci. 2008; 1143: 83-104Crossref PubMed Scopus (214) Google Scholar It has been proposed that brain parenchymal MCs can enhance brain neutrophil numbers after stroke and can exacerbate stroke pathology.19Strbian D. Karjalainen-Lindsberg M.L. Kovanen P.T. Tatlisumak T. Lindsberg P.J. Mast cell stabilization reduces hemorrhage formation and mortality after administration of thrombolytics in experimental ischemic stroke.Circulation. 2007; 116: 411-418Crossref PubMed Scopus (77) Google Scholar, 20Strbian D. Karjalainen-Lindsberg M.L. Tatlisumak T. Lindsberg P.J. Cerebral mast cells regulate early ischemic brain swelling and neutrophil accumulation.J Cereb Blood Flow Metab. 2006; 26: 605-612Crossref PubMed Scopus (126) Google Scholar, 21Strbian D. Tatlisumak T. Ramadan U.A. Lindsberg P.J. Mast cell blocking reduces brain edema and hematoma volume and improves outcome after experimental intracerebral hemorrhage.J Cereb Blood Flow Metab. 2007; 27: 795-802PubMed Google Scholar, 22Jin Y. Silverman A.J. Vannucci S.J. Mast cell stabilization limits hypoxic-ischemic brain damage in the immature rat.Dev Neurosci. 2007; 29: 373-384Crossref PubMed Scopus (73) Google Scholar, 23Biran V. Cochois V. Karroubi A. Arrang J.M. Charriaut-Marlangue C. Heron A. Stroke induces histamine accumulation and mast cell degranulation in the neonatal rat brain.Brain Pathol. 2008; 18: 1-9Crossref PubMed Scopus (64) Google Scholar, 24Hu W. Xu L. Pan J. Zheng X. Chen Z. Effect of cerebral ischemia on brain mast cells in rats.Brain Res. 2004; 1019: 275-280Crossref PubMed Scopus (29) Google Scholar However, much of the evidence to support such conclusions is indirect. For example, some of the studies that implicate MCs in stroke pathology used pharmacologic approaches to interfere with MC activation,19Strbian D. Karjalainen-Lindsberg M.L. Kovanen P.T. Tatlisumak T. Lindsberg P.J. Mast cell stabilization reduces hemorrhage formation and mortality after administration of thrombolytics in experimental ischemic stroke.Circulation. 2007; 116: 411-418Crossref PubMed Scopus (77) Google Scholar, 20Strbian D. Karjalainen-Lindsberg M.L. Tatlisumak T. Lindsberg P.J. Cerebral mast cells regulate early ischemic brain swelling and neutrophil accumulation.J Cereb Blood Flow Metab. 2006; 26: 605-612Crossref PubMed Scopus (126) Google Scholar, 22Jin Y. Silverman A.J. Vannucci S.J. Mast cell stabilization limits hypoxic-ischemic brain damage in the immature rat.Dev Neurosci. 2007; 29: 373-384Crossref PubMed Scopus (73) Google Scholar but such drugs can have effects on other cell types.25Oka T. Kalesnikoff J. Starkl P. Tsai M. Galli S.J. Evidence questioning cromolyn's effectiveness and selectivity as a ‘mast cell stabilizer' in mice.Lab Invest. 2012; 92: 1472-1482Crossref PubMed Scopus (103) Google Scholar Moreover, the role of the meningeal MCs in modulating post-stroke inflammation and pathology is unknown. Finally, little is understood about which among the many MC-derived mediators may be important in stroke pathology.17Galli S.J. Grimbaldeston M. Tsai M. Immunomodulatory mast cells: negative, as well as positive, regulators of immunity.Nat Rev Immunol. 2008; 8: 478-486Crossref PubMed Scopus (647) Google Scholar, 26Tsai M. Grimbaldeston M. Galli S.J. Mast cells and immunoregulation/immunomodulation.Adv Exp Med Biol. 2011; 716: 186-211Crossref PubMed Scopus (87) Google Scholar To address these questions, we used genetic and cell transfer approaches to study the role of MCs in the pathology of ischemic stroke in mice. Specifically, we tested a c-kit–mutant mouse model (ie, WBB6F1-KitW/W-v mice) which is profoundly MC deficient and can be repaired of this deficiency by engraftment of in vitro-derived MCs from wild-type (WT) mice. This MC knock-in approach enables the MC-dependent effects in the mutant mice to be separated from effects due to other abnormalities associated with their mutation,11Christy A.L. Walker M.E. Hessner M.J. Brown M.A. Mast cell activation and neutrophil recruitment promotes early and robust inflammation in the meninges in EAE.J Autoimmun. 