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PubMed1991

Differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain.

Brain macrophages and microglia play important roles in central nervous system (CNS) development, especially during regressive events in which particular neuronal and glial constituents are eliminated. The purpose of this study is to provide a complete map of brain macrophage and microglia distribution in all regions of the neuraxis from birth to sexual maturity. We have utilized morphology and immunostaining with the specific antibodies OX-42 and ED1 to distinguish between brain macrophages and microglia. Brain macrophages are large, round cells, 10-15 microns in diameter, with few or no cytoplasmic processes; these cells are ED1- and OX-42-immunopositive. Microglia have small cell bodies with numerous, ramified cytoplasmic processes. These cells are OX-42-positive, and ED1-negative. We found a specific pattern of distribution of brain macrophages, targeting specific cortical and subcortical areas transiently, including developing fiber tracts. These cells disappeared completely by the third postnatal week. In contrast, OX-42-positive microglia exhibited a gradual increase in number and were distributed uniformly throughout gray matter and within white matter tracts. These cells remain in the adult CNS, constituting the resident microglia population. We suggest that these two distinct phagocytic cell populations perform unique functions in the developing brain, including remodeling of restricted CNS areas by brain macrophages that is part of a normal morphological process.

Animals↗
PubMed1992

Co-cultures of microglia and astrocytes from kainic acid-lesioned adult rat hippocampus: effects of glutamate.

The long-standing question concerning the direct actions of glutamate on the membrane potential of astroglial cells in the central nervous system was addressed using the in vitro kainic acid-lesioned hippocampal slice preparation and primary cell co-cultures of astrocytes and microglia derived from such lesions. The ultrastructure of the lesioned hippocampus was examined to aid in the identification of the cells appearing in culture. In culture, microglia appeared as flat cells, less than 1 micron in thickness at the edge of the cell, but thicker (about 5 microns) near the nucleus. The cytoplasm was packed with granular inclusions. Microglia appeared in two morphological forms, amoeboid and ramified. The amoeboid form was characterized by a cell body with a single process, and was always observed 1 day after starting the cell culture. Such cells became less frequent after 1 week in culture. The ramified form appeared as a rounded cell, devoid of processes, and were frequently observed in older cultures (greater than 1 week). Microglia did not round up after exposure to dibutyrylcyclic adenosine monophosphate (cAMP), and did not stain for glial fibrillary acidic protein (GFAP). An ultrastructural examination of the lesion demonstrated that microglia were present and that they contained many cytoplasmic granules similar to lipofuscin-containing granules. No filaments were observed in the cytoplasm of microglia. By contrast, the cytoplasm of astrocytes in culture had far fewer granules, rounded up to dibutyryl-cAMP, exhibited multiple processes, and stained for GFAP. In slices, astrocytes had no lipofuscin-containing granules, but numerous cytoplasmic filaments were present.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Isolation and flow cytometric characterization of newborn mouse brain-derived microglia maintained in vitro.

Microglia have been identified in the white matter of developing and adult mouse brain using different murine macrophage markers. While several techniques for the isolation of murine microglia have been described, the small cell yields and partial purification have limited the progress of these studies. We now describe the isolation of murine microglia using a modification of McCarthy and de Vellis method. Brain tissues from 1-2 day old newborn mice were mechanically and chemically dissociated and maintained in in vitro culture for 3 weeks. In primary dissociated brain cultures, microglia are observed after 10 days migrating from small colonies. After 16-20 days, brain-derived microglia were isolated with high cell yields by continuous shaking of the cultures for 16 hr. In contrast to resident murine peritoneal macrophages, microglia express less Class II (Ia) antigen and a small percentage express L3T4 (CD4) antigen by flow cytometry.

Animals↗
PubMed1988

Major histocompatibility complex antigen expression on rat microglia following epidural kainic acid lesions.

