Microglial responses in the avascular quail retina following transection of the optic nerve

Brain Research 1023 (2004) 15 – 23 www.elsevier.com/locate/brainres

Research report

Microglial responses in the avascular quail retina following transection of the optic nerve Gye Sun Jeona, Tae-Cheon Kangb, Sang Wook Parka, Dong Woon Kima, Je Hoon Seoc, Sa Sun Choa,d,* a

Department of Anatomy, Seoul National University College of Medicine, Yongon-Dong 28, Seoul 110-799, South Korea b Department of Anatomy, College of Medicine, Hallym University, Chunchon, 200-702, South Korea c Department of Anatomy, College of Medicine, Chungbuk National University, Cheongju, South Korea d MRC Neuroscience Research Institute, Seoul National University College of Medicine, Yongon-Dong 28, Seoul 110-799, South Korea Accepted 6 January 2004 Available online 25 August 2004

Abstract This study was undertaken to investigate microglial responses in the avascular central nervous system using the quail retina that is known to be devoid of blood vessels. Following intraorbital optic nerve transection (ONT), the quail retina was examined immunohistochemically at various times up to 6 months. A few days after transection, microglia in the inner retinal layers revealed features of activation. Activated cells displayed an amoeboid shape and enhanced QH1-immunoreactivity. The numbers of these amoeboid cells were rapidly increased, first in the inner plexiform layer (IPL), and then in the ganglion cell/nerve fiber layer (GCL/NFL) of the retina where retrograde degenerating ganglion cell processes and perikarya were located. By 6 months after transection, microglia regained their resting morphology, and their cell counts returned to control levels. At early time points of microglial activation, numerous QH1+ amoeboid cells were observed along the vitreal surface of the pecten and retinal region adjacent to the insertion of the pecten, where some amoeboid cells were attached underneath the internal limiting membrane, and appeared to squeeze through the optic nerve fiber bundles. A considerable number of these amoeboid cells in the GCL/NFL and the IPL were labeled with PCNA, suggesting that active exogenous migration (from the pecten) and in situ proliferation of precursor cells contribute to the increase in microglial population of the degenerating retina. On the other hand, TUNEL-positive microglia appeared in the GCL/NFL at later time points indicate that the decrease of microglial numbers is in part due to apoptosis in these layers. Although some aspects of microglial activation in the avascular retina appear unique, their consequences were similar to those described in vascular retinae of mammals, a finding indicates that blood vessels are not a prerequisite for microglial activation, and microglial precursors could migrate long distance to reach the lesioned site, which is not accessible via blood vessels. Our data provide the first analysis of microglial activation in the avascular central nervous system (CNS), and suggest that the quail retina is a useful model for studies of microglial behavior in CNS. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Trauma Keywords: Microglial activation; Proliferation; Migration; Apoptosis; Avascular avian retina; Optic nerve transection

1. Introduction

* Corresponding author. Department of Anatomy, Seoul National University, College of Medicine, Yongon-Dong 28, Chongno-Gu, Seoul 110-799, South Korea. Tel.: +82 2 740 8204; fax: 82 2 745 9528. E-mail address: [email protected] (S.S. Cho). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.01.093

Microglia play central roles in responding to brain injury and infection. As a consequence of neuronal injury, microglia become activated and proceed through a graded but stereotypical series of morphological and molecular changes that may support neuroprotective and/