2012; 42: 50-61Crossref PubMed Scopus (127) Google Scholar, 17Galli S.J. Grimbaldeston M. Tsai M. Immunomodulatory mast cells: negative, as well as positive, regulators of immunity.Nat Rev Immunol. 2008; 8: 478-486Crossref PubMed Scopus (647) Google Scholar, 26Tsai M. Grimbaldeston M. Galli S.J. Mast cells and immunoregulation/immunomodulation.Adv Exp Med Biol. 2011; 716: 186-211Crossref PubMed Scopus (87) Google Scholar, 27Nigrovic P.A. Gray D.H. Jones T. Hallgren J. Kuo F.C. Chaletzky B. Gurish M. Mathis D. Benoist C. Lee D.M. Genetic inversion in mast cell-deficient Wsh mice interrupts corin and manifests as hematopoietic and cardiac aberrancy.Am J Pathol. 2008; 173: 1693-1701Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar because only the MC deficiency is repaired by MC engraftment. Furthermore, one can investigate the mechanisms by which MCs influence stroke pathology by engrafting MCs from transgenic mice that lack specific MC-associated products. We also tested our newly described Cpa3-Cre; Mcl-1fl/fl mice, in which MC (and basophil) numbers are reduced constitutively via Cre-mediated depletion of the anti-apoptotic factor, myeloid cell leukemia sequence 1 (Mcl-1), in the affected lineages.28Lilla J.N. Chen C.C. Mukai K. BenBarak M.J. Franco C.B. Kalesnikoff J. Yu M. Tsai M. Piliponsky A.M. Galli S.J. Reduced mast cell and basophil numbers and function in Cpa3-Cre; Mcl-1fl/fl mice.Blood. 2011; 118: 6930-6938Crossref PubMed Scopus (158) Google Scholar Cpa3-Cre; Mcl-1fl/fl mice lack the other abnormalities associated with the c-kit mutations in WBB6F1-KitW/W-v mice.28Lilla J.N. Chen C.C. Mukai K. BenBarak M.J. Franco C.B. Kalesnikoff J. Yu M. Tsai M. Piliponsky A.M. Galli S.J. Reduced mast cell and basophil numbers and function in Cpa3-Cre; Mcl-1fl/fl mice.Blood. 2011; 118: 6930-6938Crossref PubMed Scopus (158) Google Scholar With the use of these in vivo models, we identified meningeal MCs as important contributors to key features of stroke pathology, including increased numbers of brain granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two potentially proinflammatory MC-derived products, IL-6 and, to a lesser extent, chemokine (C-C motif) ligand 7 (CCL7), can contribute to pathology in this setting. Male c-kit–mutant genetically MC-deficient (WB/Rej-KitW/J × C57BL/6J-KitW-v/J)F1-KitW/Wv (WBB6F1-KitW/W-v) mice and their congenic WT (WBB6F1-Kit+/+) littermates were purchased from The Jackson Laboratory (Bar Harbor, ME). KitW/W-v mice have a profound deficiency in MCs29Grimbaldeston M.A. Chen C.C. Piliponsky A.M. Tsai M. Tam S.Y. Galli S.J. Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo.Am J Pathol. 2005; 167: 835-848Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar and certain other hematological abnormalities; however, only the MC deficiency is repaired by MC engraftment.17Galli S.J. Grimbaldeston M. Tsai M. Immunomodulatory mast cells: negative, as well as positive, regulators of immunity.Nat Rev Immunol. 2008; 8: 478-486Crossref PubMed Scopus (647) Google Scholar, 26Tsai M. Grimbaldeston M. Galli S.J. Mast cells and immunoregulation/immunomodulation.Adv Exp Med Biol. 2011; 716: 186-211Crossref PubMed Scopus (87) Google Scholar, 30Reber L.L. Marichal T. Galli S.J. New models for analyzing mast cell functions in vivo.Trends Immunol. 2012; 33: 613-625Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar KitW/W-v mice have lower levels of neutrophils than the corresponding WT mice in the bone marrow (BM), blood, and spleen and have a mild anemia.27Nigrovic P.A. Gray D.H. Jones T. Hallgren J. Kuo F.C. Chaletzky B. Gurish M. Mathis D. Benoist C. Lee D.M. Genetic inversion in mast cell-deficient Wsh mice interrupts corin and manifests as hematopoietic and cardiac aberrancy.Am J Pathol. 