Cooperative Phagocytes
2009; Elsevier BV; Volume: 174; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.090023
ISSN1525-2191
AutoresSandrine Joly, Mike Francke, Elke Ulbricht, Susanne Beck, Matthias Seeliger, Petra G. Hirrlinger, Johannes Hirrlinger, Karl S. Lang, Martin S. Zinkernagel, Bernhard Odermatt, Marijana Samardzija, Andreas Reichenbach, Christian Grimm, Charlotte E. Remé,
Tópico(s)Retinal Development and Disorders
ResumoPhagocytosis is essential for the removal of photoreceptor debris following retinal injury. We used two mouse models, mice injected with green fluorescent protein-labeled bone marrow cells or green fluorescent protein-labeled microglia, to study the origin and activation patterns of phagocytic cells after acute blue light-induced retinal lesions. We show that following injury, blood-borne macrophages enter the eye via the optic nerve and ciliary body and soon migrate into the injured retinal area. Resident microglia are also activated rapidly throughout the entire retina and adopt macrophage characteristics only in the injured region. Both blood-borne- and microglia-derived macrophages were involved in the phagocytosis of dead photoreceptors. No obvious breakdown of the blood-retinal barrier was observed. Ccl4, Ccl12, Tgfb1, Csf1, and Tnf were differentially expressed in both the isolated retina and the eyecup of wild-type mice. Debris-laden macrophages appeared to leave the retina into the general circulation, suggesting their potential to become antigen-presenting cells. These experiments provide evidence that both local and immigrant macrophages remove apoptotic photoreceptors and cell debris in the injured retina. Phagocytosis is essential for the removal of photoreceptor debris following retinal injury. We used two mouse models, mice injected with green fluorescent protein-labeled bone marrow cells or green fluorescent protein-labeled microglia, to study the origin and activation patterns of phagocytic cells after acute blue light-induced retinal lesions. We show that following injury, blood-borne macrophages enter the eye via the optic nerve and ciliary body and soon migrate into the injured retinal area. Resident microglia are also activated rapidly throughout the entire retina and adopt macrophage characteristics only in the injured region. Both blood-borne- and microglia-derived macrophages were involved in the phagocytosis of dead photoreceptors. No obvious breakdown of the blood-retinal barrier was observed. Ccl4, Ccl12, Tgfb1, Csf1, and Tnf were differentially expressed in both the isolated retina and the eyecup of wild-type mice. Debris-laden macrophages appeared to leave the retina into the general circulation, suggesting their potential to become antigen-presenting cells. These experiments provide evidence that both local and immigrant macrophages remove apoptotic photoreceptors and cell debris in the injured retina. Acute light-induced retinal degeneration is characterized by apoptosis of photoreceptor cells and by conspicuous phagocytic cells in the region of cell death.1Remé CE The dark side of light: rhodopsin and the silent death of vision the proctor lecture.Invest Ophthalmol Vis Sci. 2005; 46: 2671-2682Crossref PubMed Scopus (55) Google Scholar, 2Wenzel A Grimm C Samardzija M Remé CE Molecular mechanisms of light-induced photoreceptor apoptosis and neuroprotection for retinal degeneration.Prog Retin Eye Res. 2005; 24: 275-306Crossref PubMed Scopus (543) Google Scholar Phagocytic cells are also found in inherited dystrophies, but to a lesser degree at any given time point due to the protracted course of those diseases. The immune privileged status of the retina, similar to the brain, is thought to limit the exit of local and entry of systemic immune cells through the blood-retinal barrier.3Streilein JW Ocular immune privilege: the eye takes a dim but practical view of immunity and inflammation.J Leukoc Biol. 2003; 74: 179-185Crossref PubMed Scopus (251) Google Scholar, 4Niederkorn JY See no evil, hear no evil, do no evil: the lessons of immune privilege.Nat Immunol. 