Vigorous expression of major histocompatibility complex (MHC) class I and class I surface glycoproteins was observed on reactive microglia but not on astrocytes in the rat brain following lesions induced by epidural kainic acid (KA) on the cerebral cortex. The monoclonal antibodies used were OX18 against MHC class I, OX6 against MHC class II, OX1 against leukocyte common antigen (LCA), and W3/13 against pan-T lymphocytes. Astrocytes were marked by antibodies to glial fibrillary acidic protein (GFA) and S100b protein. The lesion differentially affected four zones: the central area of the lesion where most cells died; the peripheral zone surrounding the lesion where selective damage occurred; projection tracts from the lesioned area; and terminal fields of damaged neurons. In nonlesioned animals, class I expression was confined to vascular endothelial cells and some small glial cells. Following KA treatment, class I-positive round cells appeared in the central zone at day 1, peaked about day 5, and then slowly declined. In the peripheral zone, class I-positive microglia were present fron day 2 on. They demonstrated classical morphology for such cells, and in some cases arranged themselves in pyramidal profiles surrounding neurons. Reactive microglia were also class I positive along tracts of damaged neurons and in the terminal areas. The reaction was reduced to control levels 16-20 weeks after lesioning although some vascular endothelial cells and a few round cells still stained positively in the cystic area, which was the remnant of the central zone. Class II antigen expression first appeared in the form of round cells in the central zone of the lesion on day 1. These peaked at 5-7 days and declined thereafter. In the peripheral zone on day 5, some positive round or ameboid cells were found intermingled with typical reactive microglia. This reaction peaked at about 1-2 weeks and decreased thereafter. Class II-positive microglia appeared in fiber tracts and in the terminal areas on day 5, peaked after 2-3 weeks, and declined thereafter. Double immunostaining for class I and II antigens showed that there were significantly fewer class II- than class I-positive cells, but the morphology of the two groups was similar. No astrocytes stained positively for either group I or group II antigen. In both the primary and secondary lesioned areas, LCA staining was observed on the surface of reactive microglia. In the primary lesions there were also LCA-positive round cells in the central zone, but these were rare in the peripheral zone and the secondary lesioned areas.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Phagocytosis of myelin by microglia in vitro.

Previous experiments from this laboratory have shown that peritoneal macrophages in culture will phagocytize myelin. Myelin preopsonized with myelin antibodies is phagocytized to a much greater extent than untreated myelin, indicating that macrophages ingest myelin by an Fc receptor. The present work was undertaken to determine the characteristics of myelin phagocytosis by microglia, the resident macrophages of the central nervous system. Microglia isolated from 4-5 day primary cultures of newborn rat brains were shown to bind and phagocytize myelin labeled in the lipids by 14C-acetate. Both binding and phagocytosis as shown by the appearance of 14C-cholesterol ester were greatly increased if labeled myelin was preopsonized with antiserum to myelin basic protein or galactocerebroside. Both preopsonized and untreated myelin were phagocytized more actively by microglia than by peritoneal macrophages under the same culture conditions. Microglia cultured in the presence of GM-CSF showed slightly increased cholesterol ester production from opsonized myelin, but the effect of GM-CSF was significantly greater than myelin pretreated with control serum (34% increase) or untreated myelin (154% increase). There was no significant effect of GM-CSF on myelin phagocytosis by peritoneal macrophages. Cerebrospinal fluid containing immunoglobulin drawn from rabbits with acute EAE also opsonized myelin to increase phagocytosis by microglia, as has been previously shown with peritoneal macrophages. These results indicate that microglia may actively participate in myelin destruction in demyelinating diseases where myelin antibodies or a source of GM-CSF may be present.

Animals↗
PubMed1993

Inability to detect beta-amyloid protein precursor mRNA in Alzheimer plaque-associated microglia.

A close association between Alzheimer senile plaques and microglia (the resident mononuclear phagocytic system cells of the brain) is well documented. To determine whether microglia contain detectable beta-amyloid protein precursor (beta-APP) mRNA, the present study combined immunocytochemistry (LN3 antibody to label microglia) with in situ hybridization (full-length cRNA probe to detect all forms of beta-APP mRNA). We report that immunolabeled microglia, including those clustered around senile plaques, generally lack detectable beta-APP mRNA--suggesting that microglia are not synthesizing the plaque-associated amyloid. The possibility that, analogous to the process in other forms of amyloidosis, the resident mononuclear phagocytic cells ingest an amyloidogenic precursor and secrete amyloid was not examined. Recent demonstrations of interactions between immune-related factors and Alzheimer lesions suggest that beta-APP and its breakdown products, along with microglia and their secretory products, may work synergistically in an AD pathogenetic cascade.

