Distribution,markers, and functions of retinal microglia

Retinal microglia originate from hemopoietic cells and invade the retina from the retinal margin and the optic disc, most likely via the blood vessels of the ciliary body and iris, and the retinal vasculature, respectively. The microglial precursors that appear in the retina prior to vascularization are major histocompatibility complex (MHC) class I- and II-positive and express the CD45 marker, but lack specific macrophage markers. They differentiate into ramified parenchymal microglia in the adult retina. A second category of microglial precursors, which do express specific macrophage markers, migrate into the retina along with vascular precursors. They appear around blood vessels in the adult retina and are similar to macrophages or cells of the mononuclear phagocyte series (MPS). Microglia are distributed in the outer plexiform layer (OPL), outer nuclear layer (ONL), inner plexiform layer (IPL), ganglion cell layer (GCL), and nerve fiber layer (NFL) of the primate retina. The pattern of microglial distribution in the avascular retina of the quail indicates that blood vessels are not responsible for the final location of microglia in the retina. In the human retina, microglia express MHC class I, MHC class II, CD45, CD68, and S22 markers. In the rat and mouse retina, OX41, OX42, OX3, OX6, OX18, ED1, Mac-1, F4/80, 5D4 anti-keratan sulfate, and lectins are used to recognize microglia. Microglial cells play an important role in host defense against invading microorganisms, immunoregulation, and tissue repair. During neurodegeneration, activated microglial cells participate in the phagocytosis of debris and facilitate regenerative processes. In autoimmune disease, microglia have dual functions: initiating uveoretinitis, but also limiting subsequent inflammation. Retinal microglia may be associated with vitreoretinopathy, diabetic retinopathy, glaucoma, and age-related macular degeneration. The goal of this article was to review the present knowledge about retinal microglia and the function of retinal microglia in pathological conditions.     

Immune cells in the porcine retina: distribution, characterization and morphological features

PURPOSE: To investigate the presence of immunocompetent cells in the porcine retina and to compare the findings with those obtained earlier in human retinas. METHODS: Retinal wholemounts or cryostat sections from outbred Dutch Landrace pigs were analyzed for the presence of microglia (CD45), macrophages-monocytes (SW3, CD163, 2A10, CD14), major histocompatibility complex (MHC) class II-positive cells (MCA1335), granulocytes (MCA1219), B lymphocytes (IgM), and T lymphocytes (CD6, CD4, CD8), by using specific monoclonal antibodies followed by immunohistochemical staining. RESULTS: A uniform distribution of CD45-positive microglial cells was observed throughout the porcine retina (mean number, 289 +/- 16 cells/mm(2)). The microglia were observed along blood vessels and within the tissue between the inner limiting membrane and the inner nuclear layer. MHC class II-positive cells were mainly observed along the large- and middle-sized retinal blood vessels. Double-staining experiments showed that 54% of the microglial cells lining the larger retinal vessels were MHC class II positive. Macrophages were only incidentally observed along the larger retinal blood vessels. No T lymphocytes, B lymphocytes, monocytes, or granulocytes were observed within the retinal tissue. CONCLUSIONS: The porcine retina contains a rich network of microglial cells. Approximately half of the microglial cells lining the larger retinal vessels express MHC class II. The normal porcine retina is devoid of lymphocytes, monocytes, and granulocytes. The distribution of immunocompetent cells in the porcine retina largely resembles that observed in the human retina.     

Retinal microglia differentially express phenotypic markers of antigen-presenting cells in vitro

PURPOSE: Retinal microglial cells of newborn Lewis rats were isolated and cultured, and the effect of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-gamma (IFN-gamma) on microglial expression of the accessory molecules required for antigen presentation were studied. METHODS. Retinal microglia were isolated from newborn Lewis rats and cultured in media supplemented with either M-CSF or GM-CSF. Immunohistochemical tests using anti-macrophage complement receptor 3 (OX42) or anti-monocyte-macrophage (ED1) and DiI-ac-low-density lipoprotein (LDL) uptake were used to identify microglia. The effect on accessory molecule expression of microglial cells cultured under varying conditions (M-CSF, GM-CSF, and M-CSF plus IFN-gamma) was analyzed by fluorescence-activated cell sorter, using one of the following antibodies: anti-OX3, anti-OX6, anti-rat intercellular adhesion molecule (ICAM)-1, anti-rat B7-1, or anti-rat B7-2. RESULTS. The cultured retinal microglia were positive for macrophage-related antigens (ED1 and OX42) and also showed uptake of LDL. Furthermore, ICAM-1 and B7-2 were expressed constitutively on these cells, and MHC class II and B7-1 were also expressed after IFN-gamma stimulation. CONCLUSIONS. In vitro, the retinal microglia express the molecules required for effective antigen presentation to CD4-positive T cells. These findings suggest that microglia may play a role in local antigen presentation, especially when they are exposed to IFN-gamma.     