2006; 7: 354-359Crossref PubMed Scopus (346) Google Scholar Nevertheless, a distinct cellular response with phagocytosis of disrupted photoreceptor outer segments and apoptotic cells occurs after acute light-induced degeneration.5Hoppeler T Hendrickson P Dietrich C Remé C Morphology and time-course of defined photochemical lesions in the rabbit retina.Curr Eye Res. 1988; 7: 849-860Crossref PubMed Scopus (39) Google Scholar, 6Joly S Samardzija M Wenzel A Thiersch M Grimm C Nonessential role of β3 and β5 integrin subunits for efficient clearance of cellular debris after light-induced photoreceptor degeneration.Invest Ophthalmol Vis Sci. 2009; 50: 1423-1432Crossref PubMed Scopus (21) Google Scholar The origin of such phagocytes can potentially derive from two sources: from blood-borne macrophages or from resident retinal microglia. To track macrophage infiltration from the systemic circulation, green fluorescent protein (GFP)-tagged bone marrow precursor cells were visualized after grafting into lethally irradiated mice.7Lang KS Recher M Junt T Navarini AA Harris NL Freigang S Odermatt B Conrad C Ittner LM Bauer S Luther SA Uematsu S Akira S Hengartner H Zinkernagel RM Toll-like receptor engagement converts T-cell autoreactivity into overt autoimmune disease.Nat Med. 2005; 11: 138-145Crossref PubMed Scopus (329) Google Scholar The fractalkine receptor (CX3CR1) is expressed on central nervous system (CNS) microglia. Microglial cells expressing GFP under the Cx3cr1 promoter8Jung S Aliberti J Graemmel P Sunshine MJ Kreutzberg GW Sher A Littman DR Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.Mol Cell Biol. 2000; 20: 4106-4114Crossref PubMed Scopus (1788) Google Scholar were used to visualize resident microglia after retinal injury. Inherited retinal dystrophies and age-related macular degeneration are accompanied by glia activation.9Gupta N Brown KE Milam AH Activated microglia in human retinitis pigmentosa, late-onset retinal degeneration, and age-related macular degeneration.Exp Eye Res. 2003; 76: 463-471Crossref PubMed Scopus (416) Google Scholar Retinal microglia, as brain microglia,10Block ML Zecca L Hong JS Microglia-mediated neurotoxicity: uncovering the molecular mechanisms.Nat Rev Neurosci. 2007; 8: 57-69Crossref PubMed Scopus (2961) Google Scholar, 11Cardona AE Pioro EP Sasse ME Kostenko V Cardona SM Dijkstra IM Huang D Kidd G Dombrowski S Dutta R Lee JC Cook DN Jung S Lira SA Littman DR Ransohoff RM Control of microglial neurotoxicity by the fractalkine receptor.Nat Neurosci. 2006; 9: 917-924Crossref PubMed Scopus (1113) Google Scholar can promote photoreceptor death12Srinivasan B Roque CH Hempstead BL Al-Ubaidi MR Roque RS Microglia-derived pronerve growth factor promotes photoreceptor cell death via p75 neurotrophin receptor.J Biol Chem. 2004; 279: 41839-41845Crossref PubMed Scopus (88) Google Scholar but can also be protective13Harada T Harada C Kohsaka S Wada E Yoshida K Ohno S Mamada H Tanaka K Parada LF Wada K Microglia-Muller glia cell interactions control neurotrophic factor production during light-induced retinal degeneration.J Neurosci. 2002; 22: 9228-9236PubMed Google Scholar, 14Sasahara M Otani A Oishi A Kojima H Yodoi Y Kameda T Nakamura H Yoshimura N Activation of bone marrow-derived microglia promotes photoreceptor survival in inherited retinal degeneration.Am J Pathol. 2008; 172: 1693-1703Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar depending on the experimental conditions. Activated microglia and hematogenous macrophages might increase damage by releasing inflammatory cytokines and chemokines and trigger the formation of toxic molecules such as nitric oxide, reactive oxygen species and free radicals, collectively called molecules of oxidative stress.10Block ML Zecca L Hong JS Microglia-mediated neurotoxicity: uncovering the molecular mechanisms.Nat Rev Neurosci. 2007; 8: 57-69Crossref PubMed Scopus (2961) Google Scholar Protection may be achieved by the release of neuroprotective messengers.13Harada T Harada C Kohsaka S Wada E Yoshida K Ohno S Mamada H Tanaka K Parada LF Wada K Microglia-Muller glia cell interactions control neurotrophic factor production during light-induced retinal degeneration.J Neurosci. 