Resident Microglial Cells Research Articles

Reactive oxygen species are involved in the pathogenesis of experimental allergic encephalomyelitis in Lewis rats

During experimental allergic encephalomyelitis (EAE), both blood-borne macrophages as well as activated, resident microglial cells are considered to be involved in inflammatory reactions in the central nervous system (CNS), resulting in the neurological deficits common to EAE. Both cell types can produce multiple mediators of tissue damage, among which are the reactive oxygen species (ROS). In this study we show that macrophages and microglial cells, isolated from the CNS of Lewis rats with clinical signs of EAE, exhibited significantly elevated spontaneous and phorbol myristate acetate (PMA)-inducible levels of ROS compired to similar cells isolated from healthy controls, sham (complete Freund's adjuvant, CFA)-immunized rats as well as rats sacrificed before the manifestation of clinical signs of EAE. However, during clinical EAE, peripheral blood mononuclear cells (PBMC) did not show increased spontaneous nor PMA-inducible ROS production compared to controls. In vivo treatment of EAE with catalase, which scavenges the ROS H 2O 2, markedly suppressed the severity of the disease as compared to sham (albumin)-treated controls. In contrast, Superoxide dismutase had no effect on clinical signs. Our studies point at a putative functional role for ROS, and in particular H 2O 2, in the pathogenesis of EAE.

Evidence for activation of astrocytes via reactive microglial cells following hypoglossal nerve transection.

Following peripheral nerve injury, resident microglial cells proliferate and astrocytes undergo hypertrophy, as evidenced, e.g., by an increase in the levels of glial fibrillary acidic protein (GFAP). In a previous study we have shown that infusion of cytosine arabinoside (ARA-C) into the rat brain blocks the axotomy-induced proliferation of microglial cells. This experimental approach has been used in the present study in order to explore the issue of whether the reactive microglial cells are mediators of the increased GFAP expression in the hypoglossal nucleus of the rat following axotomy. Quantitative analysis of sections processed for immunocytochemistry or in situ hybridization demonstrated a marked increase in GFAP-like immunoreactivity and GFAP-mRNA, respectively, in the ipsilateral hypoglossal nucleus 4 and 7 days after axotomy in control experiments. These increases failed to occur in axotomized animals treated with ARA-C. Therefore, our data are compatible with the hypothesis that activation of astrocytes following axotomy as measured by increased expression of GFAP and its mRNA is induced secondarily to the microglial response.

The Relationship of Microglial Cells to Dying Neurons During Natural Neuronal Cell Death and Axotomy-induced Degeneration of the Rat Retina.

The interactions between dying neurons and phagocytotic cells within the developing and injured retina remain controversial. The present work explored the role of microglia and investigated whether so-called resident microglial cells are permanently responsible for removing cell debris whenever it is produced. As a first goal, I characterized some quantitative and morphometric features of the small ipsilateral retinocollicular projections and analysed the permanent function of phagocytosing microglia with these projections as a paradigm. To achieve this, I combined the fluorescent dyes Dil and 4Di-10ASP, both of which persist in the labelled ganglion cells after injection into the superior colliculus (SC), and retrograde labelling. After neuronal degradation, the dyes accompany the degradation products, become interiorized and then persist within the phagocytosing microglia. Consequently, early labelling of microglial cells can be assessed by injecting one dye into the SC during the first postnatal day of life, that is, prior to advanced natural neuronal cell death. Labelling of the remaining ipsilaterally projecting neurons with the second dye following intraorbital axotomy in adulthood and during subsequent neuronal death would therefore result in double labelling of some microglial cells, if these were involved in phagocytosis during both the natural and the induced phases of neuronal degradation. The ganglion cells which survived natural neuronal cell death remained fluorescent for 3 months after labelling with either dye on the day of the animal's birth, indicating that both fluorescent probes persisted within neurons. Quantitatively, 1770+/-220 ganglion cells/mm2 were labelled within the contralateral retina and a total population of 1442+/-120 cells/retina were observed within the periphery of the inferior/temporal quadrant of the ipsilateral retina. A smaller, ipsilateral projection of 150+/-24 cells/retina was uniformly scattered throughout the rest of the retinal surface. Transient projections of ganglion cells to either the contralateral or the ipsilateral colliculi and death of labelled ganglion cells during the first postnatal days resulted in labelling of 210+/-36 microglial cells/mm2 within the contralateral retina and a total number of 800+/-120 cells/retina within the inferior/temporal and 200+/-22 cells/retina within the rest of the retina. These labelled microglial cells were observed in adulthood and indicated that after taking away the neuronal cell debris they persisted within the retinal tissue. The small number of prelabelled ganglion cells which formed persistent ipsilateral projections until adulthood were axotomized by transecting the optic nerve, and resulted in additional labelling of microglial cells with the second fluorescent dye as well. Double-labelled microglia were observed within the inferior/temporal quadrant (3500+/-240 cells/retina) and to a lesser extent (340+/-40 cells/retina) scattered over the entire retinal surface. The chronotopological sequence of microglial labelling paralleled that of ganglion cell degeneration. Injection of protease inhibitors into the vitreous body during optic nerve transection retarded retrograde glial cell degeneration, probably by blocking microglial proteases. The results directly proved that the same microglial cells which remove neuronal cell debris in the postnatal retina were reactivated later in life to proteolytically degrade and then phagocytose neurons which had altered because of the axotomy.