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or cytotoxic roles [4,12,30,41]. Severe tissue injury or infection resulting in neuronal death induces conversion of microglia to phagocytic brain macrophages that remove dead cell debris [39,40]. Microglia also appear capable of interacting with circulating immune cells of the blood. They can produce cytokines and chemokines that recruit and activate immune cells [26], and they can physically engage and present antigen to lymphocytes that extravasate from blood vessels [29]. Although microglial cells are normally scattered throughout the brain parenchyma, some are situated near blood vessels (juxtavascular microglia). For example, up to ~30% of all resting parenchymal microglia in the rat hippocampus qualify as juxtavascular microglia [6,9]. A recent report demonstrated that activated juxtavascular microglia are preferentially recruited to the surfaces of blood vessels and, as a population, exhibit a more robust activation response than parenchymal (nonjuxtavascular) microglia following traumatic injury to brain tissue [13]. A close apposition to blood vessels may facilitate interaction of injury- and immune-responsive cells of the brain and blood. However, little is known about how microglia interact with vascular structures in their activation processes, and it is not clear how the activation response of microglia between the vascular and avascular CNS tissues may differ. To our knowledge, no comparable data are available since many features of microglial activation in the central nervous system (CNS) have been studied in the vascular tissues. As a first step toward addressing these questions, we used the visual system of the quail that has served as an in vivo model for studies of microglial activation in the avascular CNS. The quail retina seems to be particularly advantageous to this purpose. First, unlike its mammalian counterpart, it is completely avascular, and includes a structure called the pecten, a richly vascularized organ, which projects into the vitreous body from the optic disc [21,32]. Thus, the neural tissue (retina) and the supporting vascular structure (pecten) are spatially separated. Second, by using QH1 antibody, a specific microglial marker, the normal distribution and developmental origin of microglia in the quail retina have been well documented [18,24,25]. Third, there are many existing reports on retinal microglial activation from the mammal, which provide ample data for comparison with findings from the quail retina [1,3,14,33,36,37,42]. In addition, the system allows easy access to make a lesion (axotomy) in the CNS fiber tract that does not breach the blood– retinal barrier. Due to the naturally layered distribution of the cells inside the retina, quantitative and qualitative responses to axotomy can be observed. In this study, we will describe microglial responses in the quail retina with regard to the spatial and temporal aspects of activation, proliferation, migration, apoptosis, and compare the findings obtained with those of a mammalian counterpart.

2. Materials and methods 2.1. Animal and tissue preparation Young adult (about 6 weeks old) Japanese quails (Coturnix corturnix japonica) were used in this study. Under deep anesthesia (20 mg/kg Ketamine; 80 mg/kg Xylazine), the left optic nerve was transected intraorbitally as previously described [23]. Briefly, the left upper eyelid was cut near the orbit and left optic nerve was transected with ophthalmic scissors. The point of transection was consistent in all operations, approximately 2 mm behind the eyeball. The right retina served as a control. Quails (number of animals in parenthesis) treated in the above manner were allowed to survive for postoperative periods of four (5), seven (4), 10 (5), 14 (5), 21(5), 28 (4), 60 (4), and 180 (5) days. Under deep anesthesia, eyeballs were enucleated. The cornea, lens, and vitreous body were removed and eyecups were immersion fixed for 3 h at 4 8C in 4% paraformaldehyde. They were cryoprotected by serial sucrose treatment. The blocks containing the eyecups were rapidly frozen with liquid nitrogen and cut into 10 Am thick transverse sections on a freezing microtome. Sections were then thaw-mounted on gelatin-coated microscopic slides and stored at 20 8C until required for immunohistochemistry. All animal experiments were approved by the local Institutional Animal Care and Use Committee. 2.2. Immunohistochemistry Sections were sequentially treated with 0.3% H2O2 in methanol for 30 min, incubated in 1% normal chicken serum in PBS for 1 h, and then incubated in diluted mouse anti-QH1 antibody solution (1:300, Developmental Studies Hybridoma Bank, University of Iowa) overnight at 4 8C. They were then exposed to biotinylated horse anti-mouse IgG and streptavidin peroxidase complex (Vector, USA), and finally treated with 3,3V-diaminobenzidine (DAB; Sigma, USA) solution containing 0.003% H2O2 to visualize the antigen–antibody reaction. Controls for immunohistochemical staining included the omission of primary or secondary antibodies. To identify the proliferating microglia, we performed double-labeling immunofluorescent microscopy. The sections were incubated with monoclonal anti-proliferating cell nuclear antigen (1:100, anti-PCNA; Sigma), and biotinylated Ricinus communis agglutinin 1 (1:3,000, RCA-1; Vector) that is a specific marker for microglia [17], followed by CyTM3 labeled goat anti-mouse IgG (1:100) and CyTM2 conjugated streptavidin (1:500; Amersham, USA). Each step was followed by washing three times for 10 min with PBS. Sections were coverslipped in crystal mounts (Biomeda, USA), and observed under a Carl Zeiss fluorescence microscope. Some retinal whole mounts were