2002; 22: 9228-9236PubMed Google Scholar To date, the cellular and molecular mechanisms of microglia activation and the function in acute and chronic degeneration have not been elucidated in detail for the retina. Moreover, the relative proportion of blood-borne macrophages and of resident microglia recruited to clean the retina from dead photoreceptors has never been investigated so far. Here we show for the acutely injured retina that bone marrow-derived cells rapidly immigrate through the vascular system of the ciliary body and optic nerve and differentiate into macrophages. We demonstrate that resident retinal microglia is activated as well and both hematogenous cells and retinal microglia participate in the phagocytosis of destroyed photoreceptor cells. In vivo monitoring by scanning laser ophthalmoscopy (SLO) and indocyanine green angiography of acute-phase lesions showed immigrating blood cells without major disruption of the blood-retinal barrier. Electron microscopy indicated the exit of debris-laden macrophages into the general circulation. Inflammatory processes have recently been recognized to contribute to the pathogenesis of age-related macular degeneration and glaucoma, diseases that were previously considered mainly degenerative.15Jha P Bora PS Bora NS The role of complement system in ocular diseases including uveitis and macular degeneration.Mol Immunol. 2007; 44: 3901-3908Crossref PubMed Scopus (101) Google Scholar, 16Hollyfield JG Bonilha VL Rayborn ME Yang X Shadrach KG Lu L Ufret RL Salomon RG Perez VL Oxidative damage-induced inflammation initiates age-related macular degeneration.Nat Med. 2008; 14: 194-198Crossref PubMed Scopus (570) Google Scholar, 17Combadiere C Feumi C Raoul W Keller N Rodero M Pezard A Lavalette S Houssier M Jonet L Picard E Debre P Sirinyan M Deterre P Ferroukhi T Cohen SY Chauvaud D Jeanny JC Chemtob S Behar-Cohen F Sennlaub F CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration.J Clin Invest. 2007; 117: 2920-2928Crossref PubMed Scopus (428) Google Scholar Moreover, several cytokines and chemokines were identified to be involved in the activation process of macrophages and microglial cells in brain and retina, respectively, such as fractalkine, Tnf, Ccl4, Ccl2, and stromal-derived factor 1.14Sasahara M Otani A Oishi A Kojima H Yodoi Y Kameda T Nakamura H Yoshimura N Activation of bone marrow-derived microglia promotes photoreceptor survival in inherited retinal degeneration.Am J Pathol. 2008; 172: 1693-1703Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 18Tsou CL Peters W Si Y Slaymaker S Aslanian AM Weisberg SP Mack M Charo IF Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites.J Clin Invest. 2007; 117: 902-909Crossref PubMed Scopus (764) Google Scholar, 19Ambrosini E Aloisi F Chemokines and glial cells: a complex network in the central nervous system.Neurochem Res. 2004; 29: 1017-1038Crossref PubMed Scopus (174) Google Scholar However, these strongly regulated processes are poorly understood to date and may differ significantly in different organs20Haak S Croxford AL Kreymborg K Heppner FL Pouly S Becher B Waisman A IL-17A and IL-17F do not contribute vitally to autoimmune neuro-inflammation in mice.J Clin Invest. 2009; 119: 61-69PubMed Google Scholar or under different conditions in the same organ. Therefore, we investigated several pro- and anti-inflammatory cytokines and chemokines and found differing patterns of up-regulation in the isolated retina and eyecup in wild-type mice. Inflammatory messengers are localized specifically in macrophages within the area of damage but not in retinal neurons or Müller cells. All procedures were performed in accordance with the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research and within the guidelines of the Cantonal Veterinary Authorities of Zurich. One day after lethal irradiation (950 rad), young recipient C57BL/6 mice were transplanted by intravenous injection of 8 × 106 bone marrow cells, generated by flushing the femur and tibia of C57BL/6 donor mice carrying the green fluorescent protein (GFP+/+) gene under the chicken β-actin promoter.7Lang KS Recher M Junt T Navarini AA Harris NL Freigang S Odermatt B Conrad C Ittner LM Bauer S Luther SA Uematsu S Akira S Hengartner H Zinkernagel RM Toll-like receptor engagement converts T-cell autoreactivity into overt autoimmune disease.Nat Med. 2005; 11: 138-145Crossref PubMed Scopus (329) Google Scholar Light exposure was performed 6 to 8 weeks after transplantation. We also used transgenic heterozygous and homozygous mice where the fractalkine receptor gene (CX3CR1) was replaced by GFP (CX3CR1+/−; CX3CR1−/−).8Jung S Aliberti J Graemmel P Sunshine MJ Kreutzberg GW Sher A Littman