|Molecular Vision 2005;
Received 4 August 2004 | Accepted 6 March 2005 | Published 31 March 2005
Multilayered retinal microglial response to optic nerve transection in rats
Garcia-Valenzuela,1 Sansar C.
1Emory University Eye Center, Emory University, School of Medicine, Atlanta, GA; Departments of 2Cell Biology and Anatomy and 3Ophthalmology, New York Medical College, Valhalla, NY; 4Department of Neurosurgery, Regensburg University Clinic, Regensburg, Germany
Correspondence to: E. Garcia-Valenzuela, MD, PhD, Assistant Professor of Ophthalmology, Emory University Eye Center, 1365B Clifton Road, Atlanta, GA, 30322; Phone: (404) 778-5070; FAX: (404) 778-4380; email: email@example.com
Purpose: Microglia normally exist in several layers across the retinal thickness. When retinal ganglion cells undergo apoptosis after lesion to their axons, microglial cells proliferate and promptly clear the debris. We have previously reported on the phagocytic response following optic nerve axotomy. Here, we present how microglial cells of deeper retinal layers are affected by transection of the optic nerve.
Methods: Normal and reactive microglia in the retina of adult rats whose optic nerves had been lesioned were labeled by using antibodies OX42 and ED1. Analysis of the time course (between 1 and 180 days post-axotomy) of appearance and distribution of microglial cells in the retinal layers was performed.
Results: In normal retinas, microglia were found in the ganglion cell layer (GCL), the superficial inner nuclear layer (INL) and the outer plexiform layer (OPL). Increases in numbers of microglia occurred maximally in the GCL at day 12 post-axotomy. Increases were also detected in the superficial INL. The proliferation of these phagocytic cells led to their highest numbers in the more central eccentricities in the two most superficial layers. Microglia in the OPL remained undisturbed. Microglial normal histology is restored over a period of 6 months as dying ganglion cells disappear.
Conclusions: Histological characteristics of normal rat retinal microglia are uniform across different retinal eccentricities for each of the three laminae where they exist. Retinal microglia of various layers respond to optic nerve damage. Their increase in numbers and morphological transformation follow retinal ganglion cell death. Their morphology, density, and layered distribution slowly return to normal, confirming that retinal ganglion cells, or their densities, do not play any role in dictating microglial distribution within the different retinal layers.
Microglial cells are uniquely specialized cells encountered in the central nervous system. They can be distinguished on the basis of morphology and cytochemical characteristics [1-4]. Although microglial cells were described by a number of authors including Nissl , Robertson , Alzheimer , and Cajal [8,9], it was not until Del Rio Hortega [10,11] introduced his silver carbonate method that microglial cells could be completely visualized. Since then, microglia have been shown to be the resident phagocytic cells in the central nervous system (CNS). Although much controversy has followed functional and histological issues related to microglia, they have been shown to be derivatives of the bone marrow [12-15]. This type of phagocytic cell increases in cell numbers by two different mechanisms after CNS damage [16,17]. (1) directly by proliferation, and (2) by recruitment from the circulating monocyte pool through an intact blood-brain barrier and their subsequent differentiation into microglia. The existence of microglia that convert into macrophages upon proper stimulation has also been proposed [18-21].
Microglial cells form an integral part of the retina in adult mammals. They are present in three separate layers: ganglion cell layer (including the nerve fiber layer), inner plexiform layer (or superficial inner nuclear layer) and outer plexiform layer [22,23]. Interestingly, the latter layer has not been reported in all mammalian species studied . In the rat, microglia have traditionally been reported to be present in only the innermost two layers [23-27]. Recently however, microglia has also been described to be in the outer plexiform layer of rats after axotomy .
Two morphological types of microglia cells have been described: ramified or resting microglia; and ameboid or activated microglia . Nowadays, the identification of several markers has helped to recognize different subpopulation of microglial cells [23,28]. In this study we use two different microglia rat markers, OX42 and ED1. The antibody CD11b (or OX42 in rats), which is directed against complement iC3b receptor , has been used as a general marker for microglia/macrophage cells. ED1 antibody recognizes a phagocytic cell cytoplasmic antigen , and has been used as marker for monocyte/macrophage cells. It has been suggested that ED1 positive cells maybe a subtype of resident microglial cells with macrophage characteristics , and also that ED1 marker appear to label recently blood borne monocytes/macrophages .
