Inflammatory cell recruitment after experimental thromboembolic stroke in rats.

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INFLAMMATORY CELL RECRUITMENT AFTER EXPERIMENTAL THROMBOEMBOLIC STROKE IN RATS Q1 J. LEHMANN, a W. HA¨RTIG, b A. SEIDEL, a C. FU¨LDNER, a

a Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany

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b Paul Flechsig Institute for Brain Research, University of Leipzig, Leipzig, Germany

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c

Department of Neurology, University of Leipzig, Leipzig, Germany

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d

Institute of Anatomy, University of Leipzig, Leipzig, Germany

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expression and allocation with vessels exhibiting impaired blood–brain barrier integrity. Microglia remained numerically unaltered in ischemic hemispheres, but shifted to an activated phenotype indicated by CD45/CD86 expression and morphological changes toward an ameboid appearance in the bordering zone. Ischemia caused an increase of lymphoid cells in close vicinity to the affected vasculature, while further analyses allowed separation into natural killer cells, natural killer T cells, T cells (added by an unconventional CD11b+/CD3+ population) and two subpopulations of B cells. Taken together, our study provides novel data on the local inflammatory response to experimental thromboembolic stroke. As concomitantly present neutrophils, monocytes/macrophages and lymphoid cells in the early stage after ischemia induction correspond to changes seen in human stroke, future stroke research should preferably use animal models with relevance for clinical translation. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO.

C. HOBOHM, c J. GROSCHE, b M. KRUEGER d AND D. MICHALSKI c*

Abstract—Inflammatory mechanisms were recently identified as contributors to delayed neuronal damage after ischemic stroke. However, therapeutic strategies are still lacking, probably related to the outstanding standardization on inflammatory cell recruitment emerging from predominantly artificial stroke models, and the uncertainty on functional properties of distinct subpopulations. Using a rodent model of stroke that closely reflects human embolic ischemia, this study was focused on the local recruitment of immunoreactive cells as well as their functional and regional characterization. Wistar rats underwent thromboembolic middle cerebral artery occlusion, followed by intravenous injection of the blood–brain barrier permeability marker fluorescein Q2 isothiocyanate-albumin at 24 h. One hour later, brain tissue was subjected to multi-parameter flow cytometry and Pappenheim staining to characterize cells invaded into the ischemia-affected hemisphere, compared to the contralateral side. Immunofluorescence labeling was applied to explore the distribution patterns of recruited cells and their spatial relationships with the vasculature. One day after ischemia onset, a 6.12-fold increase of neutrophils and a 5.43-fold increase of monocytes/macrophages was found in affected hemispheres, while these cells exhibited enhanced major histocompatibility complex class II

Key words: inflammation, cellular recruitment, stroke, thromboembolic model. 14

*Corresponding author. Address: Department of Neurology, University of Leipzig, Liebigstr. 20, 04103 Leipzig, Germany. Tel: +49-3419724206; fax: +49-341-9724199. E-mail addresses: [email protected] (J. Lehmann), [email protected] (W. Ha¨rtig), seidel_andre@ gmx.net (A. Seidel), [email protected] (C. Fu¨ldner), [email protected] (C. Hobohm), [email protected] (J. Grosche), martin.krueger@ medizin.uni-leipzig.de (M. Krueger), dominik.michalski@medizin. uni-leipzig.de (D. Michalski). Q3 Abbreviations: FCS, fetal calf serum; FSC, forward scatter; FITC, fluorescein isothiocyanate; HBSS, Hank’s balanced salt solution; MCP-1, monocyte-chemoattractant protein-1; MIP-3a, macrophage inflammatory protein-3a; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; NK, natural killer; PFA, paraformaldehyde; SSC, side scatter; TBS, Tris-buffered saline. http://dx.doi.org/10.1016/j.neuroscience.2014.08.023 0306-4522/Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. 1

INTRODUCTION

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Despite intensified experimental and clinical research, stroke remains a major cause of death worldwide and contributes to an enormous financial burden (Donnan et al., 2008). On the cellular level, the gleaned pathophysiological knowledge on ischemic stroke enabled a more comprehensive perspective of tissue damage considering time-dependent mechanisms of neuronal affection (Dirnagl et al., 1999). While excitotoxicity causes cell death mainly within minutes to hours after ischemia onset, inflammation became increasingly important since this mechanism peaks later, appropriating a clinically relevant target for interventions (Dirnagl et al., 1999; Endres et al., 2008). Based on studies using predominantly histological techniques, it is generally accepted that the inflammatory response includes infiltration of myeloid cells and lymphocytes into the ischemia-affected area, complemented by local glial activation (Kochanek and Hallenbeck, 1992; Tomita and Fukuuchi, 1996; Stoll et al., 1998; Danton and Dietrich, 2003; Wang et al., 2007; Jin et al., 2010; Denes et al., 2010). Using flow cytometry and fluorescence-activated cell sorting in rodent brains subjected to experimental ischemia, Campanella et al. (2002), Stevens et al. (2002) and

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Gelderblom et al. (2009) have investigated involved cellular populations and their temporal characteristics in more detail. Thereby, rising numbers of neutrophils and macrophages in the ischemic hemisphere were found to occur within hours continuing until day 2 and 3 after ischemia onset, while lymphoid cells appeared delayed in time and in a much lower amount. After a first attempt to explore the functional relevance of cellular infiltration by Weston et al. (2007), demonstrating an association of numerically increased myeloid cells (i.e. neutrophils) with larger infarct volumes after experimental ischemic stroke in rats, two recent studies addressed the role of T lymphocytes in transient and permanent cerebral ischemia in mice, yielding contradicting neuroprotective (Liesz et al., 2009) and detrimental (Kleinschnitz et al., 2010) properties (Magnus et al., 2012). Even prior to these reports, a discussion emerged about the role of ischemia-induced inflammatory processes, while both deleterious consequences (such as cytokine-mediated cell death) and beneficial effects (i.e. the initiation of neuronal recovery and plasticity) were discussed (Stoll et al., 1998; Chamorro and Hallenbeck, 2006; Endres et al., 2008; Kriz and Lalancette-He´bert, 2009; Lakhan et al., 2009). The remaining uncertainty might hamper the development of drugs influencing the inflammatory response following ischemic stroke. The situation is further complicated by recent data indicating that the course of cellular recruitment critically depends on the applied animal model: Comparing filament-based models in mice with varying times of ischemia, Zhou et al. (2013) found an increasing number of neutrophils during the first 5 days after permanent ischemia, but a decreasing number following transient ischemia. Analyses of microglia activation at 24 h revealed that permanent ischemia led to a numerical increase of cells while transient ischemia caused cell counts not different from sham-operated animals. A very recent report by Mo¨ller et al. (2014) focused on ischemic consequences in spontaneous hypertensive rats, which became attractive due to the naturally existing arterial hypertension as a shared risk factor for stroke in humans. In these rats, permanent cerebral ischemia caused a neutrophil accumulation at day 1 followed by a decline at day 4, which was also found for monocytes – quite different from earlier studies. These observations together with the ongoing attempts to improve preclinical stroke research with emphasis on translational issues (Young et al., 2007; Fisher et al., 2009) lead to the question on details about the local inflammatory response in a rodent model of stroke that is comparable to the pathophysiology of stroke in man. The present study applied for the first time flow cytometric analyses combined with in situ immunohistochemical staining to characterize the early inflammatory cell recruitment after thromboembolic stroke in rats. This model is currently believed to best mimic the human condition due to shared mechanisms of stroke formation (Durukan and Tatlisumak, 2007; Braüninger and Kleinschnitz, 2009): a blood clot running inside of the internal carotid artery that finally occludes the middle cerebral artery.