Localization and characterization of immunocompetent cells in the human retina

PURPOSE: Recent studies have shown that experimental uveitis can be induced by the appropriate administration of various retinal antigens. Little is known about the in-situ interactions between immune cells in the retina as a prerequisite for understanding the mechanisms involving the presentation of antigens by local antigen-presenting cells (APC) to invading T cells. The study described here was therefore designed to investigate the presence of immunocompetent cells with a focus on the characterization of retinal APCs. MATERIALS AND METHODS: Retinal wholemounts, cytospins, and ocular sections were prepared from eyebank eyes obtained within 24 hours postmortem. Immunohistochemistry (single staining and double staining) was performed on the retinal wholemounts, cytospins, and the ocular sections using monoclonal antibodies specific for HLA-DR (MHC class II), CD45 (leukocytes), CD68 (macrophages), CD22 (B cells), CD3 (T cells), and CD1a (dendritic cells). RESULTS: CD68-positive macrophages were observed in one layer of the retina, whereas HLA-DR + and CD45 + cells were seen in two distinct planes: one mainly at the level of the inner nuclear layer to outer plexiform layer (deep layer) and the other mainly at the nerve fiber and ganglion cell layer (shallow layer). There was a significant difference between the different parts of the retina with regard to the density of these cells. Cell density decreased when going from the peripheral to the posterior areas of the retina. The positive cells in the deep layer were frequently associated with blood vessels, whereas the cells in the shallow layer were distributed evenly throughout the retina. Most positive cells displayed a dendritiform appearance, whereas few cells showed a pleiomorphic morphology. Very few CD1a-positive cells were noted in the retina. Neither T cells (CD3) nor B cells (CD22) could be detected in the normal human retina. Double staining showed that the majority (83.7%) of the CD45 + cell population was HLA-DR-positive, whereas approximately half (56.8%) the CD68 + cell population was HLA-DR-positive. CONCLUSIONS: This study shows that the human retina contains a number of different microglia populations, some of which express HLA-DR and could thus be involved in antigen presentation. Marked differences in cell density can be observed within the retina, with the most abundant presence seen in the peripheral retina. The normal retina contains few professional antigen-presenting cells (CD1 + ).     

Heterogeneous populations of microglia/macrophages in the retina and their activation after retinal ischemia and reperfusion injury

Activation of Microglia/macrophages has been observed in ischemia-reperfusion injury of the brain. This study was undertaken to investigate the different subpopulations of microglia/macrophages in the normal rat retina and their activation after retinal ischemia. Retinal ischemia was induced by elevation of intraocular pressure to 120 mmHg for 60 min. Microglia/macrophages were identified on frozen retinal sections by four antibodies, namely OX42, 5D4, OX6 and ED1. In the normal retina, there were heterogeneous populations of resident microglia/macrophages as characterized by their differences in morphology, antigen expression and distribution. OX42+ cells had delicate processes and were located in the inner layers of the retina, while 5D4+ cells were highly ramified and mostly scattered in the inner plexiform layer (IPL) and the outer plexiform layer. Few amoeboid ED1+ cells were also seen in the ganglion cell layer and IPL. OX6+ (MHC-II antigen presenting) cells were not detected in the normal retinas. Double labeling with OX42 and 5D4 antibodies on normal retinal sections showed few microglia exhibited positive labeling with both OX42 and 5D4, while the majority of the microglia were labeled with either OX42 or 5D4 antibodies. After retinal ischemia single labeling with these antibodies showed increased number of these antigen-expressing cells, disappearance of normal cellular processes, and rounding or amoeboid like appearance of the cell bodies. At 1 day after ischemia, there was a significant infiltration of round OX42+, ED1+ and OX6+ cells with loss of the cellular processes in the inner retina. From 3 to 14 days, all subpopulations of microglia/macrophages differentiated cellular processes and became dendritic again. Double labeling on retinas after 1 day of recovery showed OX42+ cells were co-labeled with ED1+ or OX6+ cells, but not with 5D4+ cells. Scattered amoeboid OX42+, 5D4+, and ED1+ cells were noted in the subretinal space 3-14 days after ischemia. In summary, there were heterogeneous populations of resident microglia/macrophages in the normal inner retina and they were activated early after ischemia-reperfusion injury and exhibited different antigenic expression which were further altered in the recovery phase.     