While astrocytes and Müller cells respond to retinal ganglion cell (RGC) degeneration following optic nerve transection by accumulating glial fibrillary acidic protein or upregulating some growth factors as CNTF [23,33-37], retinal microglia respond with hypertrophy, enhanced enzymatic activity and transient increase in density [2,38-41]. Phagocytosis dependent labeling of retinal microglia with lipophilic dye has illustrated that these cells are responsible for the ingestion of debris from axotomized ganglion cells [42,43]. Microglia begin proliferation a few days after retinal ganglion cells start dying following intraorbital axotomy. Their numbers reach a peak during the second week after axotomy, and then slowly drop back during the following months [28,35,43]. It is not well understood how microglial cells from different retinal layers respond to ganglion cell death consequent to optic nerve damage.
We have previously reported on the phagocytic response following optic nerve axotomy . Here we present additional data not analyzed by the first study. We analyzed quiescent microglial cells, which were found to exist in three distinct layers of the rat retina, and followed their changes across these different layers and regions during ganglion cell degeneration following optic nerve section in adult rats. Microglia were seen to undergo morphological transformation and increase in numbers in the ganglion cell layer and the superficial inner nuclear layer, but they did not change in the outer plexiform layer.
Adult Wistar rats (275-325 g; n=34) were used for all experiments. They were anesthetized with an intraperitoneal injection of a solution containing ketamine (Ketaset; 40 mg/kg, Fort Dodge Labs, Iowa, IA), xylazine (Rompum; 8 mg/kg, Bayer Corp., Shawnee, Kansas), and acepromazine (Promace, 1.2 mg/kg; Fermenta Co., Kansas City, MO). Animals were taken care of in accordance with the institutional humanitarian regulations. Animals were sacrificed with an overdose of intraperitoneal pentobarbital.
Optic nerves (ON) were approached intraorbitally by their dorso-lateral aspect. After total anesthesia of the adult rat, and under a stereomicroscope, the conjunctiva was cut open; dissection of the subconjunctival soft tissues exposed the ON; the meningeal sheath was incised longitudinally and all the optic fibers were cut 2 mm behind the eyeball. Visualization of the two ON stumps clearly separated by a gap confirmed a complete transection. Care was taken to avoid injury to the retinal blood supply. The patency of the retinal blood supply was assessed with planar ophthalmoscopy 1 day after surgery, and prior to sacrifice. Animals were sacrificed, and retinas (four eyes per time point, except the latter two in which only two eyes were used) were removed at 5, 7, 9, 12, 21, 28, 90, 180, and 360 days after axotomy. Retinas were fixed in PLP (2% paraformaldehyde, 1.5% L-lysine, 2% Na meta-periodate in 0.1 M PBS, pH 7.4) for 30 min at 25 °C.
Retinas from normal and optic nerve transected animals were washed and processed immunocytochemically by using mouse anti-rat primary antibodies OX42 (Serotec, Oxford, UK), and ED1 (Serotec, Oxford, UK). Retinas were washed three times in 0.1 M phosphate buffer saline, pH 7.4. Tissue was incubated overnight at 4 °C with primary antibody (1:100 dilution) in 4% nonfat dry milk and 0.3% Triton X-100 in PBS (blocking-permeabilizing solution). Retinas were then incubated for 2 h at 25 °C in the blocking-permeabilizing solution with the addition of 1:50-100 goat (TRITC/FITC)-anti-mouse antibodies. Following three washes in PBS, they were mounted and studied as described below. Controls were performed by following identical procedure but with omission of primary antibodies.
A group of retinas (n=12 distributed as: control, 4, 7, and 14 days after optic axotomy) were fixed, sectioned transversely with a vibratome (30 μm), and then processed for antibody staining. Sections were incubated in OX42 as mentioned above for flatmounted retinas. They were then analyzed for precise retinal depth localization of phagocytic cells.