2008; 173: 1693-1701Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar W is a null allele of Kit and Wv is a point mutation in the cytoplasmic tail of the receptor.17Galli S.J. Grimbaldeston M. Tsai M. Immunomodulatory mast cells: negative, as well as positive, regulators of immunity.Nat Rev Immunol. 2008; 8: 478-486Crossref PubMed Scopus (647) Google Scholar, 26Tsai M. Grimbaldeston M. Galli S.J. Mast cells and immunoregulation/immunomodulation.Adv Exp Med Biol. 2011; 716: 186-211Crossref PubMed Scopus (87) Google Scholar Cpa3-Cre;Mcl-1fl/fl mice are severely deficient in MCs and also have a marked deficiency in basophils.28Lilla J.N. Chen C.C. Mukai K. BenBarak M.J. Franco C.B. Kalesnikoff J. Yu M. Tsai M. Piliponsky A.M. Galli S.J. Reduced mast cell and basophil numbers and function in Cpa3-Cre; Mcl-1fl/fl mice.Blood. 2011; 118: 6930-6938Crossref PubMed Scopus (158) Google Scholar In these mice, Cre recombinase is expressed under the control of carboxypeptidase A3 (Cpa3) promoter. Mcl-1 is an intracellular anti-apoptotic factor that is required for MC survival. C57BL/6-Cpa3-Cre; Mcl-1+/+ mice were used as WT controls for Cpa3-Cre;Mcl-1fl/fl mice. IL6–knock-out (KO) mice (B6.129S2-Il6tm1Kopf/J) were purchased from The Jackson Laboratory. CCL7-KO mice31Tsou C.L. Peters W. Si Y. Slaymaker S. Aslanian A.M. Weisberg S.P. Mack M. Charo I.F. Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites.J Clin Invest. 2007; 117: 902-909Crossref PubMed Scopus (838) Google Scholar on a C57BL/6 background were initially developed and were a kind gift from Israel F. Charo (University of California San Francisco, San Francisco, CA). All of the animal procedures were approved by Stanford University Administrative Panel on Laboratory Animal Care. The MC deficiency in WBB6F1-KitW/W-v mice was selectively repaired by systemic (intravenously through retro-orbital injection under isoflurane anesthesia) or by meningeal administration of mouse BM-derived cultured MCs (BMCMCs) generated in vitro, as indicated. As described before,32Grimbaldeston M.A. Nakae S. Kalesnikoff J. Tsai M. Galli S.J. Mast cell-derived interleukin 10 limits skin pathology in contact dermatitis and chronic irradiation with ultraviolet B.Nat Immunol. 2007; 8: 1095-1104Crossref PubMed Scopus (404) Google Scholar the femoral and tibial BM cells from WBB6F1-Kit+/+, C57BL/6-Kit+/+, C57BL/6-IL6-KO, and C57BL/6-CCL7-KO mice were cultured in 20% medium conditioned by the growth of the WEHI-3 mouse myelomonocytic cell line (containing IL-3) for 4 to 5 weeks. Before engraftment, >95% of cultured cells were identified as BMCMCs by May-Grünwald-Giemsa stain. For systemic engraftment, 107 BMCMCs in 100 μL of phosphate-buffered saline were injected retro-orbitally into 9- to 11-week-old WBB6F1-KitW/W-v mice (50 μL into each retro-orbital side). For meningeal engraftment, 106 BMCMCs or vehicle alone (as a control) were injected into 9- to 11-week-old WBB6F1-KitW/W-v mice, as described.15Sayed B.A. Christy A.L. Walker M.E. Brown M.A. Meningeal mast cells affect early T cell central nervous system infiltration and blood-brain barrier integrity through TNF: a role for neutrophil recruitment?.J Immunol. 