Potential role of microglia in retinal blood vessel formation

PURPOSE: The role of microglia, present in the retina early in development before vascularization, remains ill defined. The authors investigated whether microglia are implicated in retinal blood vessel formation. METHODS: The microglia and vasculature of developing human fetal and rodent retinas were examined by labeling the endothelial cells with lectin and the microglia with CD18 antibody or green fluorescent protein driven by the promoter of the chemokine receptor CX(3)CR1. Rodent ischemic proliferative retinopathy induced by hyperoxia or hypercapnia, which model retinopathy of prematurity, and ex vivo retinal explants were used to assess microglial involvement in vascular pathology. Microglial participation in developmental retinal vessel formation was further studied in neonatal rats after pharmacologic macrophage depletion with the use of clodronate liposomes and subsequent intravitreal injection of microglia. RESULTS: Microglia intimately appose developing vessels of human and murine retinas. Ischemic retinopathy models exhibit decreased microglia concomitant with the characteristic reductions in vasculature observed in these retinopathies. Retinal explants exposed to conditions resulting in ischemic retinopathies (in vivo) reveal that antioxidants protect against microglial loss. Depletion of resident retinal microglia, but not systemic macrophages, reduced developmental vessel growth and density, which were restored by intravitreal microglial injection. CONCLUSIONS: These observations suggest that proper retinal blood vessel formation requires an adequate resident microglial population because diminished microglia are associated with decreased vascularity in models of ischemic retinopathy and retinal vascular development. In light of these findings, the traditional definition of microglia as merely immunocompetent cells should be reconsidered to encompass this new function related to blood vessel formation.     

Heterogeneous populations of microglia/macrophages in the retina and their activation after retinal ischemia and reperfusion injury

Activation of Microglia/macrophages has been observed in ischemia-reperfusion injury of the brain. This study was undertaken to investigate the different subpopulations of microglia/macrophages in the normal rat retina and their activation after retinal ischemia. Retinal ischemia was induced by elevation of intraocular pressure to 120 mmHg for 60 min. Microglia/macrophages were identified on frozen retinal sections by four antibodies, namely OX42, 5D4, OX6 and ED1. In the normal retina, there were heterogeneous populations of resident microglia/macrophages as characterized by their differences in morphology, antigen expression and distribution. OX42+ cells had delicate processes and were located in the inner layers of the retina, while 5D4+ cells were highly ramified and mostly scattered in the inner plexiform layer (IPL) and the outer plexiform layer. Few amoeboid ED1+ cells were also seen in the ganglion cell layer and IPL. OX6+ (MHC-II antigen presenting) cells were not detected in the normal retinas. Double labeling with OX42 and 5D4 antibodies on normal retinal sections showed few microglia exhibited positive labeling with both OX42 and 5D4, while the majority of the microglia were labeled with either OX42 or 5D4 antibodies. After retinal ischemia single labeling with these antibodies showed increased number of these antigen-expressing cells, disappearance of normal cellular processes, and rounding or amoeboid like appearance of the cell bodies. At 1 day after ischemia, there was a significant infiltration of round OX42+, ED1+ and OX6+ cells with loss of the cellular processes in the inner retina. From 3 to 14 days, all subpopulations of microglia/macrophages differentiated cellular processes and became dendritic again. Double labeling on retinas after 1 day of recovery showed OX42+ cells were co-labeled with ED1+ or OX6+ cells, but not with 5D4+ cells. Scattered amoeboid OX42+, 5D4+, and ED1+ cells were noted in the subretinal space 3–14 days after ischemia. In summary, there were heterogeneous populations of resident microglia/macrophages in the normal inner retina and they were activated early after ischemia-reperfusion injury and exhibited different antigenic expression which were further altered in the recovery phase.     

