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Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice

Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers... Background: Emerging evidence indicates that, similarly to what happens for peripheral macrophages, microglia can express different phenotypes depending on microenvironmental signals. In spite of the large literature on inflammation after ischemia, information on M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is lacking. The present study investigates the presence of microglia/macrophage phenotype markers, their temporal expression, whether they are concomitantly expressed by the same subpopulation, or they are expressed at distinct phases or locations in relation to the ischemic lesion. Methods: Volume of ischemic lesion, neuronal counts and TUNEL staining were assessed in C57Bl/6 mice at 6-12-24-48 h and 7d after permanent occlusion of the middle cerebral artery. At the same time points, the expression, distribution in the lesioned area, association with a definite morphology and coexpression of the microglia/macrophage markers CD11b, CD45, CD68, Ym1, CD206 were assessed by immunostaining and confocal microscopy. Results: The results show that: 1) the ischemic lesion induces the expression of selected microglia/macrophage markers that develop over time, each with a specific pattern; 2) each marker has a given localization in the lesioned area with no apparent changes during time, with the exception of CD68 that is confined in the border zone of the lesion at early times but it greatly increases and invades the ischemic core at 7d; 3) while CD68 is expressed in both ramified and globular CD11b cells, Ym1 and CD206 are exclusively expressed by globular CD11b cells. Conclusions: These data show that the ischemic lesion is accompanied by activation of specific microglia/ macrophage phenotype that presents distinctive spatial and temporal features. These different states of microglia/ macrophages reflect the complexity of these cells and their ability to differentiate towards a multitude of phenotypes depending on the surrounding micro-environmental signals that can change over time. The data presented in this study provide a basis for understanding this complex response and for developing strategies resulting in promotion of a protective inflammatory phenotype. Keywords: Inflammation, stroke, alternative activation Background retracting their processes and they are endowed with Microglia, the major cellular contributors to post-injury the capacity to rapidly respond to injury or alterations inflammation, have the potential to act as markers of in their microenvironment [1-3]. After acute brain disease onset and progression and to contribute to neu- injury, these resident cells are rapidly activated and rological outcome of acute brain injury. They are nor- undergo dramatic morphological and phenotypic mally present in the healthy brain where they actively changes. Typical morphological changes associated with survey their surrounding parenchyma by protracting and microglia activation include thickening of ramifications and of cell bodies followed by acquisition of a rounded amoeboid shape. This intrinsic response is associated to * Correspondence: desimoni@marionegri.it recruitment of blood-born macrophages which migrate Laboratory of Inflammation and Nervous System Diseases, Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Via La into the injured brain parenchyma [4,5]. This process is Masa, 19-20156 Milan, Italy © 2011 Perego et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 2 of 19 http://www.jneuroinflammation.com/content/8/1/174 accompanied by expression of novel surface antigens Methods and production of mediators that build up and maintain Animals the inflammatory response of the brain tissue. Activated Male C57Bl/6 mice (10-week old, 20-25 g, Harlan microglia and recruited macrophages (which are antige- Laboratories, Italy) were used. Procedures involving ani- nically not distinguishable, henceforth referred to as M/ mals and their care are conducted in conformity with the M), can affect neuronal function and promote neuro- institutional guidelines (Quality Management System toxicity through the release of several harmful compo- Certificate - UNI EN ISO 9001:2008 - Reg. N° 8576-A) nents such as IL-1b,TNF-a, proteases and reactive that are in compliance with national (D. Lvo. n. 116, 27 oxygen and nitrogen species [6,7]. On the other hand Gennaio 1992; Legge n° 413, 12 Ottobre 1993; Circolare they also possess protective qualities and promote neu- No. 8, 22 Aprile 1994; D.M. 29/09/95; D.M. 26/04/2000) rogenesis and lesion repair [8-10]. Indeed, microglia and international (EEC Council Directive 86/609, OJ L have been proposed to be beneficial by several mechan- 358, 1, Dec. 12, 1987; Guide for the Care and Use of isms including glutamate uptake [11] removal of cell Laboratory Animals, U.S. National Research Council, debris [12] and production of neurotrophic factors such 1996) laws and policies. Before beginning any procedure, as IGF-1 [13], GDNF [14] and BDNF [15,16]. mice were housed for at least 1 week in their home cages Studies addressing phenotypic changes occurring in at a constant temperature, with a 12 hour light-dark macrophages in peripheral inflammation and immunity cycle, and ad libitum access to food and water in a selec- have shown that these cells can undergo different forms tive pathogen-free (SPF) vivarium. of polarized activation. One is the classic or M1 activa- tion, characterized by high capacity to present antigen, Focal cerebral ischemia high production of NO and ROS and of proinflamma- Permanent ischemia was obtained by permanent middle tory cytokines. M1 cells act as potent effectors that kill cerebral artery occlusion (pMCAO) [22,23]. Briefly, mice micro-organisms and tumor cells, drive the inflamma- were anesthetized with Equitensin (pentobarbital 39 mM, tory response and may mediate detrimental effects on chloral hydrate 256 mM, MgSO4 86 mM, ethanol 10% neural cells. The second phenotype (M2) is an alterna- v/v, propyleneglycol 39.6% v/v) 100 μl/mouse administered tive apparently beneficial activation state, more related by intraperitoneal (i.p.) injection. A vertical midline inci- to a fine tuning of inflammation, scavaging of debris, sion was made between the right orbit and tragus. The promotion of angiogenesis, tissue remodeling and repair. temporal muscle was excised, and the right MCA was Specific environmental signals are able to induce these exposed through a small burr hole in the left temporal different polarization states [17]. A similar possibility bone. The dura mater was cut with a fine needle, and the has been also recently raised for microglia, by showing MCA permanently occluded by electrocoagulation just that these cells, under certain conditions, can indeed be proximal to the origin of the olfactory branch. Intraopera- pushed to both extremes of the M1 and M2 differentia- tive rectal temperature was kept at 37.0 ± 0.5°C using a tion spectrum [16,18]. More studies are needed to sub- heating pad (LSI Letica). Mortality rate was 8.5%. Sham- stantiate these observations. operated mice received identical anesthesia and surgical In this frame the present study aims at getting insight procedure without artery occlusion. on previously unexplored aspects of microglia phenotype changes induced by cerebral ischemia, namely, the pre- Experimental design and blinding senceofspecificphenotype markers, their temporal Mice were assigned to surgery and experimental groups expression, whether or not they are concomitantly with surgery distributed equally across cages and days. To expressed by the same subpopulation, whether they are minimize the variability, all surgeries were performed by expressed at distinct phases or locations in relation to the the same investigator, blinded to the experimental groups. ischemic lesion. We focussed on a few molecules that are All subsequent histological and immunohistological eva- known to be expressed by macrophages in peripheral luations were also done by blinded investigators. inflammation and that have been associated to different functions. They include: CD11b, a marker of M/M acti- Brain transcardial perfusion vation/recruitment, CD45 expressed on all nucleated At selected time points mice were deeply anesthetized hematopoietic cells [19], CD68 a marker of active phago- with Equitensin (120 μl/mouse i.p.) and transcardially per- cytosis, Ym1 a secretory protein that binds heparin and fused with 20 ml of PBS, 0.1 mol/liter, pH 7.4, followed by heparin sulphate and CD206 a C-type lectin carbohydrate 50 ml of chilled paraformaldehyde (4%) in PBS. After care- binding protein, both of them expressed by alternatively fully removing the brains from the skull, they were trans- activated macrophages and associated to recovery and ferred to 30% sucrose in PBS at 4°C overnight for function restoration [20,21]. cryoprotection. The brains were then rapidly frozen by Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 3 of 19 http://www.jneuroinflammation.com/content/8/1/174 immersion in isopentane at - 45°C for 3 min before being CD68 quantification fields at 40× magnification were sealed into vials and stored at -70°C until use. selected at 1.54 mm anterior to bregma (11 fields), at 0.50 mm anterior to bregma (11 fields) and at 0.94 mm poster- Quantification of infarct size ior to bregma (11 fields). The first raw of fields was posi- For lesion size determination, 20-μm coronal brain cryo- tioned at the lesion edge, spacing each field by 572.5 μm sections were cut serially at 320-μm intervals and stained (distance between centres of the fields). Further raws of with Cresyl Violet [8]. On each slice, the infarcted area fields were positioned distanced by 389.3 μm each. For was assessed blindly and delineated by the relative pale- TUNEL, CD45, Ym1 and CD206 twenty-four quantifica- tion fields at 20× magnification were selected. Eight fields ness of histological staining tracing the area on a video screen. The infarcted area and the percentage of brain per slice were selected and fields were separated by 572.5 swelling for edema correction were determined by sub- μm (distance between centres of fields), while distance tracting thearea ofthehealthytissueintheipsilateral between each raw was 389.3 μm. A schematic representa- hemisphere from the area of the contralateral hemisphere tion of the regions of interest and of the selected fields is on each section [24,25]. Infarct volumes were calculated depicted in Figure 1. by the integration of infarcted areas on each brain slice as quantified with computer- assisted image analyzer and Neuronal Count calculated by Analytical Image System (Imaging Research Cresyl Violet stained brain sections were used for neuro- Inc., Brock University, St. Catharines, Ontario, Canada). nal count. Thirty-three fields at 40× were analyzed for each mouse. The amount of neuronal loss was calculated Slice selection and quantitative analysis by pooling the number of stained neurons in the three Three brain coronal sections per mouse (+1.54, +0.50 and ipsilateral sections and expressed as percentage of those - 0.94 mm from bregma, KBJ Franklin and G Paxinos, The in sham- operated animals. Fields were analyzed using Mouse Brain in Stereotaxic Coordinates, Academic Press), ImageJ software http://rsbweb.nih.gov/ij/ and segmenta- were used to quantify the stained area. On each slice, ana- tion was used to discriminate neurons from glia on the tomically defined cortical regions of interest were demar- basis of cell size. cated, corresponding to the primary motor cortex, somatosensory cortex, insular cortex (granular, agranular) TUNEL staining and secondary somatosensory cortex, representing the To assess the presence of injured cells showing DNA cortical regions involved in the largest lesion extension damage, terminal deoxynucleotidyl transferaseYmediated observed at 24 h after ischemia. Field selection was per- dUTP nick end labeling (TUNEL) staining was per- formed using a BX61 Olympus microscope equipped with formed on 20-μm sections by in situ cell death detection a motorized stage acquiring the same focal plan through- kit (Roche, Mannheim, Germany) according to the man- out the samples [26]. For neuronal count, CD11b and ufacturer instructions, as previously described [27]. Figure 1 Slices selection and tissue sampling for neuronal counts and quantification of immunostained area. Fields for neuronal counts, TUNEL evaluation and quantification of stained area were positioned within the ischemic territory at defined distances (see methods for details). The regions sampled pertained to the ischemic territory (yellow) at the time points considered (6 h, 12 h, 24 h, 48 h and 7d). Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 4 of 19 http://www.jneuroinflammation.com/content/8/1/174 DNase-treated sections were used as a positive control. performed. None of the immunofluorescence reactions After staining, the sections were visualized using fluores- revealed unspecific fluorescent signal in the negative con- cent microscopy (Olympus IX70 Olympus Tokyo, trols. Immunofluorescence was acquired using a scanning Japan). Images of the area of interest were acquired sequential mode to avoid bleed-through effects by an IX81 using AnalySIS software (Olympus, Tokyo, Japan). For microscope equipped with a confocal scan unit FV500 each mouse twenty-four fields at 20× were analyzed. with 3 laser lines: Ar-Kr (488 nm), He-Ne red (646 nm), TUNEL-positive cells were counted using ImageJ soft- and He-Ne green (532 nm) (Olympus, Tokyo, Japan) and ware http://rsbweb.nih.gov/ij/ and expressed as number aUVdiode. per mm for subsequent statistical analysis [28]. Two main areas of interest were considered, namely ischemic core and border zone [29] at both 24 h and 7d Immunohistochemistry after pMCAo Three-dimensional images were acquired Immunohistochemistry was performed on 20-μmbrain over a 10 μmz-axiswitha0.23 μmstepsizeand pro- coronal sections using anti-mouse CD11b (1:700, kindly cessed using Imaris software (Bitplane, Zurich, Switzer- provided by Dr. Doni, [8] anti-mouse-CD45 (1:800, BD land) and Photoshop cs2 (Adobe Systems Europe Ltd). Biosciences Pharmigen, San Jose, CA), anti-mouse CD68 (1:200, Serotec, Kidlington, UK), anti-mouse Ym1 (1:400, Statistical analysis Stem Cell Technologies, Vancouver, Canada), anti-mouse Statistical power (1-b) was assessed as post-hoc analysis by CD206 (1:100, Serotec, Kidlington, UK). Positive cells were means of G*Power [30]. Statistical analysis was performed stained by reaction with 3, 3 diaminobenzidine tetrahy- using standard software packages GraphPad Prism drochloride (DAB, Vector laboratories, CA, USA). For (GraphPad Software Inc., San Diego, CA, USA, version negative control staining, the primary antibodies were 4.0). All data are presented as mean and standard devia- omitted and no staining was observed. CD45-positive cells tion (sd). The comparison between groups was performed displayed 2 morphologies: a leukocyte-like shape corre- using One-way ANOVA followed by appropriate post hoc sponding to cells with a rounded cell body without test. p-values lower than 0.05 were considered statistically high branches and high expression of CD45 (CD45 ), and a significant. microglia-like shape having a small cell body and several low branches and a fainter expression of CD45 (CD45 )[22]. Results high Quantification was carried out on CD45 cells. Immu- Histopathological findings at different time points from nostained area for each marker was measured using ImageJ pMCAO software http://rsbweb.nih.gov/ij/ and expressed as positive pMCAO induced an infarcted area in the ipsilateral cortex pixels/total assessed pixels and indicated as staining per- (Figure 2A) as expected [22,23]. The lesion, evaluated as centage area (as number per mm for CD206) for subse- relative paleness of cresyl violet staining and corrected for quent statistical analysis [28]. edema, at 6 h, 12 h, 24 h, 48 h and 7d, had a volume of 3 3 3 12.5 mm ±5.8,12.4mm ± 5.7, 23.8 mm ± 5.1, 22.1 3 3 Immunofluorescence and confocal analysis mm ± 3.3 and 9.6 mm ± 4.7, respectively (Figure 2B). Immunofluorescence was performed on 20-μm coronal Cortex, the brain area involved in the ischemic lesion sections according to the previously described method was considered for neuronal count (Figure 2C). Six hours [22]. Primary antibodies used were: anti-mouse CD45 after ischemia, neuronal count performed in the ipsilateral (1:800 or 1:1500); anti-mouse Ym1 (1:400, Stem Cell cortex revealed a significant cell loss when compared to Technologies, Vancouver, Canada), anti-mouse CD206 the corresponding area in the sham-operated group (1:100, Serotec, Kidlington, UK), anti-mouse CD11b (84.9%). Neuronal counts progressively but slowly (1:30000, kindly provided by Dr. Doni), anti-mouse CD68 decreased reaching 64.9% at 7d. No significant difference (1:200, Serotec, Kidlington, UK), anti-mouse NeuN (1:250, was found between ispilateral and contralateral side in Millipore, Billerica, MA, USA). Fluorconjugated secondary sham-operated animals (data not shown). antibodies used were: Alexa 546 anti-rat, Alexa 594 anti- At 6 h after pMCAO rare TUNEL-positive cells were rabbit, Alexa 488 anti-mouse (all 1:500, Invitrogen, Carls- present in the injuried cortex indicating the presence of bad, CA). Biotinilated anti-rat antibodies (1:200, Vector few dying cells (30.2 ± 14.2, expressed as cell density per Laboratories, Burlingame, CA) were also used followed by mm , Figure 2D). Number of dying cells progressively fluorescent signal coupling with streptavidine TSA ampli- increased at 12, 24 and 48 h post ischemia (278.6 ± 51.1, fication kit (cyanine 5, Perkin Elmer, MA, USA). Similarly 589.7 ± 77.3 and 708.8 ± 30.2, respectively). Seven days to what reported for immunohistochemistry DAB staining, after ischemia still several TUNEL-positive cells were pre- also in this case we considered only cell displaying CD45 sent (343.6 ± 120.0) indicating the persistence of dying high rounded morphology (CD45 , [22]). Appropriate nega- cells at this time point. Positive TUNEL staining was not tive controls without the primary antibodies were apparent in any sham-operated mice at any time points. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 5 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 2 Histopathological findings at different time points from pMCAO. A: representative brain coronal sections obtained 24 h after pMCAO. Pale demarcated areas depict the ischemic lesion. Quantification of ischemic volume (B), neuronal counts (data obtained from the mean of 33 frames/mouse and expressed as % of sham groups, C) and TUNEL-positive cells (D) in the cortex of sham and ischemic mice at different times after pMCAO. Data are reported as mean+sd, n = 8. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham; °p < 0.05, °°p < 0.01, °°°p < 0.001 versus 6 h, Bonferroni’s Multiple Comparison Test. Time-course of expression of M/M markers: CD11b, CD45, cells with hypertrophic soma endowed with thick branches CD68, Ym1, CD206 (Figure 3A/E). high The M/M markers expression was analyzed within the No CD45-positive cells (CD45 cells, see methods, ischemic area based on the tissue sampling represented Figure 3F) could be observed in sham-operated mice in Figure 1. At each time point, the sampled cortical area and in the contralateral hemisphere of ischemic mice. pertained to the ischemic territory, being the number of Six hours after ischemia these cells were clearly visible neurons in this region decreased compared to sham ani- in the area considered (0.4 ± 0.2 percent of stained mals at every time points (Figure 2C). area). The immunoreactivity was further increased 12 CD11b, a constitutive marker of microglia and macro- and 24 h after ischemia (0.6 ± 0.2 and 1.1 ± 0.3, respec- phages was expressed at every time point considered as tively). No further increase could be observed at 48 h well as in sham-operated mice (5.6 ± 1.9, percent of (1.1 ± 0.4). CD45 staining was still present at 7d (0.9 ± stained area). Starting from 6 h the immunoreactivity 0.4, Figure 3F). increased and remained elevated at every subsequent time CD68 immunoreactivity was undetectable in sham- point considered, with no major differences throughout operated mice and appeared 6 h after ischemia (0.3 ± 0.2 the experimental groups (9.5 ± 1.5, 11.7 ± 1.6, 10.1 ± 1.7, percent of stained area). It progressively increased at 13.1 ± 2.8, 13.0 ± 0.1, respectively at 6 h, 12 h, 24 h, 48 h every time point considered (0.6 ± 0.2 at 12 h; 1.7 ± 0.2 and 7d, Figure 3B). at 24 h; 3.7 ± 0.8 at 48 h). Notably, a dramatic increase in Outside the lesion, CD11b staining revealed thin ramifi- the CD68 stained area could be observed at 7d (7.4 ± 1.4, cations and small soma (Figure 3A/C). CD11b immunor- Figure 3G). eactivity was associated with a different morphology in Ym1 immunoreactivity was detectable starting from 12 relation to the cell localization in the lesioned area. Two h (0.04 ± 0.02 percent of stained area). This marker was main areas were identified, namely a lesion border show- maximally expressed at 24 h (0.84 ± 0.16) and markedly ing CD11b+ highly ramified cells (Figure 3A/D) and an decreased at later time points (0.37 ± 0.10 at 48 h and ischemic core showing both CD11b+ ameboid cells and 0.23 ± 0.15 at 7d, Figure 3H). Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 6 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 3 Immunohistochemical analysis and quantification of microglial markers: CD11b, CD45, CD68, Ym1, CD206. A: representative micrographs of CD11b immunostaining in the injured cortex at 24 hours after pMCAo. CD11b-positive cells display different morphology depending on their localization in the ischemic area (C: outside the lesion, D: border zone, E: ischemic core). B: quantification of CD11b immunostaining at different times after pMCAo. Representative micrographs of CD45 (F), CD68 (G), Ym1 (H), CD206 (I) immunoreactivity at 24 hours after ischemia, and related quantifications at different times after pMCAO (Bar 10 μm). Data are expressed as mean+sd of 33 frames/ mouse (24 frames/mouse for CD45, TUNEL, CD206), n = 8. One way Anova: p < 0.0001. *p < 0.05, **p < 0.01 vs sham; °°p < 0.01, °°°p < 0.001 vs 6 h (12 h for Ym1). Bonferroni’s Multiple Comparison Test. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 7 of 19 http://www.jneuroinflammation.com/content/8/1/174 CD206 positive cells were present in sham-operated present throughout the entire ischemic area (Figure 4). mice (7.3 ± 0.9 cell/mm ). They could be observed 6 h CD45 cells visible at 10× magnification (Figure 4) did low after pMCAO (12.2 ± 6.2) and significantly increased not reveal the presence of CD45 cells (corresponding progressively up to 24 h (23.6 ± 5.3 at 12 h and 40.0 ± to ramified microglia) appearing in the CD11b staining 14.9 at 24 h). A significant number of CD206 positive microphotograph. Conversely CD68 appeared to be cellswas still presentat48h(30.5± 10.5)and at 7d mainly concentrated in the border zone, with rare cells (32.7 ± 8.8, Figure 3I). present in the ischemic core. Notably, at longer time points (7d) along with the great increase of its expres- Localization of M/M markers with respect to the lesion sion (Figure 3G), CD68 appears both in the border and Twenty-four hours after pMCAO, the immunoreactivity in the core areas. Ym1 at 24 h after pMCAO appeared for CD11b appeared to be evenly distributed in the exclusively expressed in the ischemic core, similarly to ischemic area, being present both in the lesion border CD206 (Figure 4). With the exception of CD68, all the and in the ischemic core (Figure 4). At the same time markers considered showed a similar distribution at CD45 staining showed a similar distribution being every time point (data not shown). Figure 4 Distribution of the selected M/M markers inside the ischemic lesion at 24 h from the injury. Representative immunostaining micrographs show spatial distribution of M/M phenotype markers (CD11b, CD45, Ym1, CD206, CD68) into the ischemic area at 24 h after ischemia (upper panels). Bar = 250 μm. Drawings representing the immunostaining data (lower panels). Only immunoreactivity for CD11b could be observed outside the lesioned area in basal conditions (sham-operated mice). In the lesion, CD11b+ cells showing a ramified to globular morphology could be observed going from the border zone to the ischemic core (red). CD45 staining was present throughout the entire ischemic area (pink). Conversely CD68 appeared to be strongly concentrated in the border zone. Ym1 and CD206 were exclusively expressed in the ischemic core. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 8 of 19 http://www.jneuroinflammation.com/content/8/1/174 Coexpression of M/M markers at 24 h and 7d after CD68 was greatly increased both in globular CD11b+ pMCAO cells in the ischemic core and in ramified CD11b cells Twenty-four hours after ischemia CD68 was expressed in laying in the border zone (Figure 4 and Figure 5C-D). hypertrophic ameboid CD11b cells present in the At 24 h after pMCAO Ym1 positive cells co-labeled ischemic core and in ramified microglia in the border with CD11b globular cells within the ischemic core, zone where CD68 positive cells were mostly located (Fig- where they were exclusively located (Figure 4 and Figure ure 4 and 5A-B). A similar pattern of coexpression could 6A-B-E-F). Seven days after ischemia Ym1 and CD11b be observed at 7d. At this time point the expression of Figure 5 Coexpression of CD11b (red) and CD68 (green) 24 h (A-B) and 7d (C-D) after pMCAO. In the ischemic core at 24 h CD11b positive cells are prevalently globular and some of them are positive to CD68 (A). In the border zone (B) CD11b cells display rounded cell bodies and ramified processes positive to CD68. Globular CD11b cells in the lesioned area 7d after ischemia mostly express CD68 marker (C). A high number of CD11b cells displaying different morphology colabel with CD68 in the border zone (D). Data are representative of 3 independent experiments. Bars: 20 μm. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 9 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 6 Coexpression of CD11b (red) and Ym1 (green) at 24 h (A-B-E-F) and 7d (C-D-G-H) after pMCAO. Ym1 positive cells co-label with globular CD11b postive cells at both time points (A, C). High magnifications (E-G) and 3D rendering (F-H) show colabeling of markers further highlighting the coexpression (blu = nuclei, bar: 5 μm). Consistent with the observation that no Ym1 cells are present in the border zone (Figure 4), no immunostaining for Ym1 at neither time points could be observed in this area (B, D). Data are representative of 3 independent experiments. Bars: 20 μm. coexpression pattern was similar to that observed at 24 h At 24 h after pMCAO, the few CD68+ cells found in (Figure 6C-D-G-H). the ischemic core did not colocalize with Ym1+ cells CD206 at 24 h was present exclusively in the ischemic that were present exclusively in this area (Figure 4 and core (Figure 4) where it colocalized with globular Figure 8A-B). In the magnification of Figure 8E-F it is CD11b positive cells (Figure 7A-B-E-F). The same pat- possible to observe that even when these markers tern of coexpression was observed at 7d (Figure 7C-D- appear closely related, they actually belong to distinct G-H). cells. At longer times (7d) Ym1 cells not colocalizing Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 10 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 7 Coexpression of CD11b (red) and CD206 (green) at 24 h (A-B-E-F) and 7d (C-D-G-H) after pMCAO. At 24 h after ischemia some globular CD11b cells co-label with CD206 marker in the ischemic core (A). Seven days after ischemia CD11b cells are prevalently globular in the ischemic core and are highly positive to CD206 (C). High magnifications (E-G) and 3D rendering (F-H) show colabeling of markers further highlighting the coexpression (blu = nuclei, bar: 5 μm). Consistent with the observation that no CD206 cells are present in the border zone (Figure 4), no immunostaining for this marker at neither time points could be observed in this area (B, D). Data are representative of 3 independent experiments. Bars: 20 μm. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 11 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 8 Coexpression of CD68 (red) and Ym1 (green) at 24 h (A-B-E-F) and 7d (C-D-G-H) after pMCAO.At 24hCD68positivecells found in the ischemic core do not co-localize with Ym1 positive cells (A). In high magnifications panels (E-F) the two markers appear to belong to different cells although in close contact (blu = nuclei). Seven days after ischemia, when CD68 immunoreactivity is greately increased, Ym1 appears to be expressed also, but not exclusively in CD68 positive cells (C). Note the presence of one Ym1 positive cells (arrow) that does not co-localize with CD68. High magnifications (G) and 3D rendering (H) show colabeling of markers further highlighting the coexpression (blu = nuclei). Consistent with the observation that no Ym1 cells are present in the border zone (Figure 4), no immunoreactivity for this marker could be observed at neither time points in that area (B, D). Data are representative of 3 independent experiments. Bars: 20 μm. High magnifications and 3D rendering bar: 5 μm. with CD68 are still present, however coexpression with in CD68 positive cells is apparent in the ischemic core CD68 can also be seen (Figure 8C-D-G-H). (Figure 9C-G-H). CD206 marker is not present in the A small fraction of CD206 positive cells show coex- border zone at both time points (Figure 9B-D). pression with CD68 at 24 h after ischemia in the Ym1 and CD206 appeared to be coexpressed in the ischemic core (Figure 9A-E-F). Similar situation is ischemic core both at 24 h (Figure 10A-C-D) and 7d observed at 7d after pMCAO when a dramatic increase (Figure 10B-E-F) after pMCAO. None of the two Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 12 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 9 Coexpression of CD68 (red) and CD206 (green) at 24 h (A-B-E-F) and 7d (C-D-G-H) after pMCAO. At 24 h and 7d after ischemia, a minor part of CD68 positive cells found in the ischemic core colocalize with CD206 (A, C). High magnification (E-G) and 3D rendering (F-H) show both single- and double-positive cells in the ischemic core (blu = nuclei, bar: 5 μm). Consistent with the observation that no CD206 cells are present in the border zone (Figure 4), no immunoreactivity for this marker could be observed at neither time points in that area (B, D). Data are representative of 3 independent experiments. Bars: 20 μm. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 13 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 10 Coexpression of Ym1 (red) and CD206 (green) at 24 h (A-C-D) and 7d (B-E-F) after pMCAO. At 24 h and 7d after ischemia Ym1 positive cells co-label with CD206 positive cells in the ischemic core (A-B) Bars: 20 μm. High magnification (C-E) and 3D rendering (D-F) show coexpression of markers with the same cell nucleus (blu). Bar: 5 μm. Consistent with the observation that neither Ym1 cells nor CD206 cells are present in the border zone (Figure 4) no immunostaining for these markers could be observed at neither time points in this area (data not shown). Data are representative of 3 independent experiments. markers was present in the border zone at both time A summary of M/M markers coexpression 24 h and points considered (Figure 4). 7d after the ischemic lesion is reported in Figure 11. As expected all CD11b globular, CD68 globular, Ym1 Lastly, to provide additional details on the functional high and CD206 positive cells were all positive for CD45 status of M/M, we assessed their relationship with neu- in both ischemic core and border zone (data not rons (NeuN+). We analyzed CD11b/CD68 and CD11b/ shown), being CD45 a common marker for immune cell Ym1 double positive cells as these populations showed populations [31,32]. to increase at different time points, thus suggesting Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 14 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 11 Summary of M/M markers coexpression 24 h and 7d after the ischemic lesion. At 24 h after injury (A) immunoreactivity for CD11, which is readly increased after ischemia, is expressed in ramified and globular cells. CD68 is present in a percentage of both globular and ramified CD11b+ cells. Ym1 and CD206 that are present mostly in the core of the lesion, are expressed by a fraction of globular CD11b+ cells and can be present on the same cells. A few cells coexpressing CD206 and CD68 can be found in the area between the core and the border zone where the two markers are mainly located respectively. At 7d after injury (B) Ym1 decreases while CD68 expression greatly increases and from the border zone where it was at earlier times it invades the ischemic core (see also data in Figure 3). A few CD68+ cells appear now to express Ym1. distinct functional states. CD11b stain of M/M mem- neurons were positive for CD68 at both zones (Figure branes was chosen for documenting the morphology of 12A-B-C-D), suggesting an active phagocytosis. None of M/M when contacting neurons. We found that neurons the CD11b/Ym1 double positive cells at 24 h appeared were often enwrapped by CD11b positive cells in both to be engaging a phagocytic interaction with neurons, ischemic core and border zone at both 24 h and 7d being these cells never in contact with NeuN positive (Figure 12). In most cases CD11b cells surrounding cells (Figure 12E). At 7d, a few CD11b/Ym1 double Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 15 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 12 Coexpression of CD11b (red) and NeuN (blue) with CD68 (green) at 24 h (A-A’-B-B’) and 7d (C-C’-D-D’) or with Ym1 at 24 h (E-E’-F-F’) and 7d (G-G’-H-H’) after pMCAO. In the ischemic core CD11b/CD68 double positive cells envelop NeuN positive cells, possibly indicating phagocytosis of neurons (A; 3D rendering in A’). The same interaction was observed in the border zone (B-B’). At 7d after ischemia, when CD68 expression is enhanced (Figure 3) in both ischemic core and border zone, CD11b/CD68 double positive cells enwrap neurons, suggesting active phagocytosis also at this time point (C-C’-D-D’). At 24 h after pMCAO in the ischemic core, where Ym1 positive cells are exclusively located, CD11b/Ym1 double positive cells do not appear involved in a phagocytic interaction with neurons (NeuN positive cells, E; 3D rendering on E’). CD11b single positive cells in both ischemic core (E-E’) and border zone (where Ym1 is not expressed, F-F’) surround neurons. At 7d after ischemia part of CD11b/Ym1 double positive cells engage a phagocytic appearance by enveloping neurons in ischemic core (G-G’) coherently with their partially CD68 positive phenotype at this time point (Figure 8). In the border zone at 7d (Ym1 is absent) CD11b single positive cells still enwrap neurons (H-H’). Data are representative of 3 independent experiments. Bar: 20 μm. positive cells showed a phagocytic appearance envelop- the lesioned area with no apparent major changes during ing neurons (Figure 12G), coherently with their partially time, with the exception of CD68. CD68 positive phenotype at this time point (Figure 8). We have firstly determined the histopathological fea- In the border zone, at both time