DR Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.Mol Cell Biol. 2000; 20: 4106-4114Crossref PubMed Scopus (1788) Google Scholar Control mice were age-matched wild-type C57BL/6 mice obtained from the Central Biological Laboratory of the University of Zurich. Exposure to blue light has been previously described.21Grimm C Wenzel A Williams T Rol P Hafezi F Remé C Rhodopsin-mediated blue-light damage to the rat retina: effect of photoreversal of bleaching.Invest Ophthalmol Vis Sci. 2001; 42: 497-505PubMed Google Scholar For this study, improvements of the optical system were introduced, guiding the light under Maxwellian view into the eye with a spot of 5 mm2 focused on the cornea. When an irradiance of 30 mW/cm2 was measured at the corneal focus, the retinal irradiance within the "hot spot" was about 62 mW/cm2 with blue light bandwidth of 410 ± 10 nm. The photon flux calculated was 6.2 × 109 photons μm −2 s −1 at 410 nm. The rate of rhodopsin isomerizations was 1.45 × 109 s−1 at 410 nm.22Breton ME Schueller AW Lamb TD Pugh Jr, EN Analysis of ERG a-wave amplification and kinetics in terms of the G-protein cascade of phototransduction.Invest Ophthalmol Vis Sci. 1994; 35: 295-309PubMed Google Scholar Mice were dark-adapted for 12 hours and pupils were dilated with cyclogyl 1% (Alcon Pharmaceuticals, Hünenberg, Switzerland) and phenylephrine 5% (Bausch & Lomb Swiss AG, Steinhausen, Switzerland) in dim red light 30 minutes before anesthesia. Anesthesia consisted of a mixture of ketamine (75 mg/kg) (Parke Davis, Switzerland), xylazine 2% (23 mg/kg) (Bayer AG, Leverkusen), and sodium chloride. For light exposure, mice were placed in a special holder where their heads and eyes could be adjusted to the light beam, the distance from the cornea was 1 cm. The cornea was kept moist with a drop of Methocel 2% (OmniVision AG, Neuhausen, Switzerland) during the duration of the experiment (2 minutes of blue light/eye). With this method, a reproducible, central focus of light-induced lesions termed "hot spot" was achieved. Mice were sacrificed at 6 hours, 12 hours, 24 hours, 3 days, 6 days, 10 days, 5 weeks, and 10 weeks, respectively, after light exposure. Twenty-four hours after blue light exposure, bone marrow chimeric (BMC) mice and controls were observed by SLO. Mice were anesthetized with ketamine (66.7 mg/kg) and xylazine (11.7 mg/kg) and the pupils were dilated with tropicamide eye drops (Mydriaticum Stulln, Pharma Stulln, Stulln, Germany). SLO imaging was performed with a Heidelberg retina angiograph (HRA I, Heidelberg Engineering, Germany), a confocal scanning-laser ophthalmoscope according to previously reported procedures.23Seeliger MW Beck SC Pereyra-Munoz N Dangel S Tsai JY Luhmann UF van de Pavert SA Wijnholds J Samardzija M Wenzel A Zrenner E Narfstrom K Fahl E Tanimoto N Acar N Tonagel F In vivo confocal imaging of the retina in animal models using scanning laser ophthalmoscopy.Vision Res. 2005; 45: 3512-3519Crossref PubMed Scopus (151) Google Scholar Briefly, the HRA features two argon wavelengths (488 nm and 514 nm) in the short wavelength range and two infrared diode lasers (795 nm and 830 nm) in the long wavelength range. The confocal diaphragm of the SLO allows visualizing different planes of the posterior pole, ranging from the inner surface of the retina down to the retinal pigment epithelium and the choroid. To detect possible effects of blue light exposure on the vascular system, angiography was performed using the 795 nm laser wavelength after a subcutaneous injection of 50 mg/kg body weight indocyanine green (ICG-Pulsion, Pulsion Medical Systems AG, Munich, Germany). Mice were perfused intracardially and immunohistochemistry was performed as previously described.6Joly S Samardzija M Wenzel A Thiersch M Grimm C Nonessential role of β3 and β5 integrin subunits for efficient clearance of cellular debris after light-induced photoreceptor degeneration.Invest Ophthalmol Vis Sci. 2009; 50: 1423-1432Crossref PubMed Scopus (21) Google Scholar After blocking, different primary antibodies were applied on sections and kept overnight in a humid chamber at 4°C (Table 1). Secondary antibodies labeled with Cy3 were used (Jackson ImmunoResearch, West Grove, PA). Stainings were examined with an Axioplan2 microscope (Carl Zeiss, Jena, Germany) using a 10× (numerical aperture 0.45) or