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prepared by using the free-floating method described previously [5]. 2.3. Double labeling for TUNEL and QH1 The procedures of this techniques were similar to those used for single labelings, except that they were based on fluorescence labeling. TUNEL-labeling was performed following the protocols by Gavrieli et al. [11] with some adjustments. Sections were sequentially treated with 3% H2O 2 in methanol for 5 min, incubated in TdT buffer (Boehringer Mannheim, USA) and CoCl2 (Boehringer Mannheim) for 5 min at room temperature. Residues of biotinylated dUTP (Boehringer

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Mannheim) were catalytically added to the ends of DNA fragments with the enzyme TdT for 60 min at 37 8C. After end labeling, the sections were incubated with a streptavidin-biotinylated horseradish peroxidase complex (Vector) for 45 min at room temperature and visualized with DAB and H2O2. The same slides were sequentially treated with 0.3% H2O2 in methanol for 30 min, incubated in 1% normal chicken serum in PBS for 1 h, and then incubated in diluted mouse anti-QH1 antibody solution overnight at 4 8C. They were then exposed to biotinylated horse anti-mouse IgG and streptavidin peroxidase complex, and finally visualized with naphthol containing 3% H2O2 [8]. As the result of this techniques, QH1+ microglial cells were stained

Fig. 1. Representative histological sections of the retina stained immunohistochemically with QH1 antibody. In the normal retina (A), QH1-labeled microglia displayed the ramified morpholgy with long slender processes extending over the IPL. In the lesioned retina, QH1-labeled amoeboid cells appeared primarily in the IPL at 4 days (B), and became more numerous in the GCL/NFL (C) at 10 days after ONT. At 2–3 weeks after ONT, larger amoeboid cells with a strong immunoreactivity were localized mainly in the GCL/NFL (D, E). Thereafter, amoeboid microglia were decreased in numbers and staining intensity (F, G), and activated microglia were no longer observed 6 months after ONT (H). Photomicrographs were taken from the retinal area 1 mm anterior from the optic disc in each experimental group. Normal control, A; Postoperational day 4 (B), 10 (C), 14 (D), 21 (E), 28 (F), 60 (G), and 180 (H). NFL; nerve fiber layer, GCL; ganglion cell layer, IPL; inner plexiform layer, OPL; outer plexiform layer. Scale bar=50 Am.

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blue, while apoptotic bodies (nuclei) were stained brown.

3. Results 3.1. Morphology and distribution of microglia

2.4. Quantitative analysis Sagittal sections though the optic disc were used to evaluate cell densities of microglia in the three main layers of the retina; the ganglion cell/nerve fiber layer (GCL/NFL), inner plexiform layer (IPL), and outer plexiform layer (OPL). Using a calibrated micrometer, the number of QH1+, PCNA+, and TUNEL+ microglia in these retinal layers were counted respectively in the central part of the retina adjacent to the optic disc where a rich population of microglia is described [24]. Counts were made in two selected retinal regions (covering 1 mm length from the optic disc) of each section, at a final magnification of 100. No corrections were made for the retinal layers that become thinner due to retrograde degeneration of ganglion cells. For each experimental group, nine representative sections (three sections from three animals) were measured, and the mean was calculated. The student’s t-test was used for the statistical analysis.

In the normal retina, QH1-immunohistochemisty revealed the ramified (resting) microglia, which were characterized by small cell bodies and long slender processes extending radially over the retinal layers. The cell bodies of these microglia were found primarily in the IPL and GCL, although some were found in the OPL (Fig. 1A). These findings on the topography and shape of microglial cells in the quail retina are in accordance with previous findings in the adult quail [24] and rabbit [37]. Following optic nerve transection (ONT), the staining intensity, shape, distribution pattern and density of retinal microglia were rapidly changed. At 4 days after ONT, strongly QH1-labeld amoeboid cells appeared in the inner layers of the retina (Fig. 1B). They were more numerous in the IPL than other retinal layers. At 10 days after ONT, QH1-labeled amoeboid cells became more numerous in the GCL/NFL (Fig. 1C). By 2 weeks post axotomy, at a time corresponding to the loss of majority of retinal ganglion cell population in the quail [23], there were larger amoeboid

Fig. 2. QH1+ microglial cells in the whole-mounted retina of the control (A, C) and lesioned retina (B, D) at 14 days after ONT. In the normal retina, QH1+ cells in the IPL (A) displayed typical morphology of ramified (resting) microglia, but microglial cells in the NFL (C) were poorly ramified, with processes mainly oriented parallel to the optic nerve bundles. Following ONT, microglia in both IPL (B) and the NFL (D) were more intensely QH1-labeled, and exhibited larger cell bodies with thicker and shorter processes, although their characteristic pattern of distribution was largely preserved. Scale bar =50 Am.