points, Ym1 was not tures of the lesion induced by pMCAO. From the analysis detectable and only single CD11b positive cells did of the temporal evolution of the lesion it appears that the envelop neurons (Figure 12F-H). percentage of neuronal loss is somehow stable from 24 h up to 7d although the persistence of TUNEL-positive Discussion cells at this late point indicates that some cells may still be in degeneration at that late time. It should be noted Our study shows that the ischemic lesion is accompanied by activation of specific M/M phenotype that presents that assessing the lesion volume by the paleness of the distinctive spatial and temporal features. We have cresyl violet staining may lead to misleading conclusions demonstrated that: 1) the ischemic lesion induces the since, as detailed below, invading inflammatory cells may expression of the selected M/M markers that develop contribute to the apparent reduction of the lesioned area over time, each with a specific pattern; 2) the selected at 7d. Actually the quantification of the CD11b and high markers are associated with globular or ramified CD11b CD45 immunoreactivity indicates that inflammatory morphology, 3) each marker has a given localization in Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 16 of 19 http://www.jneuroinflammation.com/content/8/1/174 cells rapidly increase in number and/or size early after and cell surface antigens of microorganisms. Its localiza- the injury and at every time point considered. tion and predominance in phagocytic macrophages impli- M/M play a pivotal role in surveillance and response to cates CD68 in phagocytosis [43,44]. We observed that the altered CNS conditions [1,2,4]. An emerging concept is early increase in CD68 immunoreactivity is concentrated that, similarly to what happens for peripheral macro- in the border zone and expressed in ramified CD11b posi- phages, these cells can exert different antithetic functions tive cells. At later time points a dramatic increase in CD68 depending on environmental signals, acting as major expression appears both in the border zone and the players in the pro-inflammatory cytotoxic response, ischemic core and is apparent in globular CD11b cells. At both time points and in both zones, CD11b/CD68 double but also participating in the immunosuppressive and self-repair processes [6,33,34]. The phenotype markers positive cells appear to physically interact with neurons considered in this study include classical markers of M/M and show a phagocytic-like morphology characterized by activation (CD11b and CD45) and markers expressed by neuron engulfment. The phagocytic activity of alterna- alternatively activated macrophages (CD68, Ym1 and tively activated M/M is associated to clearance of cells CD206). Although evidence of M2 activation state in the debris, of damaged or dying cells and of infiltrating neu- brain has been reported in M/M in AD models [35,36], trophils thus resulting in the elimination of several poten- following global ischemia [37], in models of experimental tially cytotoxic substances [4,18,21,45,46]. However the autoimmune encephalomyelitis [38] or spinal cord injury overall functional meaning of phagocytosis in acute brain [16,39], information on M2 marker expression, coexpres- injury is still an open question. Actually the protective sion and temporal evolution in the injuried brain is lack- effect of manipulations such as stem cell infusion may be ing. We could observe that these phenotype markers are associated with a decrease in CD68 expression [47]. exclusively expressed by CD11b cells and that each of Ym1 belongs to the lectin family and is constitutively them shows distinct features in terms of time course of expressed by liver, lung and bone marrow, consistently activation and localization in relation to the ischemic with the fact that these are the original sources of myeloid lesion. cells [48]. It is synthesized and secreted by activated CD11b is expressed on the surface of many leukocytes macrophages during inflammation and exhibits a pH- and is a widely used maker of M/M. It belongs to a family dependent, specific activity towards GlcN oligomers and of cell surface receptors known as integrins. It is covalently heparin. Ym1 may control leukocyte trafficking by com- bound to a beta 2 subunit to form integrin a b (Mac-1, peting with them for binding sites on local extracellular M 2 CD11b/CD18) which is implicated in diverse responses matrix, an action resulting in down-regulation of inflam- including cell-mediated killing, phagocytosis, chemotaxis mation. Our findings show that, similarly to what reported in peripheral macrophages, Ym1 is activated transiently and cellular activation. CD11b has the ability to recognize a wide series of ligands such as fibrinogen, iC3b fragment suggesting that it may be involved in the establishment of of the third complement component, ICAM-1, denatu- an inflammatory management control of the injured rated products, blood coagulation factor X [40]. Our data region [48]. Its expression is restricted to the ischemic show that CD11b staining increases at early time points core and it colocalizes with CD11b globular cells and with after ischemia, rapidly reaching a plateau of activation. some CD68 cells at later times only. None of the CD11b/ Notably, CD11b positive cells display a different morphol- Ym1 double positive cells is associated with phagocytosis ogy in relation to the lesion, namely they are ramified in of neurons at 24 h, whilst at 7d some of them show a pha- the border zone and ameboid in the ischemic core. Simi- gocytic appearance and envelop neurons, coherently with high larlytoCD11b, alsoCD45 cells increase rapidly after their partially CD68 positive phenotype at this time point. ischemia. These cells, display a rounded morphology and An increase in Ym1 expression has been associated to the most probably correspond to recruited macrophages, neu- beneficial effect of stem cell infusion in mice subjected to trophils and lymphocytes [22,32,41]. This study does not global ischemia [37], in line with a protective role in acute specifically address the question of differentiating between brain injury. invading macrophages and resident microglia. An impor- Another marker of alternatively activated macrophages is tant future direction will be to identify specific molecular/ CD206 or mannose receptor [45,49]. This is an endocytic phenotypical markers for these two cell populations, since receptor that binds both microbial glycans and self glyco- there is evidence that they may play a different role in the proteins carrying terminal mannose, fucose and N-acetyl- progression of brain injury [9,34,42]. glucosamine by interaction with its carbohydrate CD68 or macrosialin is a member of the lysosomal/ recognition domains (CRDs). Its known function is related endosomal-associated membrane glycoprotein (LAMP) to recognition and endocytosis of the carbohydrate portion of antigens for processing and presentation [50]. Our family and a member of the scavenger receptor family results show that CD206 expression significantly increases which recognizes a wide range of anionic macromolecules over time and colocalizes with Ym1 positive cells and with such as oxidatively modified lipoprotein, apoptotic cells Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 17 of 19 http://www.jneuroinflammation.com/content/8/1/174 a fraction of CD68 positive cells that increase at later time The different states of M/M in the ischemic lesion points. reflect the complexity of these cells and their ability to Lastly, our data have been obtained in a model of per- differentiate towards a multitude of phenotypes depend- manent ischemia and may not be extended to an ischemia ing on the surrounding microenvironmental signals that with reperfusion paradigm. Notably the present data and can change over time. The inflammatory response that our previous results [22] indicate that the ratio of follows cerebral ischemia is regarded as a promising tar- high low CD45 /CD45 is dramatically different in these two get for stroke therapy. The data presented in this study provide a basis for understanding this complex response conditions, being much higher after pMCAO. This may be and for developing strategies resulting in promotion of a due to either a higher number of infiltrating cells and/or a lower survival of resident cells, thus indicating that in tran- protective inflammatory phenotype. sient ischemia the composition of the specific M/M popu- lations in the lesioned area is different. List of abbreviations BDNF: Brain Derived Neurotrophic Factor; CRDs: Carbohydrate Recognition Conclusions Domains; DAB: 3, 3’-Diaminobenzidine; GDNF: Glial cell-Derived Neurotrophic Factor; ICAM: Inter-Cellular Adhesion Molecule; IGF-1: Insulin Growth factor-1; In the ischemic lesion M/M express markers that show IL-1β: Interleukin-1β; LAMP: Lysosomal/endosomal-Associated Membrane distinct temporal expression, distribution and association Glycoprotein; M/M: Microglia/Macrophages; NO: Nitric Oxide; PBS: Phosphate with a definite cell morphology suggesting that different Buffered Saline; pMCAo: permanent Middle Cerebral Artery occlusion; ROS: Reactive Oxygen Species; TNF: Tumor Necrosis Factor; TUNEL: Terminal M/M populations are acting at the site of injury according Deoxynucleotidyl Transferase; dUTP Nick End Labeling. to well defined phenotype, time of activation and pattern of localization. Conceivably, at 24 h after insult, ramified Acknowledgements SF is a fellow of the Monzino Foundation. and phagocytic M/M surround the lesion, possibly acting as a barrier against further expansion of the lesion, whilst Authors’ contributions globular M/M committed to a protective phenotype (i.e. CP participated in the design of the experiments, carried out the experiments, acquired and interpreted the data, was involved in drafting the expressing Ym1 and CD206) populate the ischemic core manuscript. SF carried out the experiments, acquired and interpreted the with the primary function of resolving inflammation and data, was involved in drafting the manuscript. MGDS participated in the promoting wound healing. At later time points (7d) the design of the experiments, interpreted the data, was involved in drafting the manuscript. All authors read and approved the final manuscript. phagocytic behavior of M/M becomes prevalent in the whole lesioned area with numerous globular phagocytic Competing interests M/M invading the core territory. The observed switch The authors declare that they have no competing interests. towards the phagocytic phenotype is accompanied by the Received: 14 September 2011 Accepted: 10 December 2011 progressive reduction of the expression of the protective Published: 10 December 2011 Ym1. At 24 h protective Ym1 positive cells do not appear to References 1. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, be involved in neuron phagocytosis, differently from Dustin ML, Gan WB: ATP mediates rapid microglial response to local CD68 positive cells that show a close physical interac- brain injury in vivo. Nat Neurosci 2005, 8:752-758. tion with neurons. At 7d, when neuronal damage 2. Yenari MA, Kauppinen TM, Swanson RA: Microglial activation in stroke: therapeutic targets. 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Sierra A, Encinas JM, Deudero JJ, Chancey JH, Enikolopov G, Overstreet- Isolation and direct characterization of resident microglial cells from the Wadiche LS, Tsirka SE, Maletic-Savatic M: Microglia shape adult Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 19 of 19 http://www.jneuroinflammation.com/content/8/1/174 hippocampal neurogenesis through apoptosis-coupled phagocytosis. Cell Stem Cell 7:483-495. 52. Kettenmann H: Neuroscience: the brain’s garbage men. Nature 2007, 446:987-989. doi:10.1186/1742-2094-8-174 Cite this article as: Perego et al.: Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice. Journal of Neuroinflammation 2011 8:174. 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Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice

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Springer Journals
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Copyright © 2011 by Perego et al; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Neurology; Neurobiology; Immunology
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1742-2094
DOI
10.1186/1742-2094-8-174
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22152337
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Abstract

Background: Emerging evidence indicates that, similarly to what happens for peripheral macrophages, microglia can express different phenotypes depending on microenvironmental signals. In spite of the large literature on inflammation after ischemia, information on M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is lacking. The present study investigates the presence of microglia/macrophage phenotype markers, their temporal expression, whether they are concomitantly expressed by the same subpopulation, or they are expressed at distinct phases or locations in relation to the ischemic lesion. Methods: Volume of ischemic lesion, neuronal counts and TUNEL staining were assessed in C57Bl/6 mice at 6-12-24-48 h and 7d after permanent occlusion of the middle cerebral artery. At the same time points, the expression, distribution in the lesioned area, association with a definite morphology and coexpression of the microglia/macrophage markers CD11b, CD45, CD68, Ym1, CD206 were assessed by immunostaining and confocal microscopy. Results: The results show that: 1) the ischemic lesion induces the expression of selected microglia/macrophage markers that develop over time, each with a specific pattern; 2) each marker has a given localization in the lesioned area with no apparent changes during time, with the exception of CD68 that is confined in the border zone of the lesion at early times but it greatly increases and invades the ischemic core at 7d; 3) while CD68 is expressed in both ramified and globular CD11b cells, Ym1 and CD206 are exclusively expressed by globular CD11b cells. Conclusions: These data show that the ischemic lesion is accompanied by activation of specific microglia/ macrophage phenotype that presents distinctive spatial and temporal features. These different states of microglia/ macrophages reflect the complexity of these cells and their ability to differentiate towards a multitude of phenotypes depending on the surrounding micro-environmental signals that can change over time. The data presented in this study provide a basis for understanding this complex response and for developing strategies resulting in promotion of a protective inflammatory phenotype. Keywords: Inflammation, stroke, alternative activation Background retracting their processes and they are endowed with Microglia, the major cellular contributors to post-injury the capacity to rapidly respond to injury or alterations inflammation, have the potential to act as markers of in their microenvironment [1-3]. After acute brain disease onset and progression and to contribute to neu- injury, these resident cells are rapidly activated and rological outcome of acute brain injury. They are nor- undergo dramatic morphological and phenotypic mally present in the healthy brain where they actively changes. Typical morphological changes associated with survey their surrounding parenchyma by protracting and microglia activation include thickening of ramifications and of cell bodies followed by acquisition of a rounded amoeboid shape. This intrinsic response is associated to * Correspondence: desimoni@marionegri.it recruitment of blood-born macrophages which migrate Laboratory of Inflammation and Nervous System Diseases, Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Via La into the injured brain parenchyma [4,5]. This process is Masa, 19-20156 Milan, Italy © 2011 Perego et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 2 of 19 http://www.jneuroinflammation.com/content/8/1/174 accompanied by expression of novel surface antigens Methods and production of mediators that build up and maintain Animals the inflammatory response of the brain tissue. Activated Male C57Bl/6 mice (10-week old, 20-25 g, Harlan microglia and recruited macrophages (which are antige- Laboratories, Italy) were used. Procedures involving ani- nically not distinguishable, henceforth referred to as M/ mals and their care are conducted in conformity with the M), can affect neuronal function and promote neuro- institutional guidelines (Quality Management System toxicity through the release of several harmful compo- Certificate - UNI EN ISO 9001:2008 - Reg. N° 8576-A) nents such as IL-1b,TNF-a, proteases and reactive that are in compliance with national (D. Lvo. n. 116, 27 oxygen and nitrogen species [6,7]. On the other hand Gennaio 1992; Legge n° 413, 12 Ottobre 1993; Circolare they also possess protective qualities and promote neu- No. 8, 22 Aprile 1994; D.M. 29/09/95; D.M. 26/04/2000) rogenesis and lesion repair [8-10]. Indeed, microglia and international (EEC Council Directive 86/609, OJ L have been proposed to be beneficial by several mechan- 358, 1, Dec. 12, 1987; Guide for the Care and Use of isms including glutamate uptake [11] removal of cell Laboratory Animals, U.S. National Research Council, debris [12] and production of neurotrophic factors such 1996) laws and policies. Before beginning any procedure, as IGF-1 [13], GDNF [14] and BDNF [15,16]. mice were housed for at least 1 week in their home cages Studies addressing phenotypic changes occurring in at a constant temperature, with a 12 hour light-dark macrophages in peripheral inflammation and immunity cycle, and ad libitum access to food and water in a selec- have shown that these cells can undergo different forms tive pathogen-free (SPF) vivarium. of polarized activation. One is the classic or M1 activa- tion, characterized by high capacity to present antigen, Focal cerebral ischemia high production of NO and ROS and of proinflamma- Permanent ischemia was obtained by permanent middle tory cytokines. M1 cells act as potent effectors that kill cerebral artery occlusion (pMCAO) [22,23]. Briefly, mice micro-organisms and tumor cells, drive the inflamma- were anesthetized with Equitensin (pentobarbital 39 mM, tory response and may mediate detrimental effects on chloral hydrate 256 mM, MgSO4 86 mM, ethanol 10% neural cells. The second phenotype (M2) is an alterna- v/v, propyleneglycol 39.6% v/v) 100 μl/mouse administered tive apparently beneficial activation state, more related by intraperitoneal (i.p.) injection. A vertical midline inci- to a fine tuning of inflammation, scavaging of debris, sion was made between the right orbit and tragus. The promotion of angiogenesis, tissue remodeling and repair. temporal muscle was excised, and the right MCA was Specific environmental signals are able to induce these exposed through a small burr hole in the left temporal different polarization states [17]. A similar possibility bone. The dura mater was cut with a fine needle, and the has been also recently raised for microglia, by showing MCA permanently occluded by electrocoagulation just that these cells, under certain conditions, can indeed be proximal to the origin of the olfactory branch. Intraopera- pushed to both extremes of the M1 and M2 differentia- tive rectal temperature was kept at 37.0 ± 0.5°C using a tion spectrum [16,18]. More studies are needed to sub- heating pad (LSI Letica). Mortality rate was 8.5%. Sham- stantiate these observations. operated mice received identical anesthesia and surgical In this frame the present study aims at getting insight procedure without artery occlusion. on previously unexplored aspects of microglia phenotype changes induced by cerebral ischemia, namely, the pre- Experimental design and blinding senceofspecificphenotype markers, their temporal Mice were assigned to surgery and experimental groups expression, whether or not they are concomitantly with surgery distributed equally across cages and days. To expressed by the same subpopulation, whether they are minimize the variability, all surgeries were performed by expressed at distinct phases or locations in relation to the the same investigator, blinded to the experimental groups. ischemic lesion. We focussed on a few molecules that are All subsequent histological and immunohistological eva- known to be expressed by macrophages in peripheral luations were also done by blinded investigators. inflammation and that have been associated to different functions. They include: CD11b, a marker of M/M acti- Brain transcardial perfusion vation/recruitment, CD45 expressed on all nucleated At selected time points mice were deeply anesthetized hematopoietic cells [19], CD68 a marker of active phago- with Equitensin (120 μl/mouse i.p.) and transcardially per- cytosis, Ym1 a secretory protein that binds heparin and fused with 20 ml of PBS, 0.1 mol/liter, pH 7.4, followed by heparin sulphate and CD206 a C-type lectin carbohydrate 50 ml of chilled paraformaldehyde (4%) in PBS. After care- binding protein, both of them expressed by alternatively fully removing the brains from the skull, they were trans- activated macrophages and associated to recovery and ferred to 30% sucrose in PBS at 4°C overnight for function restoration [20,21]. cryoprotection. The brains were then rapidly frozen by Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 3 of 19 http://www.jneuroinflammation.com/content/8/1/174 immersion in isopentane at - 45°C for 3 min before being CD68 quantification fields at 40× magnification were sealed into vials and stored at -70°C until use. selected at 1.54 mm anterior to bregma (11 fields), at 0.50 mm anterior to bregma (11 fields) and at 0.94 mm poster- Quantification of infarct size ior to bregma (11 fields). The first raw of fields was posi- For lesion size determination, 20-μm coronal brain cryo- tioned at the lesion edge, spacing each field by 572.5 μm sections were cut serially at 320-μm intervals and stained (distance between centres of the fields). Further raws of with Cresyl Violet [8]. On each slice, the infarcted area fields were positioned distanced by 389.3 μm each. For was assessed blindly and delineated by the relative pale- TUNEL, CD45, Ym1 and CD206 twenty-four quantifica- tion fields at 20× magnification were selected. Eight fields ness of histological staining tracing the area on a video screen. The infarcted area and the percentage of brain per slice were selected and fields were separated by 572.5 swelling for edema correction were determined by sub- μm (distance between centres of fields), while distance tracting thearea ofthehealthytissueintheipsilateral between each raw was 389.3 μm. A schematic representa- hemisphere from the area of the contralateral hemisphere tion of the regions of interest and of the selected fields is on each section [24,25]. Infarct volumes were calculated depicted in Figure 1. by the integration of infarcted areas on each brain slice as quantified with computer- assisted image analyzer and Neuronal Count calculated by Analytical Image System (Imaging Research Cresyl Violet stained brain sections were used for neuro- Inc., Brock University, St. Catharines, Ontario, Canada). nal count. Thirty-three fields at 40× were analyzed for each mouse. The amount of neuronal loss was calculated Slice selection and quantitative analysis by pooling the number of stained neurons in the three Three brain coronal sections per mouse (+1.54, +0.50 and ipsilateral sections and expressed as percentage of those - 0.94 mm from bregma, KBJ Franklin and G Paxinos, The in sham- operated animals. Fields were analyzed using Mouse Brain in Stereotaxic Coordinates, Academic Press), ImageJ software http://rsbweb.nih.gov/ij/ and segmenta- were used to quantify the stained area. On each slice, ana- tion was used to discriminate neurons from glia on the tomically defined cortical regions of interest were demar- basis of cell size. cated, corresponding to the primary motor cortex, somatosensory cortex, insular cortex (granular, agranular) TUNEL staining and secondary somatosensory cortex, representing the To assess the presence of injured cells showing DNA cortical regions involved in the largest lesion extension damage, terminal deoxynucleotidyl transferaseYmediated observed at 24 h after ischemia. Field selection was per- dUTP nick end labeling (TUNEL) staining was per- formed using a BX61 Olympus microscope equipped with formed on 20-μm sections by in situ cell death detection a motorized stage acquiring the same focal plan through- kit (Roche, Mannheim, Germany) according to the man- out the samples [26]. For neuronal count, CD11b and ufacturer instructions, as previously described [27]. Figure 1 Slices selection and tissue sampling for neuronal counts and quantification of immunostained area. Fields for neuronal counts, TUNEL evaluation and quantification of stained area were positioned within the ischemic territory at defined distances (see methods for details). The regions sampled pertained to the ischemic territory (yellow) at the time points considered (6 h, 12 h, 24 h, 48 h and 7d). Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 4 of 19 http://www.jneuroinflammation.com/content/8/1/174 DNase-treated sections were used as a positive control. performed. None of the immunofluorescence reactions After staining, the sections were visualized using fluores- revealed unspecific fluorescent signal in the negative con- cent microscopy (Olympus IX70 Olympus Tokyo, trols. Immunofluorescence was acquired using a scanning Japan). Images of the area of interest were acquired sequential mode to avoid bleed-through effects by an IX81 using AnalySIS software (Olympus, Tokyo, Japan). For microscope equipped with a confocal scan unit FV500 each mouse twenty-four fields at 20× were analyzed. with 3 laser lines: Ar-Kr (488 nm), He-Ne red (646 nm), TUNEL-positive cells were counted using ImageJ soft- and He-Ne green (532 nm) (Olympus, Tokyo, Japan) and ware http://rsbweb.nih.gov/ij/ and expressed as number aUVdiode. per mm for subsequent statistical analysis [28]. Two main areas of interest were considered, namely ischemic core and border zone [29] at both 24 h and 7d Immunohistochemistry after pMCAo Three-dimensional images were acquired Immunohistochemistry was performed on 20-μmbrain over a 10 μmz-axiswitha0.23 μmstepsizeand pro- coronal sections using anti-mouse CD11b (1:700, kindly cessed using Imaris software (Bitplane, Zurich, Switzer- provided by Dr. Doni, [8] anti-mouse-CD45 (1:800, BD land) and Photoshop cs2 (Adobe Systems Europe Ltd). Biosciences Pharmigen, San Jose, CA), anti-mouse CD68 (1:200, Serotec, Kidlington, UK), anti-mouse Ym1 (1:400, Statistical analysis Stem Cell Technologies, Vancouver, Canada), anti-mouse Statistical power (1-b) was assessed as post-hoc analysis by CD206 (1:100, Serotec, Kidlington, UK). Positive cells were means of G*Power [30]. Statistical analysis was performed stained by reaction with 3, 3 diaminobenzidine tetrahy- using standard software packages GraphPad Prism drochloride (DAB, Vector laboratories, CA, USA). For (GraphPad Software Inc., San Diego, CA, USA, version negative control staining, the primary antibodies were 4.0). All data are presented as mean and standard devia- omitted and no staining was observed. CD45-positive cells tion (sd). The comparison between groups was performed displayed 2 morphologies: a leukocyte-like shape corre- using One-way ANOVA followed by appropriate post hoc sponding to cells with a rounded cell body without test. p-values lower than 0.05 were considered statistically high branches and high expression of CD45 (CD45 ), and a significant. microglia-like shape having a small cell body and several low branches and a fainter expression of CD45 (CD45 )[22]. Results high Quantification was carried out on CD45 cells. Immu- Histopathological findings at different time points from nostained area for each marker was measured using ImageJ pMCAO software http://rsbweb.nih.gov/ij/ and expressed as positive pMCAO induced an infarcted area in the ipsilateral cortex pixels/total assessed pixels and indicated as staining per- (Figure 2A) as expected [22,23]. The lesion, evaluated as centage area (as number per mm for CD206) for subse- relative paleness of cresyl violet staining and corrected for quent statistical analysis [28]. edema, at 6 h, 12 h, 24 h, 48 h and 7d, had a volume of 3 3 3 12.5 mm ±5.8,12.4mm ± 5.7, 23.8 mm ± 5.1, 22.1 3 3 Immunofluorescence and confocal analysis mm ± 3.3 and 9.6 mm ± 4.7, respectively (Figure 2B). Immunofluorescence was performed on 20-μm coronal Cortex, the brain area involved in the ischemic lesion sections according to the previously described method was considered for neuronal count (Figure 2C). Six hours [22]. Primary antibodies used were: anti-mouse CD45 after ischemia, neuronal count performed in the ipsilateral (1:800 or 1:1500); anti-mouse Ym1 (1:400, Stem Cell cortex revealed a significant cell loss when compared to Technologies, Vancouver, Canada), anti-mouse CD206 the corresponding area in the sham-operated group (1:100, Serotec, Kidlington, UK), anti-mouse CD11b (84.9%). Neuronal counts progressively but slowly (1:30000, kindly provided by Dr. Doni), anti-mouse CD68 decreased reaching 64.9% at 7d. No significant difference (1:200, Serotec, Kidlington, UK), anti-mouse NeuN (1:250, was found between ispilateral and contralateral side in Millipore, Billerica, MA, USA). Fluorconjugated secondary sham-operated animals (data not shown). antibodies used were: Alexa 546 anti-rat, Alexa 594 anti- At 6 h after pMCAO rare TUNEL-positive cells were rabbit, Alexa 488 anti-mouse (all 1:500, Invitrogen, Carls- present in the injuried cortex indicating the presence of bad, CA). Biotinilated anti-rat antibodies (1:200, Vector few dying cells (30.2 ± 14.2, expressed as cell density per Laboratories, Burlingame, CA) were also used followed by mm , Figure 2D). Number of dying cells progressively fluorescent signal coupling with streptavidine TSA ampli- increased at 12, 24 and 48 h post ischemia (278.6 ± 51.1, fication kit (cyanine 5, Perkin Elmer, MA, USA). Similarly 589.7 ± 77.3 and 708.8 ± 30.2, respectively). Seven days to what reported for immunohistochemistry DAB staining, after ischemia still several TUNEL-positive cells were pre- also in this case we considered only cell displaying CD45 sent (343.6 ± 120.0) indicating the persistence of dying high rounded morphology (CD45 , [22]). Appropriate nega- cells at this time point. Positive TUNEL staining was not tive controls without the primary antibodies were apparent in any sham-operated mice at any time points. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 5 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 2 Histopathological findings at different time points from pMCAO. A: representative brain coronal sections obtained 24 h after pMCAO. Pale demarcated areas depict the ischemic lesion. Quantification of ischemic volume (B), neuronal counts (data obtained from the mean of 33 frames/mouse and expressed as % of sham groups, C) and TUNEL-positive cells (D) in the cortex of sham and ischemic mice at different times after pMCAO. Data are reported as mean+sd, n = 8. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham; °p < 0.05, °°p < 0.01, °°°p < 0.001 versus 6 h, Bonferroni’s Multiple Comparison Test. Time-course of expression of M/M markers: CD11b, CD45, cells with hypertrophic soma endowed with thick branches CD68, Ym1, CD206 (Figure 3A/E). high The M/M markers expression was analyzed within the No CD45-positive cells (CD45 cells, see methods, ischemic area based on the tissue sampling represented Figure 3F) could be observed in sham-operated mice in Figure 1. At each time point, the sampled cortical area and in the contralateral hemisphere of ischemic mice. pertained to the ischemic territory, being the number of Six hours after ischemia these cells were clearly visible neurons in this region decreased compared to sham ani- in the area considered (0.4 ± 0.2 percent of stained mals at every time points (Figure 2C). area). The immunoreactivity was further increased 12 CD11b, a constitutive marker of microglia and macro- and 24 h after ischemia (0.6 ± 0.2 and 1.1 ± 0.3, respec- phages was expressed at every time point considered as tively). No further increase could be observed at 48 h well as in sham-operated mice (5.6 ± 1.9, percent of (1.1 ± 0.4). CD45 staining was still present at 7d (0.9 ± stained area). Starting from 6 h the immunoreactivity 0.4, Figure 3F). increased and remained elevated at every subsequent time CD68 immunoreactivity was undetectable in sham- point considered, with no major differences throughout operated mice and appeared 6 h after ischemia (0.3 ± 0.2 the experimental groups (9.5 ± 1.5, 11.7 ± 1.6, 10.1 ± 1.7, percent of stained area). It progressively increased at 13.1 ± 2.8, 13.0 ± 0.1, respectively at 6 h, 12 h, 24 h, 48 h every time point considered (0.6 ± 0.2 at 12 h; 1.7 ± 0.2 and 7d, Figure 3B). at 24 h; 3.7 ± 0.8 at 48 h). Notably, a dramatic increase in Outside the lesion, CD11b staining revealed thin ramifi- the CD68 stained area could be observed at 7d (7.4 ± 1.4, cations and small soma (Figure 3A/C). CD11b immunor- Figure 3G). eactivity was associated with a different morphology in Ym1 immunoreactivity was detectable starting from 12 relation to the cell localization in the lesioned area. Two h (0.04 ± 0.02 percent of stained area). This marker was main areas were identified, namely a lesion border show- maximally expressed at 24 h (0.84 ± 0.16) and markedly ing CD11b+ highly ramified cells (Figure 3A/D) and an decreased at later time points (0.37 ± 0.10 at 48 h and ischemic core showing both CD11b+ ameboid cells and 0.23 ± 0.15 at 7d, Figure 3H). Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 6 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 3 Immunohistochemical analysis and quantification of microglial markers: CD11b, CD45, CD68, Ym1, CD206. A: representative micrographs of CD11b immunostaining in the injured cortex at 24 hours after pMCAo. CD11b-positive cells display different morphology depending on their localization in the ischemic area (C: outside the lesion, D: border zone, E: ischemic core). B: quantification of CD11b immunostaining at different times after pMCAo. Representative micrographs of CD45 (F), CD68 (G), Ym1 (H), CD206 (I) immunoreactivity at 24 hours after ischemia, and related quantifications at different times after pMCAO (Bar 10 μm). Data are expressed as mean+sd of 33 frames/ mouse (24 frames/mouse for CD45, TUNEL, CD206), n = 8. One way Anova: p < 0.0001. *p < 0.05, **p < 0.01 vs sham; °°p < 0.01, °°°p < 0.001 vs 6 h (12 h for Ym1). Bonferroni’s Multiple Comparison Test. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 7 of 19 http://www.jneuroinflammation.com/content/8/1/174 CD206 positive cells were present in sham-operated present throughout the entire ischemic area (Figure 4). mice (7.3 ± 0.9 cell/mm ). They could be observed 6 h CD45 cells visible at 10× magnification (Figure 4) did low after pMCAO (12.2 ± 6.2) and significantly increased not reveal the presence of CD45 cells (corresponding progressively up to 24 h (23.6 ± 5.3 at 12 h and 40.0 ± to ramified microglia) appearing in the CD11b staining 14.9 at 24 h). A significant number of CD206 positive microphotograph. Conversely CD68 appeared to be cellswas still presentat48h(30.5± 10.5)and at 7d mainly concentrated in the border zone, with rare cells (32.7 ± 8.8, Figure 3I). present in the ischemic core. Notably, at longer time points (7d) along with the great increase of its expres- Localization of M/M markers with respect to the lesion sion (Figure 3G), CD68 appears both in the border and Twenty-four hours after pMCAO, the immunoreactivity in the core areas. Ym1 at 24 h after pMCAO appeared for CD11b appeared to be evenly distributed in the exclusively expressed in the ischemic core, similarly to ischemic area, being present both in the lesion border CD206 (Figure 4). With the exception of CD68, all the and in the ischemic core (Figure 4). At the same time markers considered showed a similar distribution at CD45 staining showed a similar distribution being every time point (data not shown). Figure 4 Distribution of the selected M/M markers inside the ischemic lesion at 24 h from the injury. Representative immunostaining micrographs show spatial distribution of M/M phenotype markers (CD11b, CD45, Ym1, CD206, CD68) into the ischemic area at 24 h after ischemia (upper panels). Bar = 250 μm. Drawings representing the immunostaining data (lower panels). Only immunoreactivity for CD11b could be observed outside the lesioned area in basal conditions (sham-operated mice). In the lesion, CD11b+ cells showing a ramified to globular morphology could be observed going from the border zone to the ischemic core (red). CD45 staining was present throughout the entire ischemic area (pink). Conversely CD68 appeared to be strongly concentrated in the border zone. Ym1 and CD206 were exclusively expressed in the ischemic core. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 8 of 19 http://www.jneuroinflammation.com/content/8/1/174 Coexpression of M/M markers at 24 h and 7d after CD68 was greatly increased both in globular CD11b+ pMCAO cells in the ischemic core and in ramified CD11b cells Twenty-four hours after ischemia CD68 was expressed in laying in the border zone (Figure 4 and Figure 5C-D). hypertrophic ameboid CD11b cells present in the At 24 h after pMCAO Ym1 positive cells co-labeled ischemic core and in ramified microglia in the border with CD11b globular cells within the ischemic core, zone where CD68 positive cells were mostly located (Fig- where they were exclusively located (Figure 4 and Figure ure 4 and 5A-B). A similar pattern of coexpression could 6A-B-E-F). Seven days after ischemia Ym1 and CD11b be observed at 7d. At this time point the expression of Figure 5 Coexpression of CD11b (red) and CD68 (green) 24 h (A-B) and 7d (C-D) after pMCAO. In the ischemic core at 24 h CD11b positive cells are prevalently globular and some of them are positive to CD68 (A). In the border zone (B) CD11b cells display rounded cell bodies and ramified processes positive to CD68. Globular CD11b cells in the lesioned area 7d after ischemia mostly express CD68 marker (C). A high number of CD11b cells displaying different morphology colabel with CD68 in the border zone (D). Data are representative of 3 independent experiments. Bars: 20 μm. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 9 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 6 Coexpression of CD11b (red) and Ym1 (green) at 24 h (A-B-E-F) and 7d (C-D-G-H) after pMCAO. Ym1 positive cells co-label with globular CD11b postive cells at both time points (A, C). High magnifications (E-G) and 3D rendering (F-H) show colabeling of markers further highlighting the coexpression (blu = nuclei, bar: 5 μm). Consistent with the observation that no Ym1 cells are present in the border zone (Figure 4), no immunostaining for Ym1 at neither time points could be observed in this area (B, D). Data are representative of 3 independent experiments. Bars: 20 μm. coexpression pattern was similar to that observed at 24 h At 24 h after pMCAO, the few CD68+ cells found in (Figure 6C-D-G-H). the ischemic core did not colocalize with Ym1+ cells CD206 at 24 h was present exclusively in the ischemic that were present exclusively in this area (Figure 4 and core (Figure 4) where it colocalized with globular Figure 8A-B). In the magnification of Figure 8E-F it is CD11b positive cells (Figure 7A-B-E-F). The same pat- possible to observe that even when these markers tern of coexpression was observed at 7d (Figure 7C-D- appear closely related, they actually belong to distinct G-H). cells. At longer times (7d) Ym1 cells not colocalizing Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 10 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 7 Coexpression of CD11b (red) and CD206 (green) at 24 h (A-B-E-F) and 7d (C-D-G-H) after pMCAO. At 24 h after ischemia some globular CD11b cells co-label with CD206 marker in the ischemic core (A). Seven days after ischemia CD11b cells are prevalently globular in the ischemic core and are highly positive to CD206 (C). High magnifications (E-G) and 3D rendering (F-H) show colabeling of markers further highlighting the coexpression (blu = nuclei, bar: 5 μm). Consistent with the observation that no CD206 cells are present in the border zone (Figure 4), no immunostaining for this marker at neither time points could be observed in this area (B, D). Data are representative of 3 independent experiments. Bars: 20 μm. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 11 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 8 Coexpression of CD68 (red) and Ym1 (green) at 24 h (A-B-E-F) and 7d (C-D-G-H) after pMCAO.At 24hCD68positivecells found in the ischemic core do not co-localize with Ym1 positive cells (A). In high magnifications panels (E-F) the two markers appear to belong to different cells although in close contact (blu = nuclei). Seven days after ischemia, when CD68 immunoreactivity is greately increased, Ym1 appears to be expressed also, but not exclusively in CD68 positive cells (C). Note the presence of one Ym1 positive cells (arrow) that does not co-localize with CD68. High magnifications (G) and 3D rendering (H) show colabeling of markers further highlighting the coexpression (blu = nuclei). Consistent with the observation that no Ym1 cells are present in the border zone (Figure 4), no immunoreactivity for this marker could be observed at neither time points in that area (B, D). Data are representative of 3 independent experiments. Bars: 20 μm. High magnifications and 3D rendering bar: 5 μm. with CD68 are still present, however coexpression with in CD68 positive cells is apparent in the ischemic core CD68 can also be seen (Figure 8C-D-G-H). (Figure 9C-G-H). CD206 marker is not present in the A small fraction of CD206 positive cells show coex- border zone at both time points (Figure 9B-D). pression with CD68 at 24 h after ischemia in the Ym1 and CD206 appeared to be coexpressed in the ischemic core (Figure 9A-E-F). Similar situation is ischemic core both at 24 h (Figure 10A-C-D) and 7d observed at 7d after pMCAO when a dramatic increase (Figure 10B-E-F) after pMCAO. None of the two Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 12 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 9 Coexpression of CD68 (red) and CD206 (green) at 24 h (A-B-E-F) and 7d (C-D-G-H) after pMCAO. At 24 h and 7d after ischemia, a minor part of CD68 positive cells found in the ischemic core colocalize with CD206 (A, C). High magnification (E-G) and 3D rendering (F-H) show both single- and double-positive cells in the ischemic core (blu = nuclei, bar: 5 μm). Consistent with the observation that no CD206 cells are present in the border zone (Figure 4), no immunoreactivity for this marker could be observed at neither time points in that area (B, D). Data are representative of 3 independent experiments. Bars: 20 μm. Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 13 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 10 Coexpression of Ym1 (red) and CD206 (green) at 24 h (A-C-D) and 7d (B-E-F) after pMCAO. At 24 h and 7d after ischemia Ym1 positive cells co-label with CD206 positive cells in the ischemic core (A-B) Bars: 20 μm. High magnification (C-E) and 3D rendering (D-F) show coexpression of markers with the same cell nucleus (blu). Bar: 5 μm. Consistent with the observation that neither Ym1 cells nor CD206 cells are present in the border zone (Figure 4) no immunostaining for these markers could be observed at neither time points in this area (data not shown). Data are representative of 3 independent experiments. markers was present in the border zone at both time A summary of M/M markers coexpression 24 h and points considered (Figure 4). 7d after the ischemic lesion is reported in Figure 11. As expected all CD11b globular, CD68 globular, Ym1 Lastly, to provide additional details on the functional high and CD206 positive cells were all positive for CD45 status of M/M, we assessed their relationship with neu- in both ischemic core and border zone (data not rons (NeuN+). We analyzed CD11b/CD68 and CD11b/ shown), being CD45 a common marker for immune cell Ym1 double positive cells as these populations showed populations [31,32]. to increase at different time points, thus suggesting Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 14 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 11 Summary of M/M markers coexpression 24 h and 7d after the ischemic lesion. At 24 h after injury (A) immunoreactivity for CD11, which is readly increased after ischemia, is expressed in ramified and globular cells. CD68 is present in a percentage of both globular and ramified CD11b+ cells. Ym1 and CD206 that are present mostly in the core of the lesion, are expressed by a fraction of globular CD11b+ cells and can be present on the same cells. A few cells coexpressing CD206 and CD68 can be found in the area between the core and the border zone where the two markers are mainly located respectively. At 7d after injury (B) Ym1 decreases while CD68 expression greatly increases and from the border zone where it was at earlier times it invades the ischemic core (see also data in Figure 3). A few CD68+ cells appear now to express Ym1. distinct functional states. CD11b stain of M/M mem- neurons were positive for CD68 at both zones (Figure branes was chosen for documenting the morphology of 12A-B-C-D), suggesting an active phagocytosis. None of M/M when contacting neurons. We found that neurons the CD11b/Ym1 double positive cells at 24 h appeared were often enwrapped by CD11b positive cells in both to be engaging a phagocytic interaction with neurons, ischemic core and border zone at both 24 h and 7d being these cells never in contact with NeuN positive (Figure 12). In most cases CD11b cells surrounding cells (Figure 12E). At 7d, a few CD11b/Ym1 double Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 15 of 19 http://www.jneuroinflammation.com/content/8/1/174 Figure 12 Coexpression of CD11b (red) and NeuN (blue) with CD68 (green) at 24 h (A-A’-B-B’) and 7d (C-C’-D-D’) or with Ym1 at 24 h (E-E’-F-F’) and 7d (G-G’-H-H’) after pMCAO. In the ischemic core CD11b/CD68 double positive cells envelop NeuN positive cells, possibly indicating phagocytosis of neurons (A; 3D rendering in A’). The same interaction was observed in the border zone (B-B’). At 7d after ischemia, when CD68 expression is enhanced (Figure 3) in both ischemic core and border zone, CD11b/CD68 double positive cells enwrap neurons, suggesting active phagocytosis also at this time point (C-C’-D-D’). At 24 h after pMCAO in the ischemic core, where Ym1 positive cells are exclusively located, CD11b/Ym1 double positive cells do not appear involved in a phagocytic interaction with neurons (NeuN positive cells, E; 3D rendering on E’). CD11b single positive cells in both ischemic core (E-E’) and border zone (where Ym1 is not expressed, F-F’) surround neurons. At 7d after ischemia part of CD11b/Ym1 double positive cells engage a phagocytic appearance by enveloping neurons in ischemic core (G-G’) coherently with their partially CD68 positive phenotype at this time