Retinas were assessed histologically as flatmounts or sections at a magnification of 625x, by using a fluorescein wavelength filter. Two different microscopes were used: A Leitz Dialux 20 epifluorescence microscope and a BioRad MRC-1000 confocal system coupled to a Nikon epifluorescence microscope. Retinal areas used for cell density measurements were located at three different eccentricities from the optic disc: central (about 0.5 mm), middle (about 2 mm), and peripheral (about 4 mm), in the dorsal, ventral, nasal and temporal retina. Flatmounted retinas were examined at varying depths from the nerve fiber layer (0 μm) to the outer nuclear layer (>100 μm). In each region depth examined, six microscopic fields (120x160 μm2) were chosen for cell counting, by using a predetermined systematic approach. When counting phagocytic cells in different laminae following axotomy, cells not lying within any of their normal locations (ganglion cell layer, superficial inner nuclear layer, and outer plexiform layer), were assigned to the closest lamina. The data obtained from each retina were pooled for comparison across regions. The diameter of cell soma and their extensions were measured along their longest and shortest axis. The average of these two dimensions was used thereafter for description of cell size or cellular processes.
Data obtained in all experiments was analyzed for mean and standard error of the mean. Comparisons were done between individual blocks of single variable data using two sample Student's t-test. In most cases the two tailed formula for independent samples was used. In experiments where comparisons were made of data obtained across three different retinal eccentricities, the one way ANOVA test was used. Significant differences were indicated when p values were less than 0.05.
Normal distribution of retinal microglia
Immunocytochemical staining of rat retina with OX42 antibody labels phagocytic cells and vascular structures. Examination of normal rat retina (flatmounts) revealed OX42 labeled microglial cells distributed in laminae, each being a regular array of cells fairly equidistant among themselves (Figure 1, Figure 2). They extended in the same retinal depths where capillaries form vascular plexuses with individual microglial cells often in close proximity to the blood vessels. These phagocytic cells were arranged in 3 separate layers at different depths. Their cell bodies were recognized in the ganglion cell layer (GCL), the superficial or inner aspect of the inner nuclear layer (SINL), and the outer plexiform layer (OPL). Although they had their cytoplasmic processes mostly parallel to their laminae, some also extended in the nerve fiber layer (NFL), the inner plexiform layer (IPL), and the inner nuclear layer (INL). Microglia were identified by their OX42 immunoreactivity  and their well known morphological features including small sized oblongated soma (average diameter 11.9±1.9 μm, for microglia in all retinal layers and eccentricities), and numerous, long, and highly branched processes.
Microglia in the GCL of normal retina were arranged in a single cell lamina, in homogeneous densities across different eccentricities (no significant difference): there were 219.5±18.4 cells per mm2 in central retina, 202.6±21.1 cells per mm2 in middle retina, and 207.3±15.6 cells per mm2 in peripheral retina. Similarly, the density of microglial cells was not significantly different in the various retinal quadrants. All throughout the GCL, microglia appeared to avoid contact with each other's processes, tending to remain in an equidistant array, in the same depth of the capillary network (Figure 1, Figure 2). The average soma size in this retinal layer was 12.2±1.4 μm in diameter, and the average span of their branches was 63.2±22.6 μm. GCL microglia extended their process in the GCL itself, in the NFL, and in the IPL.
The next layer of microglia had their cell bodies in the margin between the INL and the IPL (Figure 1, Figure 2). These microglial cells were more sparsely distributed (105.0±13.2 per mm2) in a single cell lamina, and were evenly distributed across different quadrants and eccentricities. They were fairly equidistant from each other, in close association with the deeper vascular plexus, and with long cytoplasmic extensions into the IPL and INL. The extent of OX42 positive cell branches in this location was significantly larger (72.6±14.9 μm in diameter) than for those in the GCL, but the average soma diameter was similarly small (11.8±1.5 μm).
We located microglia in a third lamina, more external (deeper) than the other two, with their somata at the border between the INL and the OPL. Here, OX42 positive cells were sparse, at a density of 42.1±15.8 per mm2, but the general histological organization was uniform in different eccentricities and quadrants, similar to the other two layers. Their cell bodies were small (average diameter 11.8±1.9 μm) and their processes were less extensive and ramified with an average diameter of 67.3±32.1 μm, spreading mostly into the OPL, among blood capillaries (Figure 1).
In addition to microglia, another OX42 positive cell type was visible in the flatmounts of intact retina in very low densities (4.6±2.1 per mm2), situated along the NFL and the vitreal side of the inner limiting membrane (ILL), as confirmed on transverse sections. These cells were probably macrophages since they had larger soma size (14.68±1.34 μm), and did not have elongated cytoplasmic processes. These cells were OX42 positive and ED1 positive, unlike quiescent microglia .