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Table 1 Microglial cell numbers in the retinal layers following transection of optic nerve Retinal layer

Control

Postoperative day 4

10

14

21

180

GCL/NFL IPL OPL Total

11.0F1.7 19.7F1.5 5.7F1.2 37.0F3.6

17.7F0.6* 47.0F2.6* 5.3F0.6 70.0F2.0*

42.7F1.5* 17.0F2.6 9.3F0.6 69.0F3.6*

84.3F2.1* 13.3F1.5 9.3F1.5 107.0F5.0*

39.7F1.5* 15.3F1.5 7.3F1.2 62.3F1.2*

12.0F2.0 14.7F1.5 5.7F1.2 32.3F3.8

Values are obtained from three retinal areas (1 mm) per animal (n=3 per group; meanFS.D.). Asterisks denote statistical significance (at least Pb0.001) in comparison with the corresponding value at the respective control.

cells with enhanced QH1-immunoreactivity in the GCL/ NFL (Fig. 1D). A similar finding with somewhat lesser degree of immuno-staining was noted at 3 weeks after ONT (Fig. 1E). Subsequently, amoeboid microglia gradually decreased (Fig. 1F,G), and microglial activation was no longer observed 6 months after ONT (Fig. 1H). Whole mount preparations of the retina further manifested the morphological characteristics of microglia. In the normal retina, QH1+ cells displayed a typical shape of ramified

microglia, and they were distributed in a mosaic-like fashion throughout the retina (Fig. 2A). On the other hand, microglia in the nerve fiber layer were poorly ramified, but their long slender bipolar processes were aligned with ganglion cell axons (Fig. 2C). Following ONT, microglia in both the IPL and NFL were more intensely QH1-labeled, and exhibited larger cell bodies with shorter and thicker processes, although their characteristic pattern of distribution in both layers was largely preserved during the ganglion

Fig. 3. QH1+ microglial cells in the pecten and its adjacent retinal area, showing QH1+ microglial precursors and their possible migratory path from the pecten to the retina following ONT. In the control (A), no QH1+ cells were detected on the vitreal surface of the pecten or retina. Note that the magnified pecten (inset) displayed the smooth surface without any adhering cells on it. Following ONT, however, many QH1+ cells (arrows) were observed along the vitreal surfaces of the pecten (B), and retina (C, D). In the retina, elongated QH1+ cells often appeared underneath the internal limiting membrane and appeared to squeeze into the optic neve fiber bundles (E). Normal control, A; Postoperational day 4 (B, C), 7 (D), 10 (E), and 28 (F). P; pecten, RT; retina, ON; optic nerve. Scale bars=50 Am for A, and B–F.

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Table 2 PCNA- or TUNEL-labeled microglial population (cell numbers/mm) in the retinal layers following transection of optic nerve Microglia labeling

Postoperative day 4

7

10

14

21

PCNA TUNEL

18.7F1.7a (47.0F2.6) –

9.3F0.6b (30.7F1.6) –

– 8.3F1.5b (42.7F1.5)

– 18.3F0.6b (84.3F2.1)

– 13.3F0.6b (39.7F1.5)

Values are obtained from three retinal areas (1 mm) per animal (n=3 per group; meanFS.D.). Figures in parentheses are total numbers of microglial cells in the IPL (a) and GCL/NFL (b). –: not done.

cell degeneration (Fig. 2B,D). When we counted the density of QH1+ microglia in the inner three layers (Table 1), total numbers of microglia increased rapidly in a few days after ONT ( Pb0.001). This increase peaked (2.9-fold) 2 weeks after lesion, thereafter the number of QH1+ microglial cells decreased gradually and returned to the normal value by 6 months after ONT. When compared the cell counts from three layers of the retina, a significant increase was found in both IPL and GCL/NFL layers at 4 days after ONT. Thereafter the increase was found only in the GCL/NFL, while those in the IPL and OPL remained roughly constant (Table 1).