2010; 184: 6891-6900Crossref PubMed Scopus (134) Google Scholar The mice were used for the experiments 8 to 10 weeks after either type (ie, i.v. or meningeal) of engraftment. In experiments that used such MC-engrafted mice, WT mice and MC-deficient mice used in the same experiments were the same age and housed with the same conditions of husbandry. The mice were subjected to a filament occlusion model of cerebral ischemia as described.6Arac A. Brownell S.E. Rothbard J.B. Chen C. Ko R.M. Pereira M.P. Albers G.W. Steinman L. Steinberg G.K. Systemic augmentation of αB-crystallin provides therapeutic benefit twelve hours post-stroke onset via immune modulation.Proc Natl Acad Sci U S A. 2011; 108: 13287-13292Crossref PubMed Scopus (122) Google Scholar The mice were habituated in the surgery room overnight, and all of the surgeries were initiated early in the morning. Briefly, mice were anesthetized by 1.5% to 2% isoflurane in a mixture of 1 L/minute of air and 0.2 L/minute of oxygen. The left external and common carotid arteries were permanently ligated. A hole was made in the common carotid artery, and a 7-0, silicon rubber-coated, reusable monofilament (70SPRe2045; Doccol Inc., Sharon, MA) was inserted and advanced toward the internal carotid artery 9 to 10 mm after the carotid bifurcation to occlude the left middle cerebral artery. The core body temperature was measured by a rectal probe and maintained at 37°C throughout the surgery. Thirty minutes after its insertion, the filament was removed to permit reperfusion. The surgical wound was closed, and the mice were returned to their cages with free access to water and food. We measured cerebral blood flow by using a laser Doppler flow meter. The probe was placed onto the skull 2 mm posterior and 5 to 6 mm lateral to the bregma on the left side. No differences were found in the presurgical weights of mice in any experiment. A noninvasive CODA Monitor system (Kent Scientific, Torrington, CT) was used to measure blood pressures and pulse rates. Arterial blood gas and lactate levels were measured by iSTAT CG4+ cartridges and a handheld blood analyzer (Abbott Laboratories, Abbott Park, IL). Magnetic resonance imaging (MRI) was performed at Stanford Small Animal Imaging Facility by using the GE Healthcare (Waukesha, WI) Micro-Signa software environment version 12M5 with a Varian 7 Tesla magnet, Research Resonance Instruments BFG-150 to 90 gradient insert. The mice were anesthetized with 2% isoflurane in 2 L/minute of medical grade oxygen while the respiratory rates were monitored, and the surface body temperature was also monitored and kept at 34°C throughout the imaging. An in-house 2-cm diameter surface coil was placed on the mouse skull to obtain the images. The T2-weighted (T2W) imaging protocol parameters were as follows: a two-dimensional fast spin echo sequence with echo time (TE) = 82.5 ms, repetition time (TR) = 4000 ms, echo train length = 8, axial slice thickness = 0.6 mm with no spacing, field of view = 3 cm, matrix = 128 × 128, number of excitations (NEX) = 10. The dicom files were opened in OsiriX version 3.3.2 (OsiriX Foundation, Geneva, Switzerland), and the regions of interest were manually delineated. The hyperintense areas of the stroke region and the total ipsilateral and contralateral hemisphere areas (excluding the ventricles) were measured in 11 consecutive slices, starting approximately 2.5 mm anterior and extending toward −3.5 mm posterior to the bregma. The brain swelling was calculated by the following formula that used T2W−MRI obtained at 3 days after stroke:Brainswelling=100×(totalipsilateralarea−totalcontralateralarea)/totalcontralateralarea.(1) The MRI infarct sizes were calculated by the following formula that used T2W-MRI obtained at 3 days or 2 weeks after stroke:Infarctsize=100×[totalcontralateralhemispherearea-ipsilateralhealthyarea]/(totalcontralateralhemispherearea).(2) The histological infarct size at 2 weeks after stroke was calculated by using the same formula that used measurements from silver-stained sections. For silver staining, the mice were perfused transcardially with 30 mL of cold 0.9% NaCl, followed by 30 mL of 3% balanced (pH 7.4) formalin solution. The heads were kept overnight in 3% balanced formalin solution, and then the brains were transferred into a 20% sucrose/3% formalin solution until they sank. Then, 30-μm sections were cut with a cryostat. One in every 16 sections (11 sections per brain) were stained with silver stain33Vogel J. Mobius C. Kuschinsky W. Early delineation of ischemic tissue in rat brain cryosections by high-contrast staining.Stroke. 