Changes in OX42 positive and ED1 positive retinal cells following RGC axotomy
Following intraorbital transection of the optic nerve, retinal ganglion cells begin to die within a few days, peaking at 7 days . Changes in OX42 positive microglia were first detected on flatmount retinas from optic nerve sectioned animals at day 5 after lesioning. Gradual morphological transformation occurred as cells with larger cell bodies and progressively shorter processes were noticed in more advanced stages after axotomy (Figure 1). During activation of microglia in addition to their OX42 antigenicity, most cells became ED1 positive, also.
Comparison of retinas at different stages after axotomy showed a gradual increase in the numbers of microglia in the GCL, peaking on day 12 after axotomy, a considerable time later than the peak of RGC death (Figure 1, Figure 3). Increase in the number of microglia was noted earlier in the central regions of the retina (Figure 4), where they formed multiple radial rows of OX42 positive cells, many still in physical contact with each other, resembling "waves" of cells. In middle and peripheral retina, the increase in density of these phagocytic cells was somewhat delayed (Figure 4). Although they reached maximum numbers around the same time as for central retina, their numbers were never as high as the central retina. The peak densities of microglial cells were 1008±193.2 per mm2 in central, 835.7±96.6 per mm2 in middle, and 483.1±106.5 per mm2 in peripheral retina. This difference formed a gradient in density of microglia, which correlated with the gradient of RGCs normally present and now dying due to axotomy. Microglial cells in the inner INL underwent an increase in numbers as well, though to a lesser extent, peaking at 289.9±52.9 cells per mm2 on day 12 after axotomy (Figure 1, Figure 3). In the outermost lamina where OX42 positive cells are located, there was no increase in their density (Figure 1, Figure 3).
Parallel with the different degrees of proliferation of microglia across laminae, the morphological changes following activation were also greater in these cells in the GCL, where they achieved maximum shortening of their processes. Phagocytic cells were also seen in the NFL, above the GCL, where they were probably clearing ganglion cell axonal debris. The orderly distribution of microglia across different retinal layers tended to disappear, presumably as they migrated to intermediate layers (IPL) where RGC dendritic processes were fragmented, forming debris that required clearance. After the peak in phagocytic activity, microglial numbers and morphology returned slowly to normal (Figure 1, Figure 3, Figure 4). Normal densities were approached in subsequent months. Gradually, microglia with progressively longer cytoplasmic extensions appeared, becoming spaced out into a regular array again. The separation of microglia into three distinct retinal laminae was reconstituted. Six months after optic nerve injury, the histological features of resident retinal phagocytic cells were almost normal, with mean densities of 217.8±27.3 per mm2 in the GCL, 120.8±22.5 per mm2 in the SINL (Figure 1), and no significant differences across different eccentricities in spite the fact that most retinal ganglion cells had disappeared within a month of the optic nerve transection. Density numbers and other histological characteristics of microglia remained the same from 6 to 12 months after axotomy.
The central nervous system, including the retina, is devoid of microglial cells until mid embryonic stages. As developing blood vessels grow into the retina, blood derived macrophages migrate out into the neuronal milieu [2-4,15,46]. They become distributed in a regular array and transform from round to amoeboid shaped cells [26,27]. Early on they acquire laminar disposition in the retina. In the rat, microglial-macrophagic cells increase in numbers during early postnatal stages, secondary to the developmental retinal ganglion cell death. They are responsible for the prompt clearance of cellular debris [21,47]. After this period, numbers of these phagocytic cells decrease, and the organized distribution of microglia is reestablished. Their cell bodies diminish in size and they extend long, ramified processes typical of resting microglia.
In the adult rat retina, transection of the optic nerve causes microglia to proliferate. Microglia have been reported to take up ganglion cell derived material, using phagocytosis dependent DiI labeling . Retinal microglia respond to optic nerve transection with hypertrophy, enhanced enzymatic activity in addition to the transient increase in density . In the present report, we studied immunocytochemically the morphological features of microglia in normal retina, and the changes they undergo across their laminar distribution, following retrograde degeneration of the axotomized optic nerve of adult rat retina.