EXPERIMENTAL PROCEDURES

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Experimental design

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All experiments involving animals had been conducted according to the European Communities Council Directive (86/609/EEC) and were approved by local authorities (Landesdirektion Sachsen, Leipzig, Germany, reference number TVV-34/11). Generally, efforts were made to minimize the number of animals in total and suffering of animals, which were housed in a temperature and humidity controlled room with 12 h of light/dark cycle and free access to food and water. The here presented data originated from n = 31 male Wistar rats with a mean body weight of 307 ± 20 g provided by Charles River (Sulzfeld, Germany), which underwent Q4 right-sided thromboembolic middle cerebral artery occlusion (details below). Sufficient cerebral ischemia was ensured by the presence of a relevant neurobehavioral deficit as indicated by a value of at least 1 on the Menzies score (naturally ranging from 0, no deficit, to 4, spontaneous contralateral circling; Menzies et al., 1992), which was assessed 4 and 24 h after ischemia induction. Immediately after the second neurobehavioral evaluation (at 24 h), fluorescein isothiocyanate (FITC)-albumin (20 mg/1 ml saline; Sigma, Taufkirchen, Germany) was intravenously administered, which allows fluorescence-based localization of ischemic damage at later stages by respective leakage in areas with blood–brain barrier impairment (Michalski et al., 2010). One hour after FITC-albumin injection (representing an overall circulation period of 60 min), animals were deeply anesthetized with CO2 and transcardially perfused with either 200-ml saline (n = 21; for flow cytometry and cell sorting, divided into n = 13 for model establishment and optimization of remaining cell count, and n = 8 for quantification/characterization) or 200-ml saline followed by 200-ml paraformaldehyde (PFA; up to 4%) in phosphate-buffered saline (n = 10; for immunohistological analyses). Afterward, brains were removed from the skulls and processed as described below.

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Induction of thromboembolic focal cerebral ischemia

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Unilateral middle cerebral artery occlusion was induced using a model originally described by Zhang et al. (1997) with minor modifications. Briefly, after careful preparation of right-sided cervical arteries, a polyethylene tubing with tapered end was inserted into the external carotid artery via a small incision and moved forward through the internal carotid artery up to the origin of the middle cerebral artery (distance about 1.6 cm from bifurcation). At this position, a blood clot with a medium length of about 45 mm, originating from rat blood that has been collected in a polyethylene tube at the previous day, allowed to clot on a heading pad at 37 °C for 2 h followed by overnight storage at 4 °C, was injected with about 40 ll of saline. Finally, the catheter was removed, the stump of the external carotid artery ligated and the wound closed. During surgery, additional polyethylene tubes were inserted into the femoral vein allowing future FITC-albumin administration, and femoral artery for peri-procedural measurement

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of vital parameters. Prior surgical procedures, all animals were anesthetized with 2.0–2.5% isoflurane (Isofluran Baxter, Baxter, Unterschleißheim, Germany; mixture: 70% N2O/30% O2) using a commercial vaporisator (VIP 3000, Matrix, New York, USA). To avoid anesthesiarelated cooling, the body temperature was adjusted to 37.0 °C with a thermostatically controlled warming pad (Fine Science Tools, Heidelberg, Germany) including rectal probe. Post-surgical pain control was ensured by metamizol-enriched (Novaminsulfon-ratiopharm, Ratiopharm, Ulm, Germany) drinking water.

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Flow cytometry

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For flow cytometry, brains were quickly removed from the skulls and divided into an affected and a non-affected hemisphere excluding the cerebellum. The obtained native material was primarily stored in sterile isotonic phosphate-buffered saline containing 100 U/ml penicillin and 60 U/ml streptomycin until further processing. To consider translational issues, i.e. to reflect variations between individual animals with respect to the recruitment of inflammatory cells into the infarcted area, individual brain hemispheres were analyzed by flow cytometry without pooling. Only for additional (ex vivo) morphological characterization of inflammatory cell populations, pooling of hemispheres (usually n = 5–6) was required due to limited cell numbers originating from individual brains. In order to minimize the influence of autolytic processes, tissue dissociation of the brain had to be started within 1–2 h post mortem. Individual hemispheres were mechanically and enzymatically processed as described by Campanella et al. (2002) with some modifications: Brain tissue was cut into small pieces in a Petri disk followed by enzymatic digestion in 20-ml Hank’s balanced salt solution (HBSS) containing 5 % fetal calf serum (FCS, Biochrom, Berlin, Germany), 0.2 % Collagenase II (Biochrom) and 250 ll DNase I (Fermentas, St. Leon-Rot, Germany) within a water bath under movement for 30 min at 37 °C. Afterward, the digested brain was passed through a sterile 40-lm-mesh sieve (Cell strainer, Falcon, BD Bioscience, Heidelberg, Germany) using a piston. The resulting cell suspension was centrifuged and washed twice in 10-ml HBSS/FCS (400g, 10 min, 4 °C). Finally, the cell pellet was re-suspended in 4 ml 30 % Percoll (GE Healthcare, Freiburg, Germany) solution and applied on a 3-step Percoll density gradient (3.5 ml 70% Percoll solution, 3.5 ml 37% Percoll solution) and centrifuged with low acceleration and without break (1000g, 20 min, 4 °C). After aspiration of the top layer containing predominantly myelin, the interphase layer between the 70% and the 37% Percoll phase was harvested carefully, washed twice in 10-ml HBSS/FCS (400g, 10 min, 4 °C), and finally re-suspended in 1-ml HBSS/FCS. Living cells were counted by trypan blue exclusion using a Neubauer hemocytometer (Paul Marienfeld, Lauda-Ko¨nigshofen, Germany). The cell density was usually adjusted to 1 107/ml resulting in 1 106 cells per staining variant (tube number 1–5, Table 1). Mono- and polymorphonuclear cells derived from affected and non-affected brain hemispheres were stained for flow cytometric analyses according to

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standard protocols: Briefly, cells were washed twice in 2-ml HBSS/FCS (400g, 10 min, 4 °C) followed by incubation with irrelevant murine IgG antibodies (40-lg/ ml mouse anti-human VEGF) for 20 min in order to block unspecific Fc binding. Afterward, several combinations of fluorochromated monoclonal antibodies were added to the cells and incubated for 45 min at 4 °C in the dark, washed twice again, re-suspended in 250-ll HBSS/FCS containing 1% formalin and analyzed using a conventional multi-colour flow cytometer (Navios, Beckman Coulter, Krefeld, Germany) with associated acquisition and analysis software (Kaluza, Beckman Coulter). All antibodies used for flow cytometry and their sources are listed in Table 2.

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Cell sorting and morphological characterization of separated cell populations

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Cell populations identified by flow cytometric analysis were separated by fluorescence-activated cell sorting using the high-speed cell sorter MoFlo XDP (Beckman Coulter) with the same monoclonal antibodies (Table 2) and the same gating strategy as defined by flow cytometry. Following separation, cytospin smears were prepared from the cell populations using a conventional cytocentrifuge (Cytospin 4, Thermo Scientific Shandon, Dreieich, Germany), which were then stained according to the method of Pappenheim.