ONT (Table 2). PCNA+ microglia first appeared at 4 days after ONT in the IPL (Fig. 4A,B), and occupied about 40% of total microglia in this layer. By 7 days post operation, these PCNA+ microglia were primarily found in the NFL or on the vitreal surface of the retina close to the inner limiting membrane (Fig. 4C). These cells reached about 30% of microglia in this layer. However, no significant number of PCNA-labeled cells were found at later time points than 10 days post operation. On the other hand, TUNEL-labeled microglia were first found at 10 days after ONT in the GCL/ NFL (Fig. 5A,B). They occupied about 20% of total

3.2. Migration, proliferation, apoptosis of microglia The increase of microglia in the retina could be due to proliferation of cells situated in the IPL and GCL/NFL or, alternatively, to invasion by microglial precursors. To check these possibilities, we first examined the appearance of microglial precursors from the pecten and the adjacent retinal portion, since it has been suggested that the pecten is the main source of microglial precursors that migrate into the retina during the embryonic period [18,25]. In the normal retina, QH1+ cells could not be detected on the surface of the pecten, although the parenchyma pecten and its attachment to the optic disc were darkly stained due to the inherent blood vessels and pigment cells reside in these organs (Fig. 3A). However, many QH1+ cells appeared a few days after ONT along the vitreal surface of the pecten and retinal region adjacent to the insertion of the pecten (Fig. 3B–E). These QH1+ cells were continually found up to 2 weeks postlesion, but disappeared by 4 weeks (Fig. 3F). QH1+ cells were elongated or amoeboid in shape, without any cellular processes, and intimately adhered to the vitreal surface of the pecten or midway between the pecten and the retina (Fig. 3B,C,D). In the retina, QH1+ cells were often observed underneath the internal limiting membrane, and some of them appeared to be squeezed into the optic nerve fiber bundles, and subsequently oriented radially (Fig. 3E). These findings suggest that QH1+ cells are microglial precursors that may be derived from the pecten and migrate towards the lesioned site of the reina. We also investigated whether microglial cells proliferate or die in the injured retina by double-labeled immunofluorescence microscopy using PCNA antibody or TUNEL methods. Interestingly, PCNA+ microglia appeared earlier time points while TUNEL+ microglia were found at later time points after

Fig. 4. Double-labeled retina with PCNA (red) and RCA-1 (green). PCNAlabeling was found in the nuclei of some microglial cells (arrows) in the IPL of the central (A) and peripheral (B) retina at 4 days after ONT. At 7 days after ONT, PCNA-labeling was mainly found in the microglial cells (arrows) located in the NFL (C). Note that PCNA+ microglia (arrow heads) were also present both on the vitreal side and underneath of the internal limiting membrane (C). Scale bar =50 Am.

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Fig. 5. Double-labeled retina with TUNEL (brown) and QH1 (blue). Note dark-stained nuclei of microglia by TUNEL-labeling (arrows). TUNEL-labeling first appeared in amoeboid microglia in the NFL at 10 days after ONT (A, B), and subsequently in the ramified microglia at 21 days after ONT (C, D). Scale bar =50 Am.

microglia in this layer, and this level was maintained at 21 days after ONT. By 21 days post operation, TUNEL+ microglia were found more often (Fig. 5C,D) and reached about 30% of microglia found in this layer (Table 2).

4. Discussion Our observations on the response of microglial cells to axotomy of ganglion cells in the avian retina have provided the first analysis of microglial activation in the avascular CNS tissue. Microglia in avascular quail retina changed from the ramified to the amoeboid shape a few days after ONT. Microglial activation judged by the increase rate of cell numbers in the IPL and GCL/NFL of the retina becomes peak about 2 weeks after ONT, and returned gradually to normal by 6 months post transection. This feature of microglial activation in the avascular quail retina was similar to that previously described in the vascularized mammalian counterpart [20,28,37]. It has long been known that activated microglia in the mammalian retina are involved in phagocytizing degenerating ganglion cell bodies and their processes [22]. The same may hold true for the avian retina since the time course and location of microglial activation as shown in this study are in accordance with those of ganglion cell degeneration induced axotomy in the quail retina [23]. Despite these similarities, our analyses revealed some different aspects of microglia activation in the avascular retina. Besides microglial changes in the IPL and GCL/NFL of the retina, QH1+ amoeboid cells appeared along the vitreal surface of the pecten and the retinal region adjacent to the insertion of the pecten a few days after lesion.