OX42 antibodies (which recognize the complement factor receptor iC3b) have been used by various investigators to distinguish microglial cells [21,28,48]. In confirmation of previous work, microglial cells in the intact adult rat retina are distributed homogeneously in single celled arrays at various depths within the inner retina. While their location in the GCL/NFL and in the inner-INL/IPL had been reported earlier [26,27], in agreement with a recent report by Zhang and Tso , we detected microglia in low numbers in the outer-INL/OPL, also; similar to their reported presence in human, mouse, rabbit, cat, and monkey [22,23,26,27,49-51]. In all three layers, microglia were arranged geometrically and separated from each other as implied by mutual inhibition mechanism. They showed no gradient across different eccentricities or quadrants of the retina. Their laminar organization was always in proximity to the networks of blood capillaries. This is hypothetically related to their developmental mode of entry into the retina as well as to a yet undetermined molecular communication between vessels and these blood derived cells. In the case of OX42 positive cells located in the GCL and the inner INL, the size of their branches was inversely related to their proximity to each other. This correlation did not apply to microglia in the outermost layer, where these cells were significantly more spaced out.
Following damage to optic axons, microglia increase in numbers and gradually transform into OX42 and ED1 positive amoeboid cells. We assume that the vast majority of these cells are produced by proliferation of resident microglia, since the most plausible alternative, invasion of blood borne macrophages, was found to be of limited contribution .
As expected, microglia in the GCL are the most active of the three layers during retrograde retinal degeneration. The intensity with which they react is presumably related to the numbers of dying RGC, as evidenced in the correlation of the gradient of microglial peak densities and the gradient of RGC numbers across different eccentricities. Activation of microglial cells also occurs in the inner-INL/IPL layer where displaced ganglion cell somas and dendrites are located. The precise separation of microglia in these two layers is lost during this process, probably as phagocytic cells migrate to clear the axonal and dendritic debris in the NFL and IPL. Activation of microglia spares the third and deepest layer, the outer-INL/OPL, devoid of RGCs.
The observation that peak activation of microglia (12 days after axotomy) occurs subsequent to the peak of death of axotomized RGCs (7 days after axotomy)  supports the notion that microglial activation occurs secondary to RGC death. It is interesting to note the "wave patterns" of phagocytic cells that were formed in the central regions of lesioned retinas, at the peak of activity. Microglial cells rapidly increasing in number and in direct contact with each other, aligned in parallel rows. It is assumed that these "wave patterns" were formed secondary to multiplying phagocytic cells arranging themselves along tension-resistance forces that exist in the retina due to structures such as major blood vessels and ganglion cell axons that radiate from the optic disc.
As RGC death was coming to an end, after the third week following axotomy , microglial activity markedly decreased, with gradual diminution of their numbers. This loss of phagocytic cells might be caused by apoptosis of microglia that are not needed any more. Hypothetically, surviving microglia phagocytose dying microglia. The morphology, density and layered distribution of microglial cells returned to almost perfect normal pattern over a period of 6 months, in spite of absence of most ganglion cells. This points to the lack of intercellular control that viable RGCs exert on the morphological features that phagocytic cells acquire. Microglia intercellular communication probably plays some role in keeping themselves in a geometrical array, avoiding contact from each other. It is interesting to notice that although this homogeneous interspersing is a characteristic for all three layers of microglia, each has a different predetermined density controlled by an unknown mechanism.
The experiments presented here provide evidence that retinal microglia in the two innermost laminae respond to RGC apoptosis caused by their axotomy, phagocytosing axons, dendrites and somata of these neurons only after their degradation is well under way. As their task is accomplished, microglia return almost completely to their normal morphology. The underlying molecular mechanisms guiding the intra- and intercellular processes described above remain unknown for the most part.
We thank Prof. Dr. Brawanski for all his help and support and Mrs. Marion Kubitza for her excellent technical assistance.
1. Imamoto K, Leblond CP. Presence of labeled monocytes, macrophages and microglia in a stab wound of the brain following an injection of bone marrow cells labeled with 3H-uridine into rats. J Comp Neurol 1977; 174:255-79.
2. Murabe Y, Ibata Y, Sano Y. Morphological studies on neuroglia. III. Macrophage response and "microgliocytosis" in kainic acid-induced lesions. Cell Tissue Res 1981; 218:75-86.
3. Barron KD, Dentinger MP, Krohel G, Easton SK, Mankes R. Qualitative and quantitative ultrastructural observations on retinal ganglion cell layer of rat after intraorbital optic nerve crush. J Neurocytol 1986; 15:345-62.
4. Boya J, Calvo J, Carbonell AL, Garcia-Maurino E. Nature of macrophages in rat brain. A histochemical study. Acta Anat (Basel) 1986; 127:142-5.
5. Nissl F. Uber einige Beziehungen zwischen Nervenzellenerkrankungen und gliosen Erscheinungen bei verschiedenen Psychosen. Arch Psychiatr Nervenkr 1899; 32:656-76.