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Multi-colour immunofluorescence in-situ staining, microscopy and imaging

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Brains from randomly selected animals were removed from the skulls and subsequently post-fixed with PFA overnight. Next, 30-lm-thick frontal forebrain sections were cut using a Vibratome (Leica, Wetzlar, Germany) and collected in 0.1 M Tris-buffered saline, pH 7.4 (TBS) containing sodium azide. Prior all staining procedures, free-floating sections were extensively rinsed with TBS and blocked with 5% normal donkey serum in TBS containing 0.3% Triton X-100 (NDS-TBS-T) for 1 h. To gain an overview of the whole ischemia-affected region, several sections were applied to immunolabeling of the ionized calcium binding adapter molecule-1 (Iba), known to be a highly sensitive marker of ischemia-related microglial alterations (Ito et al., 2001): Sections were incubated with rabbit-anti-Iba (Wako, Neuss, Germany; 1:400 in NDS-TBS-T) overnight, rinsed with TBS and then reacted with carbocyanine (Cy)3-conjugated donkey-antirabbit IgG (Dianova, Hamburg, Germany; 20-lg/ml TBS containing 2% bovine serum albumin = TBS-BSA) for 1 h. In addition, FITC-albumin in vivo labeling was combined with double immunofluorescence staining as listed in Table 3. After incubation with primary antibodies overnight, sections were rinsed with TBS followed by visualizing biotinylated immunoreagents with Cy3-streptavidin and detection of Iba with Cy5-donkey-anti-rabbit IgG, both from Dianova and used at 20-lg/ml TBS-BSA for 1 h. Control experiments were performed including the omission of primary antibodies, which resulted in the absence of cellular staining.

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Table 1. Marker combinations used for flow cytometry Fluorochrome

Pacific Blue (PB)

Phycoerythrin (PE) cyanin (APC)

Tube Tube Tube Tube Tube

CD11b CD11b CD11b

RT1b

#1 #2 #3 #4 #5

CD3 CD3 CD45RC

Allophyco-(AF647)

AlexaFluor 647 (APC-Cy7)

APC-Cyanin7

CD11c CD86

CD45 CD45 CD4 CD4 CD4

CD8 CD161 CD8

Table 2. List of antibodies and sources used for flow cytometry Antibody specificity/fluorochrome (clone)

Concentration

Supplier

Mouse anti-rat CD11b/c/APC (OX-42)

0.2 mg/ml

Mouse anti-rat CD11b/Pacific Blue (OX-42)

0.05 mg/ml

Mouse anti-rat CD11b/PE (WT.5)

0.2 mg/ml

Mouse Mouse Mouse Mouse

0.05 mg/ml 0.5 mg/ml 0.5 mg/ml 0.2 mg/ml

BioLegend London, UK AbD Serotec, Du¨sseldorf, Germany BD Pharmingen Heidelberg, Germany AbD Serotec BioLegend, BioLegend eBioscience, Frankfurt, Germany BioLegend BioLegend BioLegend BioLegend BioLegend BioLegend BioLegend BioLegend BioLegend BioLegend BioLegend Beckman Coulter BioLegend BioLegend eBioscience BioLegend BioLegend AbD Serotec Wako Chemicals, Neuss, Germany

anti-rat anti-rat anti-rat anti-rat

CD11c/AlexaFluor647 (8A2) CD161AlexaFluor647 (10/78) CD3/FITC (1F4) CD3/PE (G4.18)

Mouse anti-rat CD45/APC/Cy7 (OX-1) Mouse anti-rat CD45/FITC (OX-1) Mouse anti-rat CD45RC/PE (OX-22) Mouse anti-rat CD4/APC/Cy7 (W3/25) Mouse anti-rat CD4/PE (OX-35) Mouse anti-rat CD86/AlexaFluor647 (24F) Mouse anti-rat CD8a/APC (G28) Mouse IgG1 isotype/AlexaFluor647 (MOPC-21) Mouse IgG1 isotype/FITC (MOPC-21) Mouse IgG1 isotype/PE (MOPC-21) Mouse IgG2a isotype/AlexaFluor647 (MOPC-173) Mouse IgG2a isotype/APC (7T4–1F5) Mouse IgG2a isotype/PE (MOPC-173) Mouse IgM isotype/FITC (MM-30) Mouse anti-rat macrophage/PE (HIS36) Mouse anti-rat RT1b/FITC (OX-6) Mouse anti-rat RT1b/PE (OX-6) Mouse anti-rat CD11b/Biotin (OX-42) Rabbit anti-rat Iba1 (polyclonal)

0.2 mg/ml 0.5 mg/ml 0.2 mg/ml 0.2 mg/ml 0.2 mg/ml 0.5 mg/ml 0.2 mg/ml 0.5 mg/ml 0.5 mg/ml 0.5 mg/ml 0.2 mg/ml 0.5 mg/ml 0.2 mg/ml 0.5 mg/ml 0.2 mg/ml 0.5 mg/ml 0.2 mg/ml 0.1 mg/ml 0.05 mg/ml

Table 3. List of antibodies and sources used for immunofluorescence in situ staining. Double fluorescence staining of immune cells after in vivo prelabeling with FITC-albumin to allow co-localization with areas of impaired blood–brain barrier integrity. Fluorochrome-labeled secondary antibodies and Cy3-streptavidin were obtained from Dianova (Hamburg, Germany) and used at 20 lg/ml First primary antibodies

First visualizing immunoreagent

Second primary antibodies

Second visualizing immunoreagent

Biotinylated mouse-anti-RT1b (1:25; BD Pharmingen) Biotinylated mouse-anti-CD11b (1:40; AbD, Serotec Biotinylated mouse-anti-CD11b (1:40; AbD Serotec) Biotinylated mouse-anti-CD45RC (1:10; AbD Serotec)

Cy3-streptavidin

Rabbit-anti-Iba (1:200; Wako)

Cy5-donkey-anti-rabbit IgG

Cy3-streptavidin

AlexaFluor647-mouse-anti-CD11c (1:50; AbD Serotec)

–

Cy3-streptavidin

AlexaFluor647-mouse-anti-CD161 (1:40; BioLegend)

–

Cy3-streptavidin

AlexaFluor647-mouse-anti-CD11c (1:10; AbD Serotec)

–


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After all staining procedures, the sections were washed with TBS, briefly rinsed with distilled water, mounted onto slides, air-dried and coverslipped with Entellan (in toluene; Merck, Darmstadt, Germany). An Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany) served for screening microscopy, while for photo documentation an Olympus BX51 fluorescence microscope equipped with an XM10 digital camera (Olympus, Hamburg, Germany) as well as the laser-scanning microscope LSM 510 Meta (Zeiss) was used. Original images were processed with CorelDraw/Photo-Paint version 12.0 (Corel Corp., Ottawa, Canada). Brightness, contrast and sharpness of some final pictures were slightly adjusted.

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Statistical analyses

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Statistical calculations were performed with the SigmaStat software package (version 18.0, Sysstat, Erkrath, Germany). Thereby, the Mann–Whitney U test, the Wilcoxon test or – if normal distribution has been confirmed by the Shapiro–Wilk test – Student’s t-test served for testing inter-group differences. Generally, a p < 0.05 was considered as statistically significant. Data are expressed as mean ± standard deviation (SD) or as median values in the context of the 25/75 percentiles unless otherwise indicated.