Quantitative Analysis of Major Histocompatibility Complex Class II-Positive Cells in Posterior Segment of Royal College of Surgeons Rat Eyes

Potential antigen-presenting cells in the posterior segment of Royal College of Surgeons (RCS) rat eyes were analyzed quantitatively. Light microscopic immunohistochemistry was performed at postnatal days (P) 10, 20, 28, 42, 63, and 140 in the eyes of RCS rats and their congenic counterparts. Immunohistochemical studies were carried out using monoclonal antibodies against major histocompatibility complex (MHC) class II antigen (OX6), a cytoplasmic antigen in bone marrow-derived macrophages (ED1), a membrane antigen on resident tissue macrophages (ED2), and a microglia/macrophage marker (OX42). Some sections were stained by a double-labeling method using these antibodies. No MHC class II-positive cells were seen in dystrophic RCS rat retinas at P10. They were found, however, in the outer nuclear layer and debris of outer segments at P20. From P20 to P42 the number of cells increased, then decreased until P140. Congenic controls, however, showed no MHC class II-positive cells in the retina. Cells double-labeled with OX6 and ED1 were present in the outer nuclear layer at P42, but no OX6 or OX42 double-labeled cells were detected. Also, no ED2-positive cells were detected. Our results suggest that MHC class II-positive cells may play some role in retinal dystrophy.     

Lipopolysaccharide/interferon-gamma and not transforming growth factor beta inhibits retinal microglial migration from retinal explant

BACKGROUND:/aims: The retina possesses a rich network of CD45(+) positive myeloid derived cells that both surround inner retinal vessels and lie within the retina (microglia). Microglia migrate and accumulate in response to neurodegeneration and inflammation. Although microglia express MHC class II, their role remains undefined. The aims of this study are to investigate changes in human microglia phenotype, migration, and activation status in response to pro-inflammatory and anti-inflammatory stimulation. METHODS: Donor eyes were obtained from the Bristol Eye Bank with consent and whole retina was removed. 5 mm retinal trephines were cultured in glucose enhanced RPMI on cell culture insert membranes for up to 72 hours. The effects of lipopolysaccharide/interferon-gamma (LPS/IFNgamma) and transforming growth factor beta inhibits (TGFbeta) stimulation, alone or in combination, on migration, phenotype, and activation status (iNOS expression) of microglia were studied using immunofluorescence and cytokine analysis by ELISA. RESULTS: CD45(+) MHC class II(+) retinal microglia were observed within retinal explants, and in culture microglia readily migrated, adhered to culture membrane, downregulated MHC class II expression, and produced interleukin 12 (IL-12) and tumour necrosis factor alpha (TNFalpha). Following LPS/IFNgamma stimulation microglia remained MHC class II(-) iNOS(-), and secreted IL-10. Migration was suppressed and this could be reversed by neutralising IL-10 activity. TGFbeta did not affect ability of microglia to migrate and was unable to reverse LPS/IFNgamma induced suppression. CONCLUSIONS: Microglia readily migrate from retinal explants and are subsequently MHC class II(-), iNOS(-), and generate IL-12. In response to LPS/IFNgamma microglia produce IL-10, which inhibits both their migration and activation. TGFbeta was unable to counter LPS/IFNgamma effects. The data infer that microglia respond coordinately, dependent upon initial cytokine stimulation, but paradoxically respond to classic myeloid activation signals.     

Flow cytometric identification of a minority population of MHC class II positive cells in the normal rat retina distinct from CD45lowCD11b/c+CD4low parenchymal microglia.