1999; 30: 1134-1141Crossref PubMed Scopus (69) Google Scholar and scanned at 1200 dpi. The total ipsilateral, contralateral, and infarct areas were measured with ImageJ version 1.44o (NIH, Bethesda, MD). After induction of deep anesthesia, blood was collected through cardiac puncture in EDTA syringes (50 μL of 2 mmol/L EDTA for 1 mL of blood). The mice were perfused with 30 mL of cold saline, the brains were removed immediately, the hemispheres were split, and ipsilateral hemispheres were collected in phosphate-buffered saline (Gibco, Carlsbad, CA) on ice. The ipsilateral hemispheres were passed through a 70-μL cell strainer in Hanks' balanced salt solution (Gibco). Then, the homogenates were incubated in 1 mL of 2 U/mL of Liberase CI (Roche, Indianapolis, IN) in Hanks' balanced salt solution for 1 hour at 37°C, then centrifuged (490 × g for 20 minutes without brake) >30% Percoll. The cells were collected as the pellet and washed with 10% fetal bovine serum (Gibco) in Dulbecco's modified Eagle medium (Gibco). The red blood cells in blood were lyzed by lysis buffer (7.47 g of ammonium chloride and 2.04 g of Tris base in 1 L of ddH2O, pH 7.6), and the cells were washed with 10% fetal bovine serum in Dulbecco's modified Eagle medium and kept on ice until staining. Cells were first stained with 0.1% Live/Dead-Aqua (Invitrogen, Carlsbad, CA) to exclude the dead cells from the analysis and then blocked with 1% anti-mouse CD16/32 (93) (eBioscience, San Diego, CA) antibody and 10% mouse serum (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) in a staining buffer [5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) and 2 mmol/L EDTA (Sigma-Aldrich) in phosphate-buffered saline]. The cells were incubated with the following antibodies for 20 minutes at 4°C: anti-mouse CD45 (30-F11), CD11b (M1/70), Gr1 (RB6-8C5), F4/80 (BM8), CD3e (17A2), CD8b (eBioH35 to 17.2), NK1.1 (PK136) (all from eBioscience), and CD4 (RM4-5) (Invitrogen). The flow cytometric analysis was performed on a Becton Dickinson LSR-II (Stanford Shared FACS Facility), and the data were analyzed with FlowJo version 9.7.5 (TreeStar Inc., Ashland, OR). The gates were set based on the unstained cells, and the compensation was achieved by single-color stained BD-CompBeads (BD Biosciences, San Jose, CA). After perfusing each mouse under deep isoflurane anesthesia with 30 mL of cold 0.9% NaCl and 30 mL of cold 3% buffered formalin, the heads were removed and incubated overnight in 3% buffered formalin solution. Then, the cranial bones then were removed carefully so that the dura remained on the brain surface. The dura was removed from the brain surface by using a fine-tip forceps, placed onto a slide with a drop of water, and then spread out under a dissection microscope to make a whole-mount preparation. The brain sections were obtained as described in the Assessment of Infarct Size and Brain Swelling section. Toluidine blue-stained brain MCs were quantified in one of every four sections taken throughout the brain, starting approximately 1.8 mm anterior of bregma to approximately −2.5 mm posterior of bregma. This approximately spans the region from where the corpus callosum first appears in coronal sections to mid-late hippocampus. This includes the area where the highest numbers of MCs reside in the brain (the area between hippocampus and thalamus). Thirty-two sections were counted for each brain. All values are expressed as means ± SEM. The normality of the data was determined by Kolmogorov-Smirnov test for each group. For comparisons of more than two groups, we used one-way analysis of varia