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RESULTS

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The applied thromboembolic stroke model led to neurobehavioral alterations as evaluated by a mean Menzies score of 2.7 ± 0.7 at 4 h as well as 2.8 ± 0.9 at 24 h after ischemia induction (p = 0.602 between the time points). Therefore, animals fulfilled the pre-defined study inclusion criterion of a sufficient neurobehavioral deficit indicating right-hemispheric focal cerebral ischemia in the middle cerebral artery territory as a pre-condition for the intended cellular characterization at day 1.

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Overall cellular characterization

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Flow cytometry revealed an approximately 1.5-fold numerical increase of cells detectable in the ischemiaaffected hemisphere when compared to the contralateral side (means, 2.73 105 ± 2.25 versus 1.81 105 ± 1.61). For further characterization of accumulating immune cell populations, a model-specific gating strategy was established (Fig. 1): As a first step, single cells were discriminated from cell aggregates by plotting the forward scatter (FSC) against the FSC time-of-flight (TOF) signal, followed by gating on living cells in the FSC/side scatter (SSC) dot plot in order to exclude dead cells and debris. Leukocytes were successfully discriminated from other cells via CD45 expression in the CD11b-PacificBlue/CD45-APC/Cy7 dot plot. Based on the results from back-gating to the FSC/SSC dot plot and supported by morphological analyses of separated cell fractions in Pappenheim-stained cytospin smears, CD45+ subpopulations were classified as follows: CD11bhigh/CD45high designated as neutrophils,

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CD11bmedium/CD45high as monocytes/macrophages, CD11bmedium/CD45low as microglia, CD11b-/CD45high as lymphocytes 1, and CD11blow/CD45high were denoted as lymphocytes 2. Based on this classification, invading inflammatory cell populations were quantified and functionally characterized, while phenotypes of both lymphoid cell fractions (lymphocytes 1 and 2) could be further characterized in natural killer (NK) cells (CD161+), NKT cells (CD161+/CD3+), T cells (CD3+, CD4+ and CD8a+) or B cells (CD45RC+).

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Inflammatory cell recruitment associated with focal cerebral ischemia

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In ischemic hemispheres, neutrophils, monocytes/ macrophages and lymphocytes 2 were found to increase significantly concerning both the proportion of living cells (Fig. 2a) and the absolute cell numbers (Fig. 2b), while lymphocytes 1 only increased numerically. Unexpectedly, microglial cells did not show a significant inter-hemispheric difference, although the absolute number of cells tended to be increased in the ischemia-affected hemisphere (median, 5965 versus 5573 cells). When focusing on the overall amount of cellular recruitment best captured by the absolute cell number within the ischemia-affected hemisphere dominant populations were neutrophils and monocytes/ macrophages with a 6.12-fold (median, 9577 versus 1565 cells) and 5.42-fold (median, 7486 versus 1382 cells) increase when compared to the non-affected hemisphere (Fig. 2b). Notably, the applied thromboembolic stroke model allowed the detection of innate lymphoid cells exhibiting a NK or NKT cell phenotype. Typically, rat NK cells can be identified by fluorescence staining of the cell surface marker CD161, whereas NKT cells can be identified as CD161 and CD3 co-expressing cells. This staining pattern revealed three distinct cell populations in affected brain hemispheres of rats that underwent focal cerebral ischemia: a CD161+/CD3+ population representing NKT cells and 2 NK cell subpopulations, one defined as CD161high/CD3- and the other as CD161low/CD3- (Fig. 3). As indicated by back gating to the FSC/SSC dot plot, both the CD161low/CD3 and the CD161+/CD3+ population showed a relatively large size, whereas the CD161high/CD3 subpopulation was characterized by a much smaller size (Fig. 3, upper panel). Further analyses of lymphoid cells revealed a 2.48fold increase of CD3+ conventional T cells (median, 6187 versus 2497) as well as a 2.94-fold increase of the subpopulation CD3+/CD4+ (median, 4560 versus 1552) and a 2.18-fold increase of the subpopulation CD3+/ CD8+ (median, 1499 versus 688) in the ischemiaaffected hemisphere when compared to the control side: However, statistical significance was not achieved, except for the population co-expressing CD8+ (Fig. 4). In addition to conventional T cells, the applied gating strategy yielded an unconventional CD11b+/CD3+ T cell population which – to its major proportion – did neither belong to the CD4+ nor to the CD8+ T cell subpopulation. This can be concluded from the finding

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Fig. 1. Gating strategy for flow cytometric discrimination and definition/isolation by fluorescence-activated cell sorting of inflammatory cell populations from ischemia-affected rat brain hemispheres. Single, living cells were gated on the basis of their CD11b versus CD45 expression pattern using a high-quality flow cytometer (Navios, Beckman Coulter) and the respective acquisition and analysis software (Kaluza, Beckman Coulter; left panel). For further morphological characterization, defined cell populations were isolated from pooled rat brain hemispheres and immediately applied to a high-speed fluorescence-activated cell sorter instrument (MoFlo XDP, Beckman Coulter; right panel) and successively spun down onto a glass slide, fixed in methanol and stained by the May-Gru¨nwald/Giemsa staining protocol (so-called Pappenheim staining) for hematological differentiation of the harvested cells (inserted microphotographs in the middle panel). Finally, the defined and morphologically characterized inflammatory cell populations were designated as ‘neutrophils’ (CD45high/CD11bhigh), ‘monocytes/macrophages’ (CD45high/ CD11bmedium), ‘microglia’ (CD45low/CD11bmedium), ‘lymphocytes 1’ (CD45high/CD11bnegative) and ‘lymphocytes 2’ (CD45high/CD11blow).

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that only the total CD3+/CD11b+ population was found to be significantly increased in the ischemia-affected hemisphere, but neither CD4+/CD3+/CD11b+ nor CD8+/CD3+/CD11b+ subpopulations (Fig. 4). Addressing B cells by immunodetection of CD45RC, clear evidence emerged that these cells occurred in close association with ischemia. In general, the two phenotypically distinguishable B cell subpopulations CD4-/CD45RClow and CD4-/CD45RChigh were identified, designated as ‘B cells 1’ and ‘B cells 2’ (Fig. 5). Both subpopulations were found to be significantly increased in the ischemia-affected hemisphere: B cells 1 with 2283 versus 444 cells, representing a 5.14-fold increase, and B cells 2 with 1603 versus 518 cells, representing a 3.1fold increase.

Functional characterization of inflammatory cells in the ischemia-affected hemisphere

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Since microglia failed to provide significant numerical changes, emphasis was laid on their functional characterization. Using an inter-hemispheric comparison, the mean fluorescence intensity (MFI) of CD45stained microglial cells was analyzed as a correlative to the density of surface molecule expression per cell, resulting in a significant up-regulation of CD45 on microglial cells in the ischemic versus the non-ischemic side (mean, 5.31 ± 0.49 versus 4.44 ± 0.38; p = 0.003). With the intention to determine the antigen-presenting potential of microglia and invaded inflammatory cell populations, the expression of major histocompatibility

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complying with a nearly 1.5-fold decrease, respectively, indicating lower density of MHC II+ molecules on immigrated inflammatory cells (Fig. 6, lower left). In contrast, the MFI values of MHC II+ staining on microglia or lymphoid cells did not differ between ischemia- and non-affected hemispheres. Notably, microglial cells also positive for CD86 provided a 1.86fold increase of the MFI value in the ischemic versus the non-ischemic hemisphere (median, 9.7 versus 5.2), indicating a higher antigen-presenting capacity of microglia affected by ischemia (Fig. 6, lower right).