a 20× (numerical aperture 0.50) magnification. Pictures were taken with a digital imaging system (AxioCam, Carl Zeiss, Jena, Germany). In both strains, GFP autofluorescence was directly visualized without additional staining.Table 1List of Antibodies Used for Fluorescence MicroscopyAntibody nameTypeDilutionSourceCatalog numberCell type detectionIsolectinGriffonia simplicifolia1:50InvitrogenI21413Microglia, blood vesselsGS-IB4Iba-1rAb1:1000Wako019-19741Microglia and macrophagesCD68 (ED1)mAb1:100ChemiconMAB1435Activated macrophagesRhodopsin (Rho4D2)mAb1:100David HicksSee Ref. 56Hicks DBC Different rhodopsin monoclonal antibodies reveal different binding patterns on developing and adult rat retina.J Histochem Cytochem. 1987; 35: 1317-1328Crossref PubMed Scopus (128) Google ScholarRod outer segmentCCL5mAb1:20R&D SystemsIC278PTNFmAb1:13R&D SystemsAF-410NA Open table in a new tab For confocal laser scanning microscopy (LSM 510, Zeiss, Oberkochen, Germany), perfused eyecups were flat mounted on a glass slide, coverslipped and examined at different wavelengths (488 nm, 543 nm, and 633 nm). Analysis of the microglial cell processes in the CX3CR1−/− mice was performed with an upright confocal two-photon laser scanning microscope (Zeiss LSM 510NLO; Axioskop FS2M, Zeiss, Oberkochen, Germany) with Chameleon laser (Coherent, Dieburg, Germany) and C-Apochromat 40x/1.2NA water immersion objective. GFP fluorescence was excited at 890 nm (infrared); emission was recorded at 500–550 nm (green) band-pass filter and non-descanned detection. Three-dimensional image stacks with x-y frame sizes of 1024 × 1024 pixels (voxel size of 0.29 × 0.29 × 1 μm) were recorded spanning the whole thickness of the retina. Three-dimensional reconstructions were calculated using the Zeiss LSM image software and the number of microglia cells and process length in the outer retinal layers were analyzed. For the number of microglial cells and processes, the statistical analysis was performed using the nonparametric Mann Whitney rank sum test (U-test) (N = 9; **P < 0.01). Mice were euthanized by cervical dislocation. The eyes were removed, fixed in 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer overnight and the central retina was trimmed, washed, dehydrated, and Epon-embedded. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Phillips CM12 (1990) electron microscope. Mice were sacrificed under dim red light after dark adaptation or at 6 hours, 12 hours, 24 hours, or 3 days after light exposure. Retinas were removed through a slit in the cornea and the corresponding eyecups (retinal pigment epithelium, choroid, sclera) were prepared (n = 6 for dark controls, n = 3 for light-exposed specimens), immediately frozen in liquid nitrogen and stored at −80° C until further use. RNA isolation from retinas, cDNA synthesis and gene expression analysis were performed as previously described.24Thiersch M Raffelsberger W Frigg R Samardzija M Wenzel A Poch O Grimm C Analysis of the retinal gene expression profile after hypoxic preconditioning identifies candidate genes for neuroprotection.BMC Genomics. 2008; 9: 73Crossref PubMed Scopus (56) Google Scholar Primer pairs used for specific amplifications were designed to span intronic sequences or cover exon-intron boundaries (Table 2). mRNA levels were normalized to ß-actin for relative quantification of gene expression. Each reaction was done in duplicate. Statistical analysis was performed using a one-way analysis of variance (analysis of variance) followed by a Tukey post hoc test (GraphPad Software, Inc.).Table 2PCR Primers Used for Real-Time Polymerase Chain Reaction AmplificationGenesUpstreamDownstreamProduct sizeβ-actin5′-CAACGGCTCCGGCATGTGC-3′5′-CTCTTGCTCTGGGCCTCG-3′153 bpCcl45′-CAAGCCAGCTGTGGTATTC-3′5′-AGCTGCTCAGTTCAACTCC-3′109 bpCcl125′-CCTCAGGTATTGGCTGGAC-3′5′-GACACTGGCTGCTTGTGATT-3′124 bpCcl55′-GCTCCAATCTTGCAGTCGT-3′5′-CTAGAGCAAGCGATGACAGG-3′165 bpCcl95′-CAACTGCTCTTGGAATCTGG-3′5′-AGGCAGCAATCTGAAGAGTC-3′136 bpCsf15′-GCTCCAGGAACTCTCCAATA-3′5′-TCTTGATCTTCTCCAGCAGC-3′119 bpTnf5′-CCACGCTCTTCTGTCTACTGA-3′5′-GGCCATAGAACTGATGAGAGG-3′92 bpTgfb15′-GCAACATGTGGAACTCTACCAG-3′5′-CAGCCACTCAGGCGTATCA-3′94 bp Open table in a new tab To monitor the state of the blood-retinal barrier and to establish the involvement of blood-borne macrophages migrating into the retinal injury