AIMS--This study aimed to isolate and classify by flow cytometry, the cell surface phenotype of microglia in the normal rat retina with a view to identifying putative antigen presenting cells (APC) within the retina, which has to date not been possible by immunohistochemistry. METHODS--Normal rat retinal microglia were isolated and classified using a modification of an isolation technique employing graduated Percoll density gradient cell separation and flow cytometric phenotypic criteria used for CNS microglia. RESULTS--Retinal microglia can be defined by flow cytometry on the basis of their CD45lowCD11b/c+CD4low cell surface expression. Constitutive MHC class II expression in the normal rat retina was confined almost exclusively to a very minor population of cells expressing neither low (microglia) nor high levels of CD45. Three colour flow cytometric analysis confirmed that these MHC class II positive cells were ED2+. CONCLUSIONS--Using this sensitive isolation technique we have identified the cell surface characteristics of ramified, resident microglia, and found that they do not constitutively express MHC class II. There is, however, constitutive MHC class II expression on a phenotypically distinct population of cells (CD45low/highED2+). We propose these cells are the counterpart of the perivascular macrophages found in the CNS which present antigen to extravasating T cells, although their exact retinal location can only be confirmed by immunohistochemical analysis. The role of parenchymal microglia as APC remains undefined. Future isolation of microglia and putative perivascular cells using this technique will help identify the role these cells play in the initiation and perpetuation of immune responses within the retina.     

乳脂球上皮生长因子-8在大鼠神经层视网膜小胶质细胞中的表达

背景神经层视网膜小胶质细胞在视网膜胚胎后期发育过程中起“清道夫”的作用,可清除凋亡细胞。乳脂球上皮生长因子18（MFG—E8）能特异性地与凋亡细胞表面的磷酯酰丝氨酸相结合,增强巨噬细胞对凋亡细胞的吞噬作用。目的观察MFG—E8及相关细胞因子在正常大鼠神经层视网膜胚胎后期发育过程中的表达。方法取清洁级鼠龄为0、3、7、14、30、45d的正常皇家外科学院（RCS）大鼠各5只。免疫荧光双标染色标记MFG—E8和小胶质细胞标志物CD11b,荧光实时定量聚合酶链反应（real—time PCR）检测正常大鼠各组视网膜中MFG—E8、整合素135、CD11b、白细胞介素-6（IL-6）mRNA的表达变化。结果MFG—E8免疫阳性细胞分布于视网膜内层,主要为视网膜节细胞层和外丛状层,与CD11b染色部位相同。Real—time PCR检测发现,在出生后即可检测到MFG-E8、整合素β5、CD11b及IL-6 mRNA的表达,其表达量在出生后早期较低,然后逐渐增加,鼠龄14d组的mRNA表达最强烈,然后逐渐下降。鼠龄14d组的各因子mRNA表达水平明显高于其他鼠龄组,差异均有统计学意义（P〈0．01）。结论MFG—E8特异地表达于正常RCS大鼠神经层的视网膜小胶质细胞,表达量出生后呈先升高后降低,14d为高峰的规律。     

Class II antigens on retinal vascular endothelium, pericytes, macrophages, and lymphocytes of the rat

Class II histocompatibility complex antigens on the retinal vascular endothelium may allow these cells to function as antigen-presenting cells to circulating T cells. The present study investigated induction of class II antigens in vitro to characterize the response under controlled conditions. Retinal vascular endothelium from Lewis and Brown Norway rats (high versus low responders in experimental autoimmune uveitis) were exposed in vitro to recombinant rat gamma interferon, interleukin-1, interleukin-2, or Concanavalin-A spleen supernatant. Retinal pericytes, macrophages and lymphocytes were studied in comparison. A newly adapted ELISA technique was used to assay levels of antigen expression. Class II antigens (I-A OX6, I-E OX17, polymorphic I-A OX3), class I antigens (OX18), macrophage marker (OX42), macrophage and T helper cell marker (W3/25), and T suppressor/cytotoxic cell marker (OX8) were studied. Results showed that retinal vascular endothelium normally expresses very little class II antigen. However, high levels of I-A and I-E were induced by interferon or spleen supernatant. The levels of class II antigen approached that of the traditional antigen-presenting cell (macrophage) and were much higher than levels for pericytes and lymphocytes. The same doses of interferon showed larger increases in the Lewis rat compared to Brown Norway. No effect was seen with interleukin-1 or -2. Therefore, retinal vascular endothelium may be induced by gamma interferon to express class II antigens with degree of induction greater than or equal to the macrophage, and higher levels of induction were seen in the high responder strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroprotective effects of naloxone against light-induced photoreceptor degeneration through inhibiting retinal microglial activation

PURPOSE: To determine the role of microglial activation in light-induced photoreceptor degeneration and the neuroprotective effects of naloxone as a novel microglial inhibitor. METHODS: Sprague-Dawley rats were exposed to intense blue light for 24 hours. Daily intraperitoneal injection of naloxone or PBS as a control was given 2 days before exposure to light and was continued for 2 weeks. Apoptotic cells were detected by the TUNEL assay, and anti-OX42 antibody was used to label retinal microglia. Western blot was applied to evaluate the retinal interleukin (IL)-1beta protein levels. Retinal histologic examination and electroretinography (ERG) were also performed to evaluate the effects of naloxone on light-induced photoreceptor degeneration. RESULTS: TUNEL-positive cells were noted in the outer nuclear layer (ONL) of the retina as early as 2 hours and peaked at 24 hours after exposure to light. OX42-positive microglia occurred in the ONL and subretinal space at 6 hours, peaked at 3 days, and changed morphologically from the resting ramified to the activated amoeboid. Expression of IL-1beta protein was also significantly increased at 3 days. Compared with the control, the number of microglia in the outer retina was significantly decreased in the naloxone-treated group at 3 days, and the thickness of ONL and the amplitudes of dark-adapted a- and b-waves were also well preserved at 14 days. CONCLUSIONS: The activation and migration of microglia and the expression of neurotoxic factor (IL-1beta) coincide with photoreceptor apoptosis, suggesting that activated microglia play a major role in light-induced photoreceptor degeneration. Inhibiting microglial activation by naloxone significantly reduces this degeneration.     

Generation of activated sialoadhesin-positive microglia during retinal degeneration

PURPOSE: The retina contains a rich network of myeloid-derived cells (microglia) within the retinal parenchyma and surrounding vessels. Their response and behavior during inflammation and neurodegeneration remain largely undefined. In the present study, the behavior of microglia was closely examined during the onset of photoreceptor degeneration in the rds mouse, to assess their role in photoreceptor apoptosis. The results may have relevance to similar degeneration in humans (retinitis pigmentosa). METHODS: Retinas from rds and wild-type CBA mice aged 8, 14, 16, 17, 19, 21, 30, and 40 days were examined immunohistochemically, with antibodies to macrophage cell surface markers, inducible nitric oxide synthase (iNOS), and proliferating cell nuclear antigen (PCNA), during the most active phase of the disease. TUNEL was used to assess photoreceptor apoptosis. RESULTS: In the rds mouse, microglia proliferated in situ (PCNA), migrated to the subretinal space, and adopted an activated phenotype. Maximum microglial cells occurred at postnatal day (P)21, 5 days after the peak in photoreceptor apoptosis (P16). Microglia did not express iNOS, and nitrotyrosine was absent. Sialoadhesin was expressed on microglia from P14, and expression was greatest at P21. CONCLUSIONS: During retinal degeneration, microglia are activated and express sialoadhesin. The temporal relationship between photoreceptor apoptosis and microglial response suggests that microglia are not responsible for the initial wave of photoreceptor death, and this is corroborated by the absence of iNOS and nitrotyrosine. Expression of sialoadhesin may indicate blood-retinal barrier breakdown, which has immune implications for subretinal gene therapeutic strategies.     

CD45-positive cells of the retina and their responsiveness to in vivo and in vitro treatment with IFN-gamma or anti-CD40