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Spatial distribution of immunoreactive cells

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To add a morphological perspective to the observed functional characteristics of microglia, indirect immunofluorescence of Iba was applied resulting in a tripartite shell-like pattern with regard to the ischemic

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Fig. 2. Interhemispheric comparison of classified cell populations 1 day after thromboembolic focal cerebral ischemia in the rat. The cell populations that have been defined and classified by means of flow cytometry (CD11b versus CD45 expression) and subsequent Pappenheim staining, were quantified by flow cytometric analysis and demonstrated as median values of the proportional distribution (A) or the absolute cell numbers (B) in an inter-hemispheric comparison following thromboembolic stroke in rats. Thereby, neutrophils and monocytes/macrophages displayed the most drastic increase due to ischemia, which became evident both by the proportional distribution and the absolute cell numbers. Although the proportions as well as the absolute cell counts of both lymphocytic populations (lymphocytes 1 and 2) increased after ischemia, statistical significance was only found for lymphocytes 2. However, neither the proportion nor the absolute cell numbers of microglia were significantly different between the ischemia-affected and the non-affected hemisphere. Data are given as median values with added 25/75 percentiles originating from 8 animals. Level of statistical significance: ⁄p < 0.05; ⁄⁄ p < 0.01; ⁄⁄⁄p < 0.001.

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complex (MHC) class II molecules (detected by antiRT1b) or CD86 was analyzed (Fig. 6). The proportions of living cells as well as the absolute cell counts of MHC II+ and CD86+ neutrophils (median absolute cell count MHC II+, 2723 versus 599), monocytes/ macrophages (median, 3921 versus 873 cells) or lymphocytes 1 (median, 3989 versus 1237 cells), and in terms of absolute cell counts also lymphocytes 2 (median, 1813 versus 782 cells) were found to increase significantly in the ischemia-affected versus non-affected hemisphere (Fig. 6, upper panel). However, ischemia led to a significantly lower MFI value of the RT1brelated signal of neutrophils (median, 218 versus 323) and monocytes/macrophages (median, 279 versus 422)

Fig. 3. Gating strategy for identification and quantification of NK cells and NKT cells in the rat brain 1 day after thromboembolic stroke. Rat NK cells and NKT cells were fluorescently labeled using monoclonal antibodies directed against CD161 (clone 10/78) or CD161/CD3, respectively. Three distinct cell populations could be identified in hemispheres from rats after thromboembolic stroke: the CD161+/ CD3+ population representing NKT cells and 2 NK cell subpopulations defined as CD161high/CD3- and CD161low/CD3-, respectively. These 3 subpopulations were back-gated to the FSC/SSC dot plot in order to reveal their size and granularity (upper panel). The resulting box plot graph demonstrates the absolute cell numbers of the 3 NK/ NKT cell subpopulations and additionally the absolute cell number of T cells (lower panel). Data are given as the median values of absolute cell numbers added by the 25/75 percentiles originating from 8 animals. Level of statistical significance: ⁄p < 0.05.


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Fig. 4. Gating strategy for identification and quantification of distinct T cell subpopulations in the rat brain 1 day after thromboembolic stroke. T cells were fluorescence-stained using 2 different monoclonal antibodies directed against CD3 (clones 1F4 and G4.18). CD4+ and CD8+ T-cell subpopulations were identified by 2 different monoclonal antibodies directed against rat CD4 (clones W3/25, OX35) or a monoclonal antibody recognizing rat CD8a (clone G28), respectively. The simultaneous application of anti-CD3 (clone G4.18) and anti-CD11b (clone OX-42) revealed a special CD11b+/CD3+ T-cell subpopulation apart from classical CD11b-/CD3+ T cells. This subpopulation (obviously not expressing CD4 or CD8 molecules) was found to be significantly increased in the ischemia-affected hemisphere as compared to the contralateral (unaffected) hemisphere (left panel). However, the numbers of the CD11b+/CD3+ cells expressing CD4 or CD8 were comparable in ischemia-affected and non-affected hemispheres (middle and right panel). Although the classical T cell population (CD11b-/CD3+) appeared increased in the ischemic hemisphere too, this difference was not significant (left panel). Additional immunolabeling of CD4 and CD8 revealed an increase of both classical T-cell subpopulations, which was significant only in the case of CD8 T cells (middle panel). Data are given as box plots showing the median values of absolute cell numbers, added by the 25/75 percentiles originating from 8 animals. Level of statistical significance: ⁄p < 0.05.

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lesion. While the area exhibiting the most ischemiaassociated tissue damage was found to be nearly devoid of microglia, an ameboid configuration coincidental with a reduced length of cellular processes was detected in the ischemic border zone (Fig. 7). Beyond this zone, microglia appeared as ramified cells without signs of activation. Further, multiple immunofluorescence labeling was performed to provide details on the spatial pattern of myeloid and lymphoid subpopulations with reference to the ischemic area. Thereby, brain sections were screened for extravasation of FITC-album from cerebral vessels into the brain’s parenchyma indicating disruption of the blood–brain barrier due to ischemia. In addition to parenchymal leakage, FITC-albumin was found in association with cellular structures: Counterstaining of Iba and CD11b revealed a clear regional association to myeloid cells, predominantly without exact co-localization (Fig. 8A–A000 ). When focusing on the

Fig. 5. Gating strategy for identification and quantification of B cell subpopulations in the rat brain 1 day after experimental focal ischemia. Rat B cells were fluorescently labeled with a monoclonal antibody directed against CD45RC (clone OX-22). Two distinct B cell subpopulations could be identified in the hemispheres of rats after thromboembolic stroke: CD45RClow cells (designated as ‘B cells 1’) and the CD45RChigh population (designated as ‘B cells 2’; A). These subpopulations were found to be significantly increased in the ischemia-affected hemisphere compared to the non-affected hemisphere (B, C). Although the median values of both subpopulations are at the same level in both hemispheres, in some animals a higher infiltration of B cells 1 compared to B cells 2 could be observed as indicated by the higher 75 percentile in the box plots for relative as well as absolute cell counts (B, C). Data are given as median values with added 25/75 percentiles originating from 8 animals. Level of statistical significance: ⁄p < 0.05.

regional relationship to the vasculature, RT1b as marker for the antigen-presenting immunoreactive cells was found to cumulate in close association with vessels,


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Fig. 6. Antigen-presenting and co-stimulating capacity of identified inflammatory cell populations in separated hemispheres of rats 1 day after thromboembolic stroke. Using flow cytometry, antigen-presenting capacity was derived from the distribution (upper left), the absolute cell numbers (upper right), or the mean fluorescence intensity (MFI) values (lower left) of MHC class II molecule expression (stained by anti-RT1b, clone OX-6), while co-stimulating capacity of inflammatory cells was indicated by the MFI values of CD86 expression (stained by anti-rat CD86, clone 24F; lower right). The proportion and the absolute count of cells expressing MHC-class II molecules were significantly increased in the ischemic brain hemispheres in terms of neutrophils, monocytes/macrophages and lymphocytes 1. In response to ischemia, the absolute number but not the proportion of cells expressing MHC class II molecules was significantly increased also in the population designated as lymphocytes 2. The amount of MHC class II molecules per cell (indicated by MFI value) was not affected by ischemia in microglial cells and both lymphocytic populations. Neutrophils and monocytes/macrophages displayed significantly lower MFI values of MHC class II molecule expression in ischemia-affected hemispheres. A significant increase of CD86 molecules per cell (MFI) was only found in microglial cells. Data are given as median values added by the 25/75 percentiles originating from 8 animals. Level of statistical significance: ⁄p < 0.05; ⁄⁄p < 0.01.