Alzheimer Disease↗
PubMed1989

Ferritin immunohistochemistry as a marker for microglia.

An immunohistochemical analysis of formalin-fixed, paraffin-embedded brain sections was performed with antisera against holoferritin and the light(L)-subunit of ferritin. Sections immunostained using anti-glial fibrillary acidic protein (GFAP), Ricinus communis agglutinin-1 (RCA-1) stain for microglia and iron stain (Berlin blue stain) were compared. The L-subunit of ferritin was purified from normal human spleen according to the modified scrapie-associated fibrils purification, and the anti-serum was raised in a rabbit. Both ferritin antisera positively stained resting and, more markedly, reactive microglia, both of which were also stained with RCA-1 but not with GFAP. Ferritin-positive resting microglia were seen more abundantly in cerebral and cerebellar cortices than in white matter. The advantages of ferritin antisera over RCA-1 are as follows. (1) RCA-1 heavily stains blood vessels, while anti-ferritin does not, hence the microglial cells are more readily visualized with ferritin immunohistochemistry. (2) Reactive microglia and macrophages are more strongly stained with anti-ferritin. (3) The staining intensity of ferritin is independent of the length of tissue fixation in formalin. However, anti-ferritin is inferior to RCA-1 in staining resting microglia with a scanty cytoplasm, especially in the white matter, probably because the former recognizes cytoplasmic components, while the latter recognizes cell membrane. Iron stain only gave a reaction to microglial cells in brains with neurosyphilis and to hemosiderin-laden macrophages. Thus, in addition to RCA-1, ferritin antisera are useful as a microglia marker in formalin-fixed, paraffin-embedded sections.

Brain↗
PubMed1993

Microglia is a component of the prion protein amyloid plaque in the Gerstmann-Sträussler-Scheinker syndrome.

The microglial cell has been demonstrated as component of the cerebral amyloid plaque of Alzheimer's disease. Involvement of microglia in plaques of another cerebral amyloidosis, the Gerstmann-Sträussler-Scheinker syndrome (GSS), has found little attention. We examine here the presence of microglia in GSS plaques by immunohistochemistry and transmission electron microscopy. Paraffin sections from five brains of patients with GSS were immunolabelled with antibodies against prion protein, A4/beta amyloid protein, ferritin, leukocyte common antigen, HLA-DR, CD 68, and the MAC387 epitope for microglia and monocytes/macrophages; microglia was also labelled with the Ricinus communis agglutinin-1 lectin. Such (immuno)labelling demonstrated many delicate cell processes and occasional somata within and around prion protein plaques in all GSS brains. Microglial immunoreactivity was strongest with anti-ferritin and variable with anti-macrophage antibodies. Ultrastructural examination of brain tissue from one autopsy and one biopsy of GSS identified microglial cells in close proximity of amyloid plaque fibrils. Our observations demonstrate microglia as an important component of the amyloid plaque in GSS and suggest a major role for microglia in processing and deposition, or at least organization, of prion protein amyloid. Thus, plaques in both transmissible and non-transmissible cerebral amyloidoses seem to develop via similar pathogenetic mechanisms, irrespective of differences in etiology and molecular composition of the amyloid.

Brain↗
PubMed1990

Activation and proliferation of the isolated microglia by colony stimulating factor-1 and possible involvement of protein kinase C.