PURPOSE: The ability to mount antigen-specific immune reactions in the retina demonstrates local recognition of retinal antigens. However, properties of antigen-presenting cells (APCs) of the retina are uncertain. The current study was undertaken to look for evidence of CD45(+) cells with APC potential in the retina and to examine their in situ and in vitro responses to IFN-gamma and anti-CD40, two stimuli known to upregulate activities associated with antigen presentation. METHODS: Mice were pretreated with systemic or intracameral (IC) inoculations of IFN-gamma or anti-CD40. Retinas were harvested, enzymatically dissociated, positively selected with anti-CD45, and analyzed by flow cytometry with antibodies known to identify APCs. RESULTS: The most common CD45(+) retinal cells were CD11b(+), F4/80(+), CD8 alpha(+), CD80(+), and major histocompatibility complex (MHC) class II(lo), a phenotype characteristic of central nervous system (CNS) microglia (MG). There was also a small population of DEC-205(+) cells and a smaller number of CD11c(+) cells, both markers of dendritic cells (DCs). IC inoculation of IFN-gamma led to an increase in the number of CD45(+) cells and a modest upregulation of MHC class II on CD11b(+) cells. IC inoculation of anti-CD40 also increased the total number of CD45(+) cells and the number of CD11b(+) cells, but increases in CD80 and MHC class II expression on CD11b(+) cells were insignificant. After anti-CD40 treatment, CD45(hi)11c(+) cells increased in number and altered their expression of CD11b. CONCLUSIONS: Retinal MG were readily identified as the most numerous population. A small population of cells with perivascular cell (PVC)-like properties was found. They were CD45(hi)11c(+), some had elevated MHC class II, and they were affected by anti-CD40 treatment in vivo. No conventional DCs were found, although there was a distinct DEC-205(+) population. Overall, the effects of IFN-gamma and anti-CD40 treatment were attenuated in the retina in vivo and also on CD45(+) cells in culture, compared with the control.     

Glia–neuron interactions in the mammalian retina

The mammalian retina provides an excellent opportunity to study glia–neuron interactions and the interactions of glia with blood vessels. Three main types of glial cells are found in the mammalian retina that serve to maintain retinal homeostasis: astrocytes, Müller cells and resident microglia. Müller cells, astrocytes and microglia not only provide structural support but they are also involved in metabolism, the phagocytosis of neuronal debris, the release of certain transmitters and trophic factors and K⁺ uptake. Astrocytes are mostly located in the nerve fibre layer and they accompany the blood vessels in the inner nuclear layer. Indeed, like Müller cells, astrocytic processes cover the blood vessels forming the retinal blood barrier and they fulfil a significant role in ion homeostasis. Among other activities, microglia can be stimulated to fulfil a macrophage function, as well as to interact with other glial cells and neurons by secreting growth factors. This review summarizes the main functional relationships between retinal glial cells and neurons, presenting a general picture of the retina recently modified based on experimental observations. The preferential involvement of the distinct glia cells in terms of the activity in the retina is discussed, for example, while Müller cells may serve as progenitors of retinal neurons, astrocytes and microglia are responsible for synaptic pruning. Since different types of glia participate together in certain activities in the retina, it is imperative to explore the order of redundancy and to explore the heterogeneity among these cells. Recent studies revealed the association of glia cell heterogeneity with specific functions. Finally, the neuroprotective effects of glia on photoreceptors and ganglion cells under normal and adverse conditions will also be explored.     

Enzymhistochemical identification of microglial cells in the rat retina: light and electron microscopic studies

We used the method of thiamine pyrophosphatase (TPPase) enzyme histochemistry and flat-mounted and transverse-sectioned retinas to identify microglial cells. Light microscopically, TPPase activity was demonstrated on the outer surfaces of glial cells located in the inner plexiform layer (IPL) and the ganglion cell layer (GCL) of the entire retinal regions, and also on the outer surfaces of blood vessels. Electron microscopically, TPPase activity was observed on the plasma membranes of the glial cells, the endothelial cells of microvessels and the pericytes. The TPPase-positive glial cells had a dark nucleus with large clumps of chromatin beneath the nuclear envelope. These findings strongly suggest that the glial cells with TPPase activity observed in the IPL and the GCL of the rat retina were microglial cells.     

小胶质细胞活化与rd小鼠遗传性视网膜变性

目的研究小胶质细胞活化与rd小鼠遗传性视网膜变性的关系。方法对出生后8、10、12、14、16及18d的rd小鼠及对照小鼠视网膜进行感光细胞凋亡TUNEL法检测及形态计量学分析。CD11b免疫组织化学染色标记视网膜小胶质细胞。结果rd小鼠出生后10d视网膜感光细胞层开始出现TUNEL染色阳性细胞,第16d达到高峰。视网膜小胶质细胞在rd小鼠出生后10d开始活化,第14d达到高峰。小胶质细胞向感光细胞层的迁移与感光细胞凋亡之间存在紧密的时间和空间关系。结论rd小鼠视网膜变性以感光细胞凋亡为主。小胶质细胞活化可能在视网膜变性过程中发挥重要作用。     

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.