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whereas microglia as visualized by Iba-immunoreactivity provided a homogenous allocation in the perivascular zone (Fig. 8B). Additional analyses at higher magnifications indicate that antigen-presenting immunoreactive cells (identified by anti-RT1b) are located in vessels with decreased blood–brain barrier integrity (demonstrated by FITC-albumin leakage) and in the parenchyma in close vicinity to these vessels (Fig. 8C). This finding strongly indicates a predominantly vessel-associated route of inflammatory cell recruitment into the ischemia-affected tissue. Additional experiments focused on the regional association between extravasated FITC-albumin (either if associated with cellular structures or the vasculature per se) and markers addressing the subpopulations of NK/NKT cells (CD161), T/B cells (CD45RC) and dendritic cells (CD11c), while the latter one is considered to represent subpopulations of both monocytes and lymphocytes (Fig. 9). Thereby, CD11b, representing predominantly myeloid cells, was found to be regularly co-localized with CD11c (Figure 9A0 –A000 ), while CD161+ cells provided a much lower overlap with

CD11b (Figure 9B000 –B000 ). Remarkably, a partial overlap of CD45RC and CD11c was observed in close relationship to ischemia-affected vessels (Figure 9C000 , colour-coded in purple), whereas dendritic cells clearly dominated the intravascular compartment and lymphoid (B/T) cells the juxta-/perivascular space.

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DISCUSSION

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The present study aimed on the cellular inflammatory response during the first day after cerebral ischemia using a rat model of thromboembolic stroke, which has been chosen to closely mimic the human pathophysiology (Durukan and Tatlisumak, 2007; Young et al., 2007). With respect to earlier studies which applied flow cytometry for characterization of invading cells into the ischemic brain as induced by filament insertion or electrocoagulation (Campanella et al., 2002; Stevens et al., 2002; Gelderblom et al., 2009), this is – to the best of our knowledge – the first work on consequences of experimental stroke caused by a blood clot-mediated cerebral vessel occlusion.

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Fig. 7. Morphological changes of microglial cells in a coronal forebrain section from a rat 1 day after thromboembolic stroke. Indirect immunofluorescence labeling of Iba allows the discrimination between unaffected striatal tissues containing ramified microglia from the infarct border zone displaying activated, more ameboid microglial cells with shortened processes whereas the area of maximum tissue damage is apparently devoid of Iba-immunoreactivity. The delineation of the different zones is clarified by dashed lines. Scale bar: 200 lm.

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Technical considerations

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A major challenge of the performed approach was the limited availability of monoclonal antibodies to rat cell surface molecules. Therefore, this study required a variety of antibody combinations to allow identification of different cell populations, naturally not purporting to be complete. Thus, future studies are requested to use the here found specifications in addition to newly elaborated antibodies in order to explore cellular differentiation in more detail. Our concept for identifying leukocyte and microglial populations predominantly based on the staining patterns for CD11b and CD45 as suggested by Sedgwick et al. (1991), who demonstrated discrimination of microglia from monocytes/macrophages/neutrophils and lymphocytes. This strategy was confirmed by Ford et al. (1995), who identified CD45low/CD11bhigh expressing cells as microglia and CD45high/CD11bhigh expressing cells as myeloid cells. However, after cytometry-based separation and histological characterization by Pappenheim staining, we showed that in our model CD45low/ CD11bmedium expressing cells represent microglia. Additionally, neutrophils and monocytes/macrophages (both expressing high amounts of CD11b) were discriminated by their MFI, while neutrophils as identified by a typical morphology visualized by the Pappenheim staining provided higher MFI values. Therefore, we designated this population (CD45high/CD11bhigh) as neutrophils and the other cell population with a somewhat lower MFI value (CD45high/CD11bmedium, similar MFI level as microglia) as monocytes/macrophages. Since the CD45/CD11b profile also allowed the differentiation of lymphocytes in terms of a CD45high/CD11b and a CD45high/CD11blow

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population, we decided to differentiate between lymphocytes 1 and 2. Based on their morphological appearance, a few of the lymphocytes in the CD11blow group were characterized by a small nucleus/plasma ratio and some granularity, while the majority exhibited a typical morphology – i.e. big nucleus/plasma ratio and highdense nucleus. Therefore, some cells of the lymphocytes 2 population might represent monocytes/macrophages or even specific subtypes of lymphocytes (e.g. NK or other innate cells), which need to be investigated by further studies using multiple labeling with antibodies directed against CD45, CD11b, CD45RC, CD3 and CD161. As a further limitation of this study, observations were restricted to a single time point, in detail 25 h after ischemia induction. However, in this first approach of characterizing the inflammatory response in the thromboembolic model the authors focused on tissues with constantly initiated inflammatory mechanisms occurring within hours or days after ischemia (Dirnagl, 1999). Consequently, future studies are required to add data on the time-dependency of cellular reactions described here.

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Changes in microglia

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Microglial alterations in response to ischemic stroke have been described comprehensively (Sedgwick et al., 1991; Aloisi, 2001; Campanella et al., 2002; Stevens et al., 2002; Lalancette-Hebert et al., 2007; Wang et al., 2007; Hu et al., 2012). Thereby, previous studies provided evidence that the up-regulation of CD45 represents a sensitive signal for the activated state of microglial cells in conjunction with inflammatory processes (Campanella et al., 2002; Stevens et al., 2002). A conclusive observation in the present work is based on inter-hemispheric comparisons: the CD45 expression was found to be upregulated on microglia cells in the ischemic hemisphere. This finding indicates comparable mechanisms of microglial activation also in the thromboembolic rat model of stroke. Remarkably, in models with transient cerebral ischemia the number of microglial cells and the extent of tissue damage concomitantly increased (Denker et al., 2007; Gelderblom et al., 2009), except for a report by Denes et al. (2007) who found an inverse association after 30 min of transient focal cerebral ischemia in mice. Thus, the quite low and non-significant numerical increase of microglial cells in the ischemia-affected hemisphere observed in the present study might be related to the used thromboembolic model, typically devoid of a controlled recanalization, thereby leading to more severe tissue damage than in transient models. Although the number of microglial cells was not significantly changed after ischemia, in addition to CD45 levels also the degree of CD86 expression per cell was significantly increased in the ischemic hemisphere suggesting an activated state, which was supported by the observed morphological alterations. Although previous studies reported morphological changes toward an ameboid appearance in the ischemic border zone (Walberer et al., 2010), future efforts are requested to explore the functional relevance of this observation, affirmed by a recent study from Hu

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Fig. 8. Concomitant fluorescence labeling of FITC-albumin and different markers of immune cells in ischemia-affected rat brain tissue 1 day after experimentally induced stroke, captured by confocal laser-scanning microscopy. FITC-albumin (FITC-alb.) as leakage marker indicating an impaired blood–brain barrier integrity – found to be concentrated in cells of the striatal parenchyma (A) – is counterstained in (A0 ) with indirect Cy5immunolabeling of Iba (colour-coded in blue) and in (A000 with CD11b-immunostaining based on a biotinylated mouse antibody and red fluorescent Cy3-streptavidin. The merged picture (A00 0 ) clearly shows the occurrence of stained markers in regionally allocated, but typically not the same cells. (B) The simultaneous visualization of FITC-alb., Iba-immunoreactivity (blue) and RT1b, a marker for MHC class II molecules (revealed by a biotinylated mouse antibody and Cy3-streptavidin, red) indicates the cellular distribution associated to the vasculature within the striatal border zone of ischemia (C). Scale bars: 50 lm (in A000 and B) and 100 lm (in C). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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et al. (2012) who demonstrated a shift in the cellular polarization of microglia after transient experimental stroke in mice that indicates different phenotypes of activated microglia. In this context, concomitant morphological alterations need to be matched with the currently discussed pro- and/or anti-inflammatory properties of microglia (Biber et al., 2014).

Infiltration of monocytes/macrophages and neutrophils

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The limited proliferation of microglial cells in contrast to the massive recruitment of monocytes/macrophages and neutrophils into ischemia-affected brain is one of the major findings of the present work. This observation

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Fig. 9. Triple fluorescence labeling of FITC-albumin as leakage marker for ischemia-induced blood–brain barrier disruption and its co-localization with diverse markers of immunoreactive cells in the rat brain 1 day after thromboembolic stroke. CD11b-immunoreactivity revealed by biotinylated mouse antibodies and red fluorescent Cy3-streptavidin (A0 and B0 ) was combined with the immunodecoration of CD11c (A00 ) and CD161 (B00 ) based on mouse antibodies conjugated to Alexa647 (infrared immunosignal, colour-coded in blue). CD45RC also visualized by biotinylated mouse antibodies and Cy3-streptavidin (C0 ) was stained simultaneously with CD11c, visualized by Alexa647 (C00 ). The merged pictures A000 and B000 exclusively indicate co-expression of markers for immunoreactive cells (most prominent structures are marked by arrows), added by insets showing selected cells at higher magnifications while co-localization of CD11b and CD11c as well as CD11b and CD161 appeared in purple. The merged picture C000 reveals predominantly in the marked vessel zone cellular elements containing both FITC-alb. and CD45RC (yellow), as well as coexpression of CD45RC and CD11c (purple), and a few structures with all 3 markers (white). Scale bars = 25 lm (in A000 and B000 ) and 50 lm (in C000 ). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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appears impressively robust, because earlier studies based on different techniques of ischemia induction revealed comparable results (Campanella et al., 2002; Stevens et al., 2002; Schilling et al., 2003; Tanaka et al., 2003; Kokovay et al., 2006; Gelderblom et al., 2009). Remarkably, monocytes/macrophages recruited to the ischemic brain were found to express MHC class II molecules indicating an activated phenotype, which leads to the hypothesis that these cells may play a significant role in antigen presentation and T-cell

activation. Moreover, invaded monocytes/macrophages were considered to directly trigger the local inflammation by secreting pro-inflammatory cytokines such as IL-1b, TNF-a and IL-23 (Fu et al., 2004; Liesz et al., 2009; Shichita et al., 2009). Concerning the mechanisms of monocyte accumulation, earlier studies indicated a critical role for the CC-chemokine monocyte-chemoattractant protein-1 (MCP-1). Accordingly, MCP-1 – not detectable in the unaffected brain – was found in the cerebrospinal fluid of stroke patients (Losy and Zaremba, 2001), while animal data suggested that this chemokine contributes


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to the progression of brain damage by recruitment of monocytes/macrophages (Hughes et al., 2002). Consequently, MCP-1/ mice exposed to focal cerebral ischemia exhibited smaller lesion sizes, decreased macrophage invasion as well as IL-1 b, IL-6 and G-CSF secretion (Hughes et al., 2002; Strecker et al., 2011). In addition to MCP-1, the CC-chemokine macrophage inflammatory protein-3a (MIP-3a) was identified by Terao et al. (2009) as a further regulating protein involved in monocyte/macrophage recruitment after experimental ischemic stroke in rats. In this context, flow cytometrybased quantification of monocytes/ macrophages in the thromboembolic stroke model might represent a useful way, first, to confirm the impact of regulatory proteins like MCP-1 or MIP-3a in this human-like model, and second, to explore therapeutic strategies dealing with drug-based modulations of these proteins. Infiltration of neutrophils into the infarcted brain was demonstrated in several studies as for instance by Garcia et al. (1994), who used a permanent model of focal cerebral ischemia in the rat, and found accumulation of neutrophils already 30 min after ischemia induction. Recently, neutrophils became interesting since both animal and human studies of cerebral ischemia indicated a close relationship between blood–brain barrier breakdown and matrix metalloproteinases (i.e. MMP-9), a group of enzymes predominantly secreted by neutrophils (Justicia et al., 2003; Rosell et al., 2008). Further studies confirmed this observation by adding a significant correlation between the degree of neutrophil accumulation and the ischemic lesion size (Price et al., 2004). Thus, MMP-9 expression together with the capacity to produce reactive oxygen species mainly implies an exacerbating rather than a protective role of neutrophils during the inflammatory response after ischemia. In our translationoriented study the thromboembolic stroke model in the rat resulted in a striking invasion of neutrophils into the ischemia-affected brain within the first 25 h. These data are in line with previous observations in ischemic stroke models using (electro)coagulation or filament-based vessel occlusion (Stevens et al., 2002; Gelderblom et al., 2009; Zhou et al., 2013; Mo¨ller et al., 2014). A strong indication for the activated state of invaded neutrophils in our model was their MHC class II expression. Although neutrophils are typically devoid of MHC class II molecules under physiological conditions, certain pathological processes including the accumulation of cytokines like IFNc, IL-3 or GM-CSF lead to its expression (Smith et al., 1995; Wright et al., 2010; Abi Abdallah et al., 2011). Based on the assumed complex interactions, also a role for neutrophils as antigen-presenting cells with consecutive T-cell activation appears obvious. This perspective is supported by immunofluorescence labeling shown in this work, since RT1b+ cells were closely associated with the ischemic area, especially in vicinity to the vasculature with impaired blood–brain barrier integrity. The recruitment of neutrophils from the blood seems to be predominantly induced by the CXC chemokine cytokine-induced neutrophil chemoattractant protein-1/chemokine (C–X–C motif) ligand 1 (CINC-1/CXCL1) since this chemokine was found to be quickly up-regulated in response to

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ischemic stroke (Barone et al., 1991; Liu et al., 1993; Garcia et al., 1994; Denker et al., 2007; Yamagami et al., 1999). While focusing on the infiltration process from the vasculature into the brain’s parenchyma, a variety of involved adhesion molecules – e.g., the intercellular adhesion molecule-1 (ICAM-1) and the vascular adhesion molecule-1 (VCAM-1) – had been identified (Danton and Dietrich, 2003; Wang et al., 2007; Denes et al., 2010). It is noteworthy that our data on the thromboembolic model of stroke differ in some aspects from the findings reported for other models. Gelderblom et al. (2009) have shown that infiltration of the ischemic hemisphere by macrophages, lymphocytes and DCs preceded the neutrophil influx in mice suffering from a transient (1 h) focal cerebral ischemia. In contrast, we were able to demonstrate a strong and apparently simultaneous recruitment of neutrophils together with monocytes/macrophages and lymphocytes within 25 h after ischemia induction. Moreover, Stevens et al. (2002) could show a 2.5-fold increase of neutrophils in the ischemic versus the non-ischemic hemisphere after 24 h in a mouse model of transient (1 h) focal cerebral ischemia, which peaked after 72 h to a 7.5-fold increase. The present study revealed a drastic sixfold accumulation of neutrophils already at 25 h, which is in line with the results from Mo¨ller et al. (2014) who performed a permanent distal occlusion of the middle cerebral artery in spontaneous hypertensive rats. These discrepancies might be attributed to the degree of tissue damage caused by transient as well as permanent cerebral ischemia, probably accompanied by species-related peculiarities. However, a recent study by Zhou et al. (2013) investigated the recruitment of neutrophils after 30 and 90 min of transient versus permanent focal cerebral ischemia in mice. Thereby, transient ischemia caused more invading neutrophils within the first day than permanent ischemia. When considering histological data from human stroke to verify the findings from animal studies, Chuaqui and Tapia (1993) described the occurrence of neutrophils starting already at day one after the ischemic event, which could be recently confirmed by Enzmann et al. (2013). Regarding translational issues, flow cytometry-based quantification of neutrophils in the rat model of thromboembolic stroke might therefore represent a useful setting to investigate the related pathophysiology in more detail, and to test potential treatment strategies influencing the degree of neutrophil influx. Remarkably, all three cellular populations (neutrophils, monocytes/macrophages and microglia) revealed only low levels of CD11c in flow cytometric analyses (data not shown). Thus, it was not possible to clearly identify dendritic cells by the applied gating strategy, but immunofluorescence labeling identified CD11c-expressing cells allocated with the ischemic area, suggesting the recruitment and/or differentiation of dendritic cells also in the rat model of thromboembolic stroke. This finding is in agreement with previous observations: Kostulas et al. (2001) reported the pres- Q5 ence of dendritic cells in ischemic brain hemispheres after filament-based permanent focal cerebral ischemia in rats, and Mo¨ller et al. (2014) demonstrated a transient increase of dendritic cells in the ischemia-affected hemisphere at


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one day after thermocoagulation-induced occlusion of the middle cerebral artery in rats. However, the functional role of dendritic cells in stroke-related cerebral inflammation is still a matter of debate.

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Infiltration of lymphocytes, NK cells and NKT cells

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In our model, a significant infiltration of lymphocytes as well as cells with lymphocyte-like morphology (i.e. the populations designated as lymphocytes 1 and lymphocytes 2) into the ischemia-affected hemisphere was observed at 25 h after stroke induction. Immunophenotyping revealed that these cell populations are predominantly T cells, B cells, NK cells and NKT cells. This finding is in agreement with observations in other stroke models (Jander et al., 1995; Campanella et al., 2002; Stevens et al., 2002; Gelderblom et al., 2009). Although the functional role of T cells in strokeinduced inflammation remains subject of further investigation, there is increasing evidence for a diseasepromoting role of lymphocytes in stroke, since mice devoid of functional T and B cells developed significantly smaller infarct sizes and neurological deficits than wild-type mice (Kleinschnitz et al., 2010). Adoptive transfer experiments using purified B and T cells suggest that T cells are the most critical lymphocyte subpopulation exacerbating cerebral damage after stroke (Kleinschnitz et al., 2010; Yilmaz et al., 2010). Additionally, Shichita et al. (2009) have shown that c d T cells, a primordial T cell population which belong to innate lymphoid cells like NKT cells as well, might significantly trigger the cerebral inflammation following stroke by secreting IL-17 in an IL-23-dependent manner (as demonstrated for IL-17-/- mice or by administration of a neutralizing anti-IL-17 antibody) in a transient mouse model of stroke. The pivotal role of c d T cells including related cytokines like IL-17 with specific effects on neutrophils should be verified in the thromboembolic rat model of stroke by future studies. In contrast to the disease-promoting role of c d T cells, CD4+/CD25+/Foxp3+ regulatory T cells (so-called Treg cells) were found to play a crucial role as counterregulators of cerebral inflammation after experimental stroke in mice (Chamorro et al., 2012; Magnus et al., 2012). Analyses of underlying mechanisms revealed anti-inflammatory properties of IL-10, down-regulating TNF-a and IFN-c as the probably most potent inflammatory cytokines (Liesz et al., 2009; Chamorro et al., 2012). Although the present work did not focus on Treg cells, their course and functional impact needs to be verified in the thromboembolic model of stroke in order to gain translationally relevant details. Interestingly, in our first approach to capture the lymphoid influx after thromboembolic stroke, we observed an untypical T cell subpopulation expressing CD11b, which might be a special feature of this model. Since the functional relevance of this special population remains unclear at this stage, additional analyses should focus on a regional characterization and potential co-localization with other markers. In addition to T cells, we were able to identify B cells with subsequent differentiation into two distinct

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subpopulations. Resolving the uncertainty regarding the function of B lymphocytes, a recent study delivered evidence for a protective role of regulatory B cells mediated by IL-10 (Ren et al., 2011). With respect to translational aspects, further studies have to explore whether B cell subpopulations found in our study might express a regulatory phenotype as characterized by IL-10 production. Finally, we could show that two distinct NK cell subpopulations (CD161high/CD3 and CD161low/CD3) as well as NKT cells (CD161+/CD3+) infiltrated the ischemia-affected hemisphere following thromboembolic stroke. As indicated by back-gating to the FSC/SSC dot plot, both the CD161low/CD3- and the CD161+/CD3+ population displayed a relatively large cell size, suggesting that these populations exhibited an activated phenotype. Probably one or even both of these cell populations might represent a phenotype that was previously described as large granular lymphocytes, which were later identified as NK cells (Born et al., 1983; Sheehy et al., 1983). Further immunohistochemical and flow cytometric analyses are requested to clarify whether these cells – in the setting of stroke – may secrete perforin and/or granzymes as typical features of NK cells. Future studies have to elucidate whether these cells are involved in stroke-induced inflammation, for example by the secretion of IFN-c – known to be involved in the early inflammatory response (Liesz et al., 2011; Magnus et al., 2012).

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CONCLUSIONS

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Our study provides for the first time details on the inflammatory cell recruitment within ischemia-affected brain regions after thromboembolic stroke in rats, a model that has become attractive concerning translational aspects. While microglia failed to increase numerically in the ischemia-affected hemisphere, flow cytometry indicated a clear shift to an immunoreactive state, which was supported by histochemically visualized morphological alterations toward an ameboid configuration in the ischemic border zone. These findings strengthened the relevance of microglial cells in the local inflammatory response after stroke, irrespective of numerical changes. In contrast to earlier reports, our data suggested a simultaneous influx of monocytes/macrophages and neutrophils within the first 25 h after focal cerebral ischemia, since we were able to demonstrate their occurrence after experimental thromboembolic stroke, which is in line with observations following human stroke. This similarity impressively underlines the need for further investigations addressing the inflammatory response in translationally relevant rodent models. As a confirmation and an extension of previous studies using different stroke models, the present work elucidated a significant increase of T and B cells in the ischemia-affected hemisphere, allowed discrimination between NK and NKT cells, and further revealed a newly described cell population co-expressing CD11b and CD3 as well as 2 subpopulations of B cells. The functional impact of

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these subtypes needs to be further addressed in future studies.

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CONFLICT OF INTEREST

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The authors declare that there is no conflict of interest.

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Acknowledgments—The authors would like to thank Dr. Petra Fink-Sterba and Ms. Sigrid Weisheit (Medical Experimental Centre, University of Leipzig, Leipzig) for animal care. Ms. Ulrike Scholz (Fraunhofer Institute for Cell Therapy and Immunology, Dept. Cell Engineering/GLP, Leipzig) is acknowledged for excellent technical assistance.

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(Accepted 17 August 2014) (Available online xxxx)


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