Subsequently, QH1+ cells showing the elongated shape were found underneath the internal limiting membrane and appeared to squeeze into the optic never fiber bundles in the retina. Migration of microglial precursors from the pecten into the retina has been reported in the developing quail retina [18,25]. Besides, infiltrating hematogenous macrophages from the blood vessels into the lesioned site were demonstrated in the mammalian CNS [10,16,27,35]. Given the fact that the pecten is a richly vascularized organ [21,32], these QH1+ amoeboid cells may be migrating macrophages from blood vessels in the pecten into the lesioned retina. It is known that hematogenous macrophages are able to acquire a ramified morphology indistinguishable from resident microglia while microglial cells can develop into a phagocytic phenotype indistinguishable from infiltrating macrophages [10,16,27,35]. However, clear distinction between these cells in injured CNS tissue is close to impossible due to the lack of discriminating cellular markers. In this respect, due to unique anatomical features of avian retina in which the neural tissue (retina) and the supporting vascular structure (pecten) are spatially separated, populations of infiltrating phagocytic cells become well separated at least before they move into the retina and completely mix with resident microglia. As several previous studies of the postischemic microglial/macrophage response demonstrated that the increase of microglial cell number in the injured brain area was largely due to activating local microglia [16,35], we analyzed microglial proliferation by PCNA/RCA-1 double-labeling experiments. As expected, a significant number of PCNA+ microglial cells were found mainly in the retinal layers where the increases in microglial cell number

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occurred. PCNA+ microglia occupied about 40% of microglia in the IPL at 4 days, and 30% of microglia in the GCL/NFL at 7 days after ONT. Appearance of PCNA+ microglia and concomitant changes of microglial population in IPL and GCL/NFL suggest that microglial proliferation contribute in part the massive increase of this glial population in these retinal layers. Earlier studies also reported glial proliferation in the retina of the degenerating optic nerve in the rat [38] and rabbit [34,37], but neither studies presented quantitative data on glial proliferation. Interestingly, the increase of microglia numbers in the IPL was confined to the early time point while prolonged increase of microglial number was noted in the GCL/NFL. The reason of this finding is not known at present. In the brain, it has been reported that microglia proliferate far away from the lesion site [31,43], and become recruited to perineuronal sites, where they actively displace the afferent synaptic terminals during synaptic stripping [2]. Therefore, it is conceivable that local microglia including newly proliferated cells in the IPL may finally migrate into the GCL/NFL where active retrograde degeneration of ganglion cell bodies and axons occur. On the other hand, PCNA+ amoeboid cells on the vitreal surface of the lesioned retina may reflect active proliferation of exogenous macrophages migrating from the pecten to the retina where they may take part in the microglial increase. This notion is in accordance with previous reports in that cell division of microglia precursors occurs in location where they are actively migrating during development of the quail retina and cerebellum [7,19]. After ONT, the transient increase in microglial cell number is followed by a slow, gradual decline, suggesting apoptosis in the regulation of the postmitotic population of microglia. In an apparent confirmation of this hypothesis, the current study revealed the presence of a subpopulation of TUNEL+ microglia, beginning at day 10 and peaking 2 weeks after ONT. Our results are in agreement with a previous study where the induced apoptosis in microglia was observed after transection of the facial nerve [15], although this condition induces microglial apoptosis more rapidly than ONT damage in our avian model. On the other hand, it should be noted that presence of TUNEL+ microglia in the retinal layer after ONT was not completely coincident with a microglial decrease. For example, no significant numbers of TUNEL+ microglia were found in the IPL where a rapid decrease in microglial numbers was noted at 10 days after ONT. This suggests that apoptosis is not only factor, but others, such as microglial migration may work together in regulation of microglial cell numbers in the lesioned area. Further functional studies are needed to reveal the dynamic phenomena of microglial activation. In conclusion, our data provide the first comprehensive analysis of microglial activation in the avascular CNS, and suggest that the quail retina is a useful model for further studies of microglial behavior in injured CNS tissue.

Acknowledgements The QH1 antibody, developed by Dr. F. Dieterlen, was obtained from the Developmental Studies Hybridoma Bank maintained by The University of Iowa, Department of Biological Science, Iowa City, IA 52242, USA under contract NO1-HD-7-3263 from the NICHD. This study was supported by the Korea Research Foundation Grant (KRF-2000-015-FP0002) and in part by the BK 21 Project for Medicine, Dentistry and Pharmacy.

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