Microglia were isolated from primary mixed brain cell culture of normal newborn mice and then cultivated. They were able to be maintained in vitro for 1-2 months, but incorporated little [3H]thymidine under normal culture conditions. When treated with the conditioned medium of L929 mouse fibroblast cells as a crude CSF-1 (mouse macrophage-colony stimulating factor) or purified CSF-1, microglia showed morphological changes and increased in both cell number and [3H]thymidine uptake. In addition, crude CSF-1 increased lysosomal enzyme activity and superoxide anion formation of microglia up to 2 and 3.8 fold as control value, respectively. These effects of CSF-1 were not observed in the purified astrocyte culture. Purified microglia had CSF-1 receptors which were recognized by the anti-CSF-1 receptor antibody that arose from a peptide of a product of proto-oncogene, c-fms. 12-O-Tetradecanoylphorbol-13-acetate (TPA) also increased microglia cell number and their biochemical activities, suggesting the possible involvement of protein kinase C activation. Protein kinase inhibitors, such as staurosporine or H-7, inhibited the effects of both CSF-1 and TPA. These results indicate that microglia may be regulated in its biochemical and proliferation activities by CSF-1 and that this may occur via activation of protein kinase C.

Animals↗
PubMed1992

Specific transcellular staining of microglia in the adult rat after traumatic degeneration of carbocyanine-filled retinal ganglion cells.

The present work was undertaken to assess the fate of ganglion cell debris in the axotomized retina of adult rats and employed a new technique to label phagocytosing microglia via the internalized material. In the main experiment, transection axotomy was performed on the intraorbital segment of the optic nerve, and a fast-transported, vital fluorescent styryl dye (4Di-10ASP) was deposited at the ocular stump of the nerve in order to pre-label retrogradely the ganglion cells destined to die because of the axotomy. Optic nerve transection resulted in progressive degradation of ganglion cell axons, perikarya, and dendrites within the retina and in release of fluorescent material, which was then incorporated into cells identified as microglia. No other retinal cells stained, although astrocytes and Müller's cells also responded to neuron degeneration by accumulating glial fibrillary acidic protein. Incorporation of labelled material into microglia topo-chronologically paralleled the ganglion cell degeneration starting within the optic fibre layer (OFL) and proceeding towards the ganglion cell layer (GCL) and the inner plexiform layer (IPL) of the affected retina. Long-term labelling of microglia monitored up to 3 months after optic nerve transection indicated that labelled microglial cells persisted within the retina. Microglia displayed a strong territorial arrangement within the GCL and IPL, and staggered, bilaminated distribution in both layers. These studies directly prove that microglia in the retina can be transcellularly labelled during traumatic degeneration of ganglion cells. The findings suggest that microglial cells play an important role in axotomy-induced wound healing and removal of cell debris.

Animals↗
PubMed1991

Isolation and characterization of newborn rabbit brain-derived microglia.

We isolated brain microglia from newborn rabbits and maintained these cells in in vitro culture. Enriched populations of rabbit microglia share several characteristics of mononuclear phagocytes including intracellular staining for nonspecific esterase and acid phosphatase. Microglia express Fc receptors, generate superoxide anion, and stain positive with the lectin Ricinus communis. Rabbit brain microglia develop multinucleated giant cells and small colonies in in vitro culture. The cells are highly phagocytic in culture. Other investigators have recently demonstrated that rabbits can be infected with HIV-1 in vivo and that neurological symptoms occur only when HIV-1 infection was carried out in HTLV-1-infected rabbits. Brain microglia most likely play a central role in HIV-1 encephalopathy. The availability of rabbit brain microglia in in vitro culture, offers a valuable potential cell model to study HIV-1 infection in the central nervous system.

Animals↗
PubMed1990

Brain microglia constitutively express beta-2 integrins.

Localization of beta-2 integrins in normal and Alzheimer disease temporal cortex was studied immunohistochemically. Resting microglia were found to express constitutively CD11a (LFA-1), CD11b (Mac-1, CR3), CD11c (P150, 95; CR4), and CD18 (beta-2). They were also found to express constitutively leukocyte common antigen and the immunoglobulin receptor Fc gamma RI. The intensity of expression of each of these antigens was enhanced on reactive microglia in Alzheimer disease tissue. HLA-DR was detected on only a few microglia in control tissue, but was intensely expressed on large numbers of reactive microglia in Alzheimer tissue. These data are consistent with a leukocyte origin and a phagocytic role for microglia. They provide further evidence of an inflammatory response of brain tissue in Alzheimer disease. The microglia were found to make up 9-12% of the total glial population in gray matter and 7.5-9% in white matter.