Characterisation of two new monoclonal antibodies directed against rat microglia

With the aid of cultured rat microglial cells as immunogen, we raised two monoclonal antibodies, designated murine clone (MUC) 101 and 102, which recognised subsets of resident microglial cells in the normal central nervous system and cells of the mononuclear phagocyte system in peripheral organs. These antibodies were characterised by immunoperoxidase immunocytochemistry, immunoelectron microscopy, and immunoblotting. The immunostained cells were identified as microglial cells by double-immunofluorescence labelling with the B4-isolectin from Griffonia simplicifolia, an established microglial cell marker. Under normal conditions, both antibodies labeled resident microglia but with different distribution patterns. Under pathological conditions, e.g., after facial nerve transection, they labeled activated, perineuronal microglia in the operated facial nucleus. Immunoelectron microscopy demonstrated a membrane localisation of the antigen recognised by MUC 102. In peripheral organs, MUC 101 and 102 reacted with different cell populations of the mononuclear phagocyte system, particularly in thymus, spleen, and peripheral lymph node. Western blot experiments showed that MUC 101 recognised two proteins of 116 and 95 kD in fractions obtained from operated facial nucleus while MUC 102 reacted with two proteins of 62 and 70 kD molecular weight. These immunocytochemical results 1) confirm the antigenic similarity between microglia and cells of the monocyte-macrophage cell lineage, and 2) indicate that considerable antigen heterogeneity might exist among resident microglia. MUC 101 and 102 could thus become useful for studying the function of microglial cells both under normal and pathological conditions.

Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.

In addition to the major population of infiltrating leukocytes recovered from inflamed rat central nervous system (CNS), all of which expressed high levels of leukocyte common antigen CD45, many cells were coisolated that were MRC OX42+ (complement receptor 3/CD11b) but expressed low-to-moderate levels of CD45 and major histocompatibility complex (MHC) class I molecules. Most cells from normal CNS, in contrast, lay within this latter, CD45low population. From previous in situ immunohistochemical studies, the fortuitously isolated CD45low cells were probably resident (ramified) microglia. Using irradiation chimeras, we show that resident microglia respond to inflammation by upregulating CD45, CD4, and MHC class I molecules with a minority of these cells increasing their expression of MHC class II molecules. A 3- to 4-fold increase in the number of microglia isolated from inflamed CNS provided indirect evidence that the cells had proliferated. In normal CNS, a very small population of blood-derived CD45high-expressing cells are present; most MHC class II expression is associated with these few cells and not with the resident microglia.