point (Figure 8). In the border zone at 7d (Ym1 is absent) CD11b single positive cells still enwrap neurons (H-H’). Data are representative of 3 independent experiments. Bar: 20 μm. positive cells showed a phagocytic appearance envelop- the lesioned area with no apparent major changes during ing neurons (Figure 12G), coherently with their partially time, with the exception of CD68. CD68 positive phenotype at this time point (Figure 8). We have firstly determined the histopathological fea- In the border zone, at both time points, Ym1 was not tures of the lesion induced by pMCAO. From the analysis detectable and only single CD11b positive cells did of the temporal evolution of the lesion it appears that the envelop neurons (Figure 12F-H). percentage of neuronal loss is somehow stable from 24 h up to 7d although the persistence of TUNEL-positive Discussion cells at this late point indicates that some cells may still be in degeneration at that late time. It should be noted Our study shows that the ischemic lesion is accompanied by activation of specific M/M phenotype that presents that assessing the lesion volume by the paleness of the distinctive spatial and temporal features. We have cresyl violet staining may lead to misleading conclusions demonstrated that: 1) the ischemic lesion induces the since, as detailed below, invading inflammatory cells may expression of the selected M/M markers that develop contribute to the apparent reduction of the lesioned area over time, each with a specific pattern; 2) the selected at 7d. Actually the quantification of the CD11b and high markers are associated with globular or ramified CD11b CD45 immunoreactivity indicates that inflammatory morphology, 3) each marker has a given localization in Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 16 of 19 http://www.jneuroinflammation.com/content/8/1/174 cells rapidly increase in number and/or size early after and cell surface antigens of microorganisms. Its localiza- the injury and at every time point considered. tion and predominance in phagocytic macrophages impli- M/M play a pivotal role in surveillance and response to cates CD68 in phagocytosis [43,44]. We observed that the altered CNS conditions [1,2,4]. An emerging concept is early increase in CD68 immunoreactivity is concentrated that, similarly to what happens for peripheral macro- in the border zone and expressed in ramified CD11b posi- phages, these cells can exert different antithetic functions tive cells. At later time points a dramatic increase in CD68 depending on environmental signals, acting as major expression appears both in the border zone and the players in the pro-inflammatory cytotoxic response, ischemic core and is apparent in globular CD11b cells. At both time points and in both zones, CD11b/CD68 double but also participating in the immunosuppressive and self-repair processes [6,33,34]. The phenotype markers positive cells appear to physically interact with neurons considered in this study include classical markers of M/M and show a phagocytic-like morphology characterized by activation (CD11b and CD45) and markers expressed by neuron engulfment. The phagocytic activity of alterna- alternatively activated macrophages (CD68, Ym1 and tively activated M/M is associated to clearance of cells CD206). Although evidence of M2 activation state in the debris, of damaged or dying cells and of infiltrating neu- brain has been reported in M/M in AD models [35,36], trophils thus resulting in the elimination of several poten- following global ischemia [37], in models of experimental tially cytotoxic substances [4,18,21,45,46]. However the autoimmune encephalomyelitis [38] or spinal cord injury overall functional meaning of phagocytosis in acute brain [16,39], information on M2 marker expression, coexpres- injury is still an open question. Actually the protective sion and temporal evolution in the injuried brain is lack- effect of manipulations such as stem cell infusion may be ing. We could observe that these phenotype markers are associated with a decrease in CD68 expression [47]. exclusively expressed by CD11b cells and that each of Ym1 belongs to the lectin family and is constitutively them shows distinct features in terms of time course of expressed by liver, lung and bone marrow, consistently activation and localization in relation to the ischemic with the fact that these are the original sources of myeloid lesion. cells [48]. It is synthesized and secreted by activated CD11b is expressed on the surface of many leukocytes macrophages during inflammation and exhibits a pH- and is a widely used maker of M/M. It belongs to a family dependent, specific activity towards GlcN oligomers and of cell surface receptors known as integrins. It is covalently heparin. Ym1 may control leukocyte trafficking by com- bound to a beta 2 subunit to form integrin a b (Mac-1, peting with them for binding sites on local extracellular M 2 CD11b/CD18) which is implicated in diverse responses matrix, an action resulting in down-regulation of inflam- including cell-mediated killing, phagocytosis, chemotaxis mation. Our findings show that, similarly to what reported in peripheral macrophages, Ym1 is activated transiently and cellular activation. CD11b has the ability to recognize a wide series of ligands such as fibrinogen, iC3b fragment suggesting that it may be involved in the establishment of of the third complement component, ICAM-1, denatu- an inflammatory management control of the injured rated products, blood coagulation factor X [40]. Our data region [48]. Its expression is restricted to the ischemic show that CD11b staining increases at early time points core and it colocalizes with CD11b globular cells and with after ischemia, rapidly reaching a plateau of activation. some CD68 cells at later times only. None of the CD11b/ Notably, CD11b positive cells display a different morphol- Ym1 double positive cells is associated with phagocytosis ogy in relation to the lesion, namely they are ramified in of neurons at 24 h, whilst at 7d some of them show a pha- the border zone and ameboid in the ischemic core. Simi- gocytic appearance and envelop neurons, coherently with high larlytoCD11b, alsoCD45 cells increase rapidly after their partially CD68 positive phenotype at this time point. ischemia. These cells, display a rounded morphology and An increase in Ym1 expression has been associated to the most probably correspond to recruited macrophages, neu- beneficial effect of stem cell infusion in mice subjected to trophils and lymphocytes [22,32,41]. This study does not global ischemia [37], in line with a protective role in acute specifically address the question of differentiating between brain injury. invading macrophages and resident microglia. An impor- Another marker of alternatively activated macrophages is tant future direction will be to identify specific molecular/ CD206 or mannose receptor [45,49]. This is an endocytic phenotypical markers for these two cell populations, since receptor that binds both microbial glycans and self glyco- there is evidence that they may play a different role in the proteins carrying terminal mannose, fucose and N-acetyl- progression of brain injury [9,34,42]. glucosamine by interaction with its carbohydrate CD68 or macrosialin is a member of the lysosomal/ recognition domains (CRDs). Its known function is related endosomal-associated membrane glycoprotein (LAMP) to recognition and endocytosis of the carbohydrate portion of antigens for processing and presentation [50]. Our family and a member of the scavenger receptor family results show that CD206 expression significantly increases which recognizes a wide range of anionic macromolecules over time and colocalizes with Ym1 positive cells and with such as oxidatively modified lipoprotein, apoptotic cells Perego et al. Journal of Neuroinflammation 2011, 8:174 Page 17 of 19 http://www.jneuroinflammation.com/content/8/1/174 a fraction of CD68 positive cells that increase at later time The different states of M/M in the ischemic lesion points. reflect the complexity of these cells and their ability to Lastly, our data have been obtained in a model of per- differentiate towards a multitude of phenotypes depend- manent ischemia and may not be extended to an ischemia ing on the surrounding microenvironmental signals that with reperfusion paradigm. Notably the present data and can change over time. The inflammatory response that our previous results [22] indicate that the ratio of follows cerebral ischemia is regarded as a promising tar- high low CD45 /CD45 is dramatically different in these two get for stroke therapy. The data presented in this study provide a basis for understanding this complex response conditions, being much higher after pMCAO. This may be and for developing strategies resulting in promotion of a due to either a higher number of infiltrating cells and/or a lower survival of resident cells, thus indicating that in tran- protective inflammatory phenotype. sient ischemia the composition of the specific M/M popu- lations in the lesioned area is different. List of abbreviations BDNF: Brain Derived Neurotrophic Factor; CRDs: Carbohydrate Recognition Conclusions Domains; DAB: 3, 3’-Diaminobenzidine; GDNF: Glial cell-Derived Neurotrophic Factor; ICAM: Inter-Cellular Adhesion Molecule; IGF-1: Insulin Growth factor-1; In the ischemic lesion M/M express markers that show IL-1β: Interleukin-1β; LAMP: Lysosomal/endosomal-Associated Membrane distinct temporal expression, distribution and association Glycoprotein; M/M: Microglia/Macrophages; NO: Nitric Oxide; PBS: Phosphate with a definite cell morphology suggesting that different Buffered Saline; pMCAo: permanent Middle Cerebral Artery occlusion; ROS: Reactive Oxygen Species; TNF: Tumor Necrosis Factor; TUNEL: Terminal M/M populations are acting at the site of injury according Deoxynucleotidyl Transferase; dUTP Nick End Labeling. to well defined phenotype, time of activation and pattern of localization. Conceivably, at 24 h after insult, ramified Acknowledgements SF is a fellow of the Monzino Foundation. and phagocytic M/M surround the lesion, possibly acting as a barrier against further expansion of the lesion, whilst Authors’ contributions globular M/M committed to a protective phenotype (i.e. CP participated in the design of the experiments, carried out the experiments, acquired and interpreted the data, was involved in drafting the expressing Ym1 and CD206) populate the ischemic core manuscript. SF carried out the experiments, acquired and interpreted the with the primary function of resolving inflammation and data, was involved in drafting the manuscript. MGDS participated in the promoting wound healing. At later time points (7d) the design of the experiments, interpreted the data, was involved in drafting the manuscript. All authors read and approved the final manuscript. phagocytic behavior of M/M becomes prevalent in the whole lesioned area with numerous globular phagocytic Competing interests M/M invading the core territory. The observed switch The authors declare that they have no competing interests. towards the phagocytic phenotype is accompanied by the Received: 14 September 2011 Accepted: 10 December 2011 progressive reduction of the expression of the protective Published: 10 December 2011 Ym1. At 24 h protective Ym1 positive cells do not appear to References 1. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, be involved in neuron phagocytosis, differently from Dustin ML, Gan WB: ATP mediates rapid microglial response to local CD68 positive cells that show a close physical interac- brain injury in vivo. Nat Neurosci 2005, 8:752-758. tion with neurons. At 7d, when neuronal damage 2. Yenari MA, Kauppinen TM, Swanson RA: Microglial activation in stroke: therapeutic targets. 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