6. Robertson WF. A microscopic demonstration of the normal and pathological histology of mesoglia cells. J Ment Sci 1900; 46:733-52.
7. Alzheimer A. Histologische Studien zur Diferenzialdiagnose der progressiven Paralyse. Nissl's histologische und histopathologische Arbeiten 1904; 1:18-314.
8. Ramón y Cajal S. Histologie du Système Nerveux de l'Homme et des Vertébrés [Translated by L. Azoulay]. Vol. I. Paris: Maloine; 1952.
9. Ramón y Cajal S. Histologie du Système Nerveux de l'Homme et des Vertébrés [Translated by L. Azoulay]. Vol. II. Paris: Maloine; 1955.
10. del Rio-Hortega P. El "tercer elemento" de los centros nerviosos. Bol Soc Espan Biol 1919; 9:69-120.
11. del Rio-Hortega P. Microglia. In: Penfield W, editor. Cytology and cellular pathology of the nervous system. Vol 2, New York: Hoeber. 1932. p. 482-534.
12. Perry VH, Gordon S. Macrophages and microglia in the nervous system. Trends Neurosci 1988; 11:273-7.
13. Hickey WF, Kimura H. Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo. Science 1988; 239:290-2.
14. de Groot CJ, Huppes W, Sminia T, Kraal G, Dijkstra CD. Determination of the origin and nature of brain macrophages and microglial cells in mouse central nervous system, using non-radioactive in situ hybridization and immunoperoxidase techniques. Glia 1992; 6:301-9.
15. Kaur C, Hao AJ, Wu CH, Ling EA. Origin of microglia. Microsc Res Tech 2001; 54:2-9.
16. Perry VH, Brown MC, Gordon S. The macrophage response to central and peripheral nerve injury. A possible role for macrophages in regeneration. J Exp Med 1987; 165:1218-23.
17. Lawson LJ, Perry VH, Gordon S. Turnover of resident microglia in the normal adult mouse brain. Neuroscience 1992; 48:405-15.
18. Innocenti GM, Clarke S, Koppel H. Transitory macrophages in the white matter of the developing visual cortex. II. Development and relations with axonal pathways. Brain Res 1983; 313:55-66.
19. Streit WJ, Kreutzberg GW. Response of endogenous glial cells to motor neuron degeneration induced by toxic ricin. J Comp Neurol 1988; 268:248-63.
20. Streit WJ, Graeber MB, Kreutzberg GW. Functional plasticity of microglia: a review. Glia 1988; 1:301-7.
21. Milligan CE, Levitt P, Cunningham TJ. Brain macrophages and microglia respond differently to lesions of the developing and adult visual system. J Comp Neurol 1991; 314:136-46.
22. Hume DA, Perry VH, Gordon S. Immunohistochemical localization of a macrophage-specific antigen in developing mouse retina: phagocytosis of dying neurons and differentiation of microglial cells to form a regular array in the plexiform layers. J Cell Biol 1983; 97:253-7.
23. Chen L, Yang P, Kijlstra A. Distribution, markers, and functions of retinal microglia. Ocul Immunol Inflamm 2002; 10:27-39.
24. Kohno T, Inomata H, Taniguchi Y. Identification of microglia cell of the rat retina by light and electron microscopy. Jpn J Ophthalmol 1982; 26:53-68.
25. Terubayashi H, Murabe Y, Fujisawa H, Itoi M, Ibata Y. Enzymhistochemical identification of microglial cells in the rat retina: light and electron microscopic studies. Exp Eye Res 1984; 39:595-603.
26. Ashwell K. Development of microglia in the albino rabbit retina. J Comp Neurol 1989; 287:286-301.
27. Ashwell KW, Hollander H, Streit W, Stone J. The appearance and distribution of microglia in the developing retina of the rat. Vis Neurosci 1989; 2:437-48.
28. Zhang C, Tso MO. Characterization of activated retinal microglia following optic axotomy. J Neurosci Res 2003; 73:840-5.
29. Streit WJ, Walter SA, Pennell NA. Reactive microgliosis. Prog Neurobiol 1999; 57:563-81.
30. Ling EA, Kaur LC, Yick TY, Wong WC. Immunocytochemical localization of CR3 complement receptors with OX-42 in amoeboid microglia in postnatal rats. Anat Embryol (Berl) 1990; 182:481-6.