Aged↗
PubMed1992

Microglia in the mature and developing quail brain as revealed by a monoclonal antibody recognizing hemopoietic cells.

The monoclonal antibody QH1, which recognizes quail endothelial and hemopoietic cells, was found to label microglia in the developing and mature brain of the quail. Forms of microglia similar to those described in mammals were labelled. Ameboid microglia predominated at embryonic stages, became less numerous in late embryonic development, and disappeared completely by day 10 post-hatch (P10). Poorly ramified microglia were present as early as day 5 of incubation (E5), and were progressively replaced by mature ramified microglia from E14 onwards. From P10 onwards, ramified microglia were the only microglial form seen in the quail brain.

Aging↗
PubMed1989

Microglia in retinae transplanted to the central nervous system.

Retinae from fetal rats and mice were transplanted to the brains of neonatal rats: retinae from fetal rats were transplanted to the brains of adult immunodeficient nude mice. Using immunocytochemistry with monoclonal antibodies directed against cell surface antigens on macrophages and lymphocytes, we examined the leukocyte populations in transplants. We have shown that the transplants become populated by macrophages which have the morphology and phenotype of microglia. Furthermore, we have shown that in xenogeneic transplants most, if not all, of these cells are derived from the host. The microglia in the transplant retinae are more numerous and less precisely distributed when compared to normal retinae. Some microglia, particularly those associated with large blood vessels, express antigens typical of reactive microglia, including Class II antigens. We find that large numbers of macrophages and microglia are associated with the outer segments of the photoreceptors. In the absence of the retinal epithelium the macrophages may phagocytose discs shed from the outer segments of rods. We suggest that microglia derived from the host may be an important component of the instability of xenogeneic grafts.

Animals↗
PubMed1989

The appearance and distribution of microglia in the developing retina of the rat.

We have examined the development of microglia in the rat retina, using a peroxidase-conjugated lectin derived from Griffonia simplicifolia. Retinas were studied from animals aged from E(embryonic day)12, just after the invagination of the optic cup and prior to the closure of the optic fissure, to adulthood. The lectin also proved a sensitive label for the endothelial cells of the developing retina. Our results provide some support for the view that microglia are derived from the monocyte-macrophage series of blood cells. At E12, most labeled cells were found at the vitreal surface, suggesting that they had come from the hyaloid circulation, while some had entered the retina and appeared to be migrating towards its ventricular surface. From E14 to early postnatal ages, most labeled cells had processes and resembled the amoeboid microglial cells described in silver carbonate staining studies (Ling, 1982). The number of labeled cells rose from about 700 to E14 to a peak of about 27,000 at P(postnatal day)7, and fell to about 19,600 by P12. As early as E16, a regularity was apparent in the distribution of microglial cells over the surface of the retina, the cells tending to avoid each other. Microglial cells are found throughout the thickness of the very young retina, but as the layers of the retina differentiate, they are increasingly restricted to the inner half of the retina. Our findings indicate that microglia enter the retina well before the period of neuronal death, making it unlikely that they invade the retina solely in response to cell death. Our results confirm however that, once in the retina, microglia become associated with, and appear to phagocytose, the pyknotic debris which appears during the period of neuronal death. They also become closely associated with the retinal vasculature. In the adult, the intensity of the labeling of microglia was much reduced. Those cells which were labeled appeared more differentiated, resembling the "resting microglia" described in earlier studies.

Animals↗
PubMed1992

The effects of pre-natal exposure to methylazoxymethanol acetate on microglia.

The effects of exposure to a cytotoxic agent, methylazoxymethanol acetate (MAM Ac), on the distribution, density and quantitative morphology of microglia in the rat forebrain have been examined with the aid of a peroxidase-conjugated lectin derived from Griffonia simplicifolia. Following exposure to MAM Ac (25 mg/kg maternal body weight) on embryonic day 13 (E13), round microglia were concentrated around the areas of induced cell death at the outer margins of the ventricular germinal zone, particularly in the striatopallidal angle and dorsal thalamus. By E19, there were no detectable differences in microglia distribution between experimental and control animals. The increase in number of microglial cells in the neocortex and caudatoputamen on exposure to MAM Ac lasted for only 4 to 6 days. Subsequently, the number of microglia dropped below control values in both regions. The density of microglia in these areas was similar in control and experimental animals from 6 days after exposure. The proportion of microglia relative to all other cells was also similar at post-natal day 17 (P17) in both experimental and control animals. These results suggest that the distribution and final size of the microglial population is determined by the microenvironment and not by the extent of cell death which may have acted as the initial stimulus to microglial invasion.

Animals↗
PubMed1991

Processing of beta-amyloid precursor protein in microglia and astrocytes favors an internal localization over constitutive secretion.

Microglial cells and astrocytes are closely associated with nearly all compact deposits of the amyloid beta-protein found in the senile plaques characteristic of Alzheimer's disease and trisomy 21. The biosynthesis and metabolic fate of the beta-amyloid precursor protein (beta APP) in astrocytes has not been characterized, and its identification in microglia has not been described. Here, we report the expression of beta APP by astrocytes and microglia in primary cultures of cerebral cortex from newborn rats. Using metabolic labeling followed by immunoprecipitation, we show that both astrocytes and microglia express substantial amounts of the major isoforms of beta APP. This is confirmed by PCR-mediated amplification of the corresponding mRNAs, showing that all three major transcripts (beta APP695, beta APP751, and beta APP770) are present in relatively equal amounts. Despite rapid turnover of the precursor, astrocytes and microglia show a reduced production of soluble fragments of beta APP compared to cells transfected with beta APP cDNAs. The relative amount of soluble beta APP molecules generated is both cell type and isoform specific. Immunocytochemistry reveals that full-length beta APP is located in internal membranous vesicles, with only very little insertion at the cell surface. The latter data are in agreement with the reduced ability of microglia and astrocytes to cleave the beta APP into soluble derivatives. Our findings indicate that both astrocytes and microglia strongly express all three major forms of beta APP but apparently process these molecules by an alternative pathway that generates very small amounts of soluble beta APP. The immunocytochemical localization and the biochemical data lead to the suggestion that beta APP may not function principally as a cell surface or secreted protein in vivo but may have an important intracellular function.

Amyloid beta-Protein Precursor↗
PubMed1996

Eicosanoids and nitric oxide influence induction of reactive gliosis from spreading depression in microglia but not astrocytes.

Microglia and astrocytes are transformed into reactive glia (RG) by brain disease and normal function. Eicosanoids and nitric oxide (NO), two intercellular mediators, may influence gliosis. We investigated how drugs that alter production of these paracrine signals effect induction of glial reactivity from spreading depression. Unilateral (left) neocortical spreading depression was induced in 95 halothane anesthetized rats by intracortical injections of 0.5 M KCl, with or without drug treatment (five animals/group). Immunohistochemical staining (IS) intensity using the OX-42 and anti-glial fibrillary acidic protein (GFAP) antibodies determined reactivity in microglia and astrocytes, respectively. After 3 days, brains were processed for OX-42 and GFAP-IS and mean optical densities (OD) of IS were measured. Average OD's (for OX-42) and the log ratio (left/right) of OD's (OX-42 and GFAP) were compared to normal animals. Spreading depression induced significant log ratios for both OX-42- and GFAP-IS (P's < 0.01). However, dexamethasone (a glucocorticoid), nordihydroguaiaretic acid (a lipoxygenase inhibitor), and nitroprusside (a NO donor) prevented significant left sided and log ratio OD values for microglia (P's > 0.05). L-Name, a NO synthase inhibitor, caused significant increases in left and right OD's for microglia (P's < 0.05). Mepacrine, a phospholipase A2 inhibitor, Indomethacin, a cyclooxygenase inhibitor, and phenylephrine, an adrenergic agonist, did not prevent induction of significant OX-42 log ratios (P's < 0.01, 0.05, 0.01), and resulted in increases in left side OD's (P's < 0.01, 0.05, 0.05). Significant GFAP log ratios occurred after spreading depression in all drug groups, P's < 0.01. Thus, induction of reactivity in microglia is more sensitive to eicosanoids and NO than in astrocytes.

Animals↗