Our study shows that the ischemic lesion is accompanied by activation of specific M/M phenotype that presents distinctive spatial and temporal features. We have demonstrated that: 1) the ischemic lesion induces the expression of the selected M/M markers that develop over time, each with a specific pattern; 2) the selected markers are associated with globular or ramified CD11b morphology, 3) each marker has a given localization in the lesioned area with no apparent major changes during time, with the exception of CD68.
We have firstly determined the histopathological features of the lesion induced by pMCAO. From the analysis of the temporal evolution of the lesion it appears that the percentage of neuronal loss is somehow stable from 24 h up to 7d although the persistence of TUNEL-positive cells at this late point indicates that some cells may still be in degeneration at that late time. It should be noted that assessing the lesion volume by the paleness of the cresyl violet staining may lead to misleading conclusions since, as detailed below, invading inflammatory cells may contribute to the apparent reduction of the lesioned area at 7d. Actually the quantification of the CD11b and CD45high immunoreactivity indicates that inflammatory cells rapidly increase in number and/or size early after the injury and at every time point considered.
M/M play a pivotal role in surveillance and response to altered CNS conditions [1, 2, 4]. An emerging concept is that, similarly to what happens for peripheral macrophages, these cells can exert different antithetic functions depending on environmental signals, acting as major players in the pro-inflammatory cytotoxic response, but also participating in the immunosuppressive and self-repair processes [6, 33, 34]. The phenotype markers considered in this study include classical markers of M/M activation (CD11b and CD45) and markers expressed by alternatively activated macrophages (CD68, Ym1 and CD206). Although evidence of M2 activation state in the brain has been reported in M/M in AD models [35, 36], following global ischemia , in models of experimental autoimmune encephalomyelitis  or spinal cord injury [16, 39], information on M2 marker expression, coexpression and temporal evolution in the injuried brain is lacking. We could observe that these phenotype markers are exclusively expressed by CD11b cells and that each of them shows distinct features in terms of time course of activation and localization in relation to the ischemic lesion.
CD11b is expressed on the surface of many leukocytes and is a widely used maker of M/M. It belongs to a family of cell surface receptors known as integrins. It is covalently bound to a beta 2 subunit to form integrin αMβ2 (Mac-1, CD11b/CD18) which is implicated in diverse responses including cell-mediated killing, phagocytosis, chemotaxis and cellular activation. CD11b has the ability to recognize a wide series of ligands such as fibrinogen, iC3b fragment of the third complement component, ICAM-1, denaturated products, blood coagulation factor X . Our data show that CD11b staining increases at early time points after ischemia, rapidly reaching a plateau of activation. Notably, CD11b positive cells display a different morphology in relation to the lesion, namely they are ramified in the border zone and ameboid in the ischemic core. Similarly to CD11b, also CD45high cells increase rapidly after ischemia. These cells, display a rounded morphology and most probably correspond to recruited macrophages, neutrophils and lymphocytes [22, 32, 41]. This study does not specifically address the question of differentiating between invading macrophages and resident microglia. An important future direction will be to identify specific molecular/phenotypical markers for these two cell populations, since there is evidence that they may play a different role in the progression of brain injury [9, 34, 42].
CD68 or macrosialin is a member of the lysosomal/endosomal-associated membrane glycoprotein (LAMP) family and a member of the scavenger receptor family which recognizes a wide range of anionic macromolecules such as oxidatively modified lipoprotein, apoptotic cells and cell surface antigens of microorganisms. Its localization and predominance in phagocytic macrophages implicates CD68 in phagocytosis [43, 44]. We observed that the early increase in CD68 immunoreactivity is concentrated in the border zone and expressed in ramified CD11b positive cells. At later time points a dramatic increase in CD68 expression appears both in the border zone and the ischemic core and is apparent in globular CD11b cells. At both time points and in both zones, CD11b/CD68 double positive cells appear to physically interact with neurons and show a phagocytic-like morphology characterized by neuron engulfment. The phagocytic activity of alternatively activated M/M is associated to clearance of cells debris, of damaged or dying cells and of infiltrating neutrophils thus resulting in the elimination of several potentially cytotoxic substances [4, 18, 21, 45, 46]. However the overall functional meaning of phagocytosis in acute brain injury is still an open question. Actually the protective effect of manipulations such as stem cell infusion may be associated with a decrease in CD68 expression .
Ym1 belongs to the lectin family and is constitutively expressed by liver, lung and bone marrow, consistently with the fact that these are the original sources of myeloid cells . It is synthesized and secreted by activated macrophages during inflammation and exhibits a pH-dependent, specific activity towards GlcN oligomers and heparin. Ym1 may control leukocyte trafficking by competing with them for binding sites on local extracellular matrix, an action resulting in down-regulation of inflammation. Our findings show that, similarly to what reported in peripheral macrophages, Ym1 is activated transiently suggesting that it may be involved in the establishment of an inflammatory management control of the injured region . Its expression is restricted to the ischemic core and it colocalizes with CD11b globular cells and with some CD68 cells at later times only. None of the CD11b/Ym1 double positive cells is associated with phagocytosis of neurons at 24 h, whilst at 7d some of them show a phagocytic appearance and envelop neurons, coherently with their partially CD68 positive phenotype at this time point. An increase in Ym1 expression has been associated to the beneficial effect of stem cell infusion in mice subjected to global ischemia , in line with a protective role in acute brain injury.
Another marker of alternatively activated macrophages is CD206 or mannose receptor [45, 49]. This is an endocytic receptor that binds both microbial glycans and self glycoproteins carrying terminal mannose, fucose and N-acetylglucosamine by interaction with its carbohydrate recognition domains (CRDs). Its known function is related to recognition and endocytosis of the carbohydrate portion of antigens for processing and presentation . Our results show that CD206 expression significantly increases over time and colocalizes with Ym1 positive cells and with a fraction of CD68 positive cells that increase at later time points.
Lastly, our data have been obtained in a model of permanent ischemia and may not be extended to an ischemia with reperfusion paradigm. Notably the present data and our previous results  indicate that the ratio of CD45high/CD45low is dramatically different in these two conditions, being much higher after pMCAO. This may be due to either a higher number of infiltrating cells and/or a lower survival of resident cells, thus indicating that in transient ischemia the composition of the specific M/M populations in the lesioned area is different.