31. Dijkstra CD, Dopp EA, Joling P, Kraal G. The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in rat recognized by monoclonal antibodies ED1, ED2 and ED3. Adv Exp Med Biol 1985; 186:409-19.
32. Zhang J, Wu GS, Ishimoto S, Pararajasegaram G, Rao NA. Expression of major histocompatibility complex molecules in rodent retina. Immunohistochemical study. Invest Ophthalmol Vis Sci 1997; 38:1848-57.
33. Bignami A, Dahl D. The radial glia of Muller in the rat retina and their response to injury. An immunofluorescence study with antibodies to the glial fibrillary acidic (GFA) protein. Exp Eye Res 1979; 28:63-9.
34. Miller NM, Oberdorfer M. Neuronal and neuroglial responses following retinal lesions in the neonatal rats. J Comp Neurol 1981; 202:493-504.
35. Thanos S. The Relationship of Microglial Cells to Dying Neurons During Natural Neuronal Cell Death and Axotomy-induced Degeneration of the Rat Retina. Eur J Neurosci 1991; 3:1189-1207.
36. Bodeutsch N, Siebert H, Dermon C, Thanos S. Unilateral injury to the adult rat optic nerve causes multiple cellular responses in the contralateral site. J Neurobiol 1999; 38:116-28.
37. Chen H, Weber AJ. Expression of glial fibrillary acidic protein and glutamine synthetase by Muller cells after optic nerve damage and intravitreal application of brain-derived neurotrophic factor. Glia 2002; 38:115-25.
38. Battisti WP, Wang J, Bozek K, Murray M. Macrophages, microglia, and astrocytes are rapidly activated after crush injury of the goldfish optic nerve: a light and electron microscopic analysis. J Comp Neurol 1995; 354:306-20.
39. Zeev-Brann AB, Lazarov-Spiegler O, Brenner T, Schwartz M. Differential effects of central and peripheral nerves on macrophages and microglia. Glia 1998; 23:181-90.
40. Origasa M, Tanaka S, Suzuki K, Tone S, Lim B, Koike T. Activation of a novel microglial gene encoding a lysosomal membrane protein in response to neuronal apoptosis. Brain Res Mol Brain Res 2001; 88:1-13.
41. Selles-Navarro I, Ellezam B, Fajardo R, Latour M, McKerracher L. Retinal ganglion cell and nonneuronal cell responses to a microcrush lesion of adult rat optic nerve. Exp Neurol 2001; 167:282-9.
42. Thanos S. Specific transcellular carbocyanine-labelling of rat retinal microglia during injury-induced neuronal degeneration. Neurosci Lett 1991; 127:108-12.
43. Thanos S, Mey J, Wild M. Treatment of the adult retina with microglia-suppressing factors retards axotomy-induced neuronal degradation and enhances axonal regeneration in vivo and in vitro. J Neurosci 1993; 13:455-66.
44. Garcia-Valenzuela E, Sharma SC. Laminar restriction of retinal macrophagic response to optic nerve axotomy in the rat. J Neurobiol 1999; 40:55-66.
45. Garcia-Valenzuela E, Gorczyca W, Darzynkiewicz Z, Sharma SC. Apoptosis in adult retinal ganglion cells after axotomy. J Neurobiol 1994; 25:431-8.
46. Ling EA. Ultrastructure and peroxidase cytochemistry of macrophages present in the retina of postnatal rats. Arch Histol Jpn 1981; 44:167-76.
47. Pearson HE, Payne BR, Cunningham TJ. Microglial invasion and activation in response to naturally occurring neuronal degeneration in the ganglion cell layer of the postnatal cat retina. Brain Res Dev Brain Res 1993; 76:249-55.
48. Rao K, Lund RD. Optic nerve degeneration induces the expression of MHC antigens in the rat visual system. J Comp Neurol 1993; 336:613-27.
49. Boycott BB, Hopkins JM. Microglia in the retina of monkey and other mammals: its distinction from other types of glia and horizontal cells. Neuroscience 1981; 6:679-88.
50. Schnitzer J, Scherer J. Microglial cell responses in the rabbit retina following transection of the optic nerve. J Comp Neurol 1990; 302:779-91.
51. Diaz-Araya CM, Provis JM, Penfold PL, Billson FA. Development of microglial topography in human retina. J Comp Neurol 1995; 363:53-68.