site in vivo, we used BMC mice that were lethally irradiated and transplanted with GFP-expressing bone marrow cells. The ocular fundus (excitation at 514 nm), fundus autofluorescence (excitation at 488 nm and 795 nm) and indocyanine green angiography (excitation at 795 nm) was monitored by SLO in wild-type control and BMC mice 1, 7, and 14 days after blue light exposure (Figure 1). No leakage of retinal vessels was apparent at different time points in control and BMC mice, indicating that no major breakdown of the blood-retinal barrier occurred (Figure 1C). At 514 nm funduscopy, a circumscribed area in the ocular fundus, previously termed hot spot,21Grimm C Wenzel A Williams T Rol P Hafezi F Remé C Rhodopsin-mediated blue-light damage to the rat retina: effect of photoreversal of bleaching.Invest Ophthalmol Vis Sci. 2001; 42: 497-505PubMed Google Scholar was easily detectable in retinas of control and BMC mice 1 day after blue light exposure (Figure 1A, arrows). The hot spot, which appears lighter than the remaining retina in the 514-nm funduscopy, masked the choroidal vasculature and is likely due to a subretinal fluid accumulation. At all time points analyzed, no major difference in the fundus imaging was observed between control and BMC mice (Figure 1B and 1C). At an excitation of 488 nm, fundus examination revealed the presence of GFP-positive autofluorescent bone marrow cells in vessels and retinal tissue of BMC mice 1 day after light (Figure 1A, star). No autofluorescence was detected in control wild-type mice. Autofluorescent cells remained in the retina of BMC mice until the end of the observation period (Figure 1, B, and C, star). In both control and BMC mice we observed an accumulation of autofluorescent material within the hot spot area at both excitation wavelengths (488 nm and 795 nm) at 7 and 14 days (Figure 1B and 1C, arrowheads). These findings suggest that retinoid-containing debris with the corresponding absorption of 488 nm and debris of to date unknown molecular composition with an absorption of 795 nm accumulate within the hot spot region (subretinal space and pigment epithelium). Retinoids are contained in breakdown products of photoreceptor outer segments that contain the visual pigment chromophore 11-cis-retinal. In addition, however, macrophages having engulfed photoreceptor material may still be present in the hot spot region. To localize and to identify blood-borne macrophages, GFP autofluorescence was observed on retinal cryosections alone or combined with antibodies for microglia (GSA-IB4 and Iba1) or macrophages (Iba1 and CD68) (Figure 2). Unexposed retinas from BMC mice presented green fluorescent cells within choroidal and conjunctival blood vessels, within presumptive meninges and connective tissues around the optic nerve (Figure 2A, left panel) but not in the retina itself. Only few cells appeared in retinal vessels, whereas the regions of the ciliary body and overlying conjunctiva were more populated (Figure 2B, left panel, arrows). Three days after light exposure, the optic nerve head and the ciliary body were strongly colonized by green bone marrow cells (Figure 2, A, B, arrow, right panels). The migration of green bone marrow cells was more pronounced into the area of the hot spot (Figure 2A, right panel, arrow) than in the contralateral side that underwent no damage in the light-exposed eye (Figure 2A, right panel, arrowhead). Immunofluorescence was performed on retinal tissues to identify blood-borne macrophages that may or may not express glia markers. In BMC mice, the hot spot region revealed different cell populations 3 and 6 days after light exposure, respectively (Figure 2C): 1) green fluorescent macrophages only expressing the GFP protein; 2) GFP-expressing macrophages additionally stained with one of the glia markers GSA-IB4 or Iba1 (yellow); and 3) a minority of cells expressing only glia markers (red). Staining of GFP cells (green) with anti-CD68 (red) confirmed the presence of activated macrophages in the hot spot zone of BMC retinas 6 days after exposure (Figure 2D). These different expression patterns were only seen within the hot spot region but not in the contralateral side of the light-exposed eye and indicate different populations of macrophages with and without microglial characteristics, respectively. At 5 and 10 weeