Specific Immunolabeling of Brain Macrophages and Microglial Cells in the Developing and Mature Chick Central Nervous System

Abstract

The present study showed that the HIS-C7 monoclonal antibody, which recognizes the chick form of CD45, is a specific marker for macrophages/microglial cells in the developing and mature chick central nervous system (CNS). HIS-C7-positive cells were characterized according to their morphological features and chronotopographical distribution patterns within developing and adult CNS, similar to those of macrophages/microglial cells in the quail CNS and confirmed by their histochemical labeling with Ricinus communis agglutinin I, a lectin that recognizes chick microglial cells. Therefore, the HIS-C7 antibody is a valuable tool to identify brain macrophage and microglial cells in studies of the function, development, and pathology of the chick brain. CD45 expression differed between chick microglia (as revealed with HIS-C7 antibody) and mouse microglial cells (as revealed with an antibody against mouse form of CD45). Thus, a discontinuous label was seen on mouse microglial cells with the anti-mouse CD45 immunostaining, whereas the entire surface of chick microglial cells was labeled with the anti-chick CD45 staining. The functional relevance of these differences between species has yet to be determined.

Development of microglia in the chick embryo spinal cord: Implications in the regulation of motoneuronal survival and death

“…The appearance of some CD45-labeled cells in the lumen of the central canal coincides with the increase of these cells in the ependymal layer, suggesting that microglia are able to traverse the thick neuroepithelium of spinal cord and use the central canal as an additional route of migration. These observations are consistent with previous studies performed in other regions of the vertebrate CNS, including avians (Kawaguchi, 1980;Kurz and Christ, 1998;Martín-Partido and Navascués, 1990;Ashwell, 1991;Cuadros et al, 1993Cuadros et al, , 2006. It is interesting to note the high density of CD45-positive macrophages observed in the ventral and dorsal spinal nerve roots, close to areas in which microglia concentrate within the developing spinal cord.…”

“…From E3 to E10, CD45-positive cells in the spinal cord and surrounding tissues appeared as a homogeneous cellular population mainly displaying a round or ovoid (ameboid) shape. A similar morphology has also been described in the developing brain and retina of chicks (Cuadros et al, 2006) with CD45 immunocytochemistry. On E3, some CD45-positive cells were observed in paravertebral mesenchyme, particularly around the developing ventral and dorsal nerve roots, and in the meningeal primordium surrounding the spinal cord.…”

“…The following primary antibodies were used for immunocytochemistry: 1) mouse monoclonal antibody anti-chicken CD45, HIS-C7 (CD45, diluted 1/40; Cedi Diagnostics, Lelystad, The Netherlands; catalog 7500970; lot 05M005), which recognizes the avian form of CD45, a specific marker for macrophages/microglial cells in the developing and mature CNS (Cuadros et al, 2006); 2) mouse monoclonal antibody 1611 anti-HuC/D (Hu, diluted 1/50; Molecular Probes, Eugene, OR; catalog A21271, lot 45232A), raised against human neuronal protein HuC/HuD; anti-Hu antibody specifically labels neuronal cells in different vertebrate species, including chicken (Wakamatsu and Weston, 1997;Brunet et al, 2007); 3) rabbit anti-cleaved caspase-3 (Asp175) polyclonal antibody (diluted 1/50; Cell Signaling, Beverly, MA; catalog 9661, lot 18); 4) mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (diluted 1/400; Chemicon; catalog MAB369, lot 24091577); and 5) rat anti-BrdU monoclonal antibody (diluted 1/40; Abcam, Cambridge, United Kingdom; catalog ab6326, lot 263784).…”

Embryonic and postnatal development of microglial cells in the mouse retina

“…Anti-CD45 and anti-CD68 antibodies recognize mouse homologs of CD45 and CD68 molecules, respectively. CD45 is a tyrosine phosphatase protein present in the membrane of cells of monocyte/macrophage lineage (Penninger et al, 2001) and the antibody used, whose immunogen was purified B cells from mouse lymph nodes, recognizes a single band of Ͼ170 kDa in Western blot of mouse brain extracts (Cuadros et al, 2006). CD68 (macrosialin) is a membrane glycoprotein present in lysosomes of macrophage-lineage cells (Da Silva and Gordon, 1999); the antibody used in this study, raised against concavalin A acceptor protein from P815 cell line (manufacturer's technical information), recognizes bands of 87-115 kDa corresponding to different degrees of protein glycosylation related to cell activation and phagocytosis (Da Silva and Gordon, 1999 After permeabilization in 0.1% Triton X-100 (Sigma, St. Louis, MO) in PBS (pH 7.4), sections were incubated with normal goat serum (Sigma) diluted 1:30 in PBS-1% bovine serum albumin (PBS-BSA) for 45-60 minutes.…”

“…The distribution of microglial cells in the adult retina has been described in fish (Dowding et al, 1991;Salvador-Silva et al, 2000), amphibians (Goodbrand and Gaze, 1991), birds (Navascués et al, 1994;Won et al, 2000;Cuadros et al, 2006), and mammals, including rabbits (Ashwell, 1989;Schnitzer, 1989;Humphrey and Moore, 1996), mice (Zhang et al, 2005b), rats Harada et al, 2002;Zhang et al, 2005a), monkeys (Vrabec, 1970;Boycott and Hopkins, 1981), and humans (Penfold et al, 1991(Penfold et al, , 2001Provis et al, 1995;Yang et al, 2000;Gupta et al, 2003). It has been observed in these species that microglial cells in the adult normal retina appear in the ganglion cell layer (GCL) and all fibrous layers, whereas they are scarce in the inner nuclear layer (INL) and absent in the outer nuclear layer (ONL).…”