Human microglial cells: Characterization in cerebral tissue and in primary culture, and study of their susceptibility to HIV‐1 infection

Neuropathological studies have shown that human immunodeficiency virus type 1-infected cells within the brain express several markers characteristic of macrophages and could either be microglial cells, or monocytes invading the CNS, or both. To better define the target cells of human immunodeficiency virus type 1 within the brain, we have studied human microglial cells, both in vivo and in vitro, and compared them to monocytes for their antigenic markers and their susceptibility to human immunodeficiency virus type 1 infection. Brain-derived macrophages were isolated from primary cortical and spinal cord cultures obtained from 8 to 12-week-old human embryos. The isolated cells presented esterase activity, phagocyted zymosan particles, expressed several (Fc receptors, and CD68/Ki-M7 and CD11b/CR3 receptors) of the macrophagic antigenic markers, and appeared to be resident microglial cells from human embryonic brain. Conversely, brain-derived macrophages did not express antigens CD4, CD14, or CD68/Ki-M6, which are easily detected on freshly isolated monocytes. Using these antigenic differences between isolated microglial cells and monocytes, we have observed that two populations of macrophages could be individualized. In the normal adult brain, microglial cells were numerous in both the gray and the white matter. The infrequent cells sharing antigens with monocytes were found almost exclusively around vessels. In 8 to 12-week-old human embryos, microglial cells were found in both the parenchyma and the germinative layer. Cells sharing antigens with monocytes were only found at the top of and inside the germinative layer. In brain tissue from patients with human immunodeficiency virus type 1 encephalitis, cells sharing antigens with monocytes are abundant not only around the vessels but also in the parenchyma. In double-labeling experiments, human immunodeficiency virus type 1-infected cells showed monocyte antigens. Finally, microglial cells also differ from monocytes in their in vitro susceptibility to human immunodeficiency virus type 1 infection; after stimulation by r-TNF alpha or GmCSF, monocytes but not microglial cells can replicate human immunodeficiency virus type 1. This in vitro difference in human immunodeficiency virus type 1 susceptibility between monocytes and microglial cells together with the presence of monocytic antigens within the brain tissue of human immunodeficiency virus type 1-infected patients suggest that human immunodeficiency virus type 1-infected cells within the brain are either monocytes that have crossed the blood-brain barrier and spread through the tissue or perivascular microglial cells that, after phagocyting infected blood lymphocytes, subsequently contain viral antigen and migrate to brain tissue.

Soluble factor(s) produced in injured fish optic nerve regulate the postinjury number of oligodendrocytes: Possible role of macrophages

Mammalian central nervous system (CNS) axons are virtually incapable of regenerating after injury. However, CNS neurons of lower vertebrates, such as fish and amphibians, are endowed with a high regenerative capacity. Lately, the glial cells have been credited with the regenerative ability of any specific CNS. We have previously demonstrated that many oligodendrocytes are recovered in cultures of injured rat optic nerve, while only a few oligodendrocytes are recovered from injured fish optic nerve in culture. We further demonstrated that medium conditioned by regenerating fish optic nerves (CM), which has been shown to cause axonal elongation in injured rabbit optic nerves, causes a decrease in the number of oligodendrocytes in rat glial cultures. In the present study, we demonstrate that soluble factors in the CM are capable of reducing the number of fish oligodendrocytes in fish optic nerve cultures. In addition, an inverse relationship was found between the number of macrophages and the number of oligodendrocytes. These results thus suggest that macrophages and/or activated resident microglial cells are directly or indirectly responsible for the presence of these soluble factor(s) that regulate the postinjury number of oligodendrocytes in the fish optic nerves.

Monokine production by microglial cell clones

Cytokines have been suggested to act as intermediates between the immune and the central nervous system, but little is known about the type of cells synthesizing them in the brain. We have immortalized with oncogenic retroviruses primary brain cell cultures from mouse embryos and have generated clones of microglial cells that have been characterized. Three of the clones studied produce interleukin 1 (IL 1); IL 6 and tumor necrosis factor-alpha as assessed by biological assays and by Northern blot analysis. Our data raise the question on the role of these cytokines in the brain and suggest that early resident microglial cells might play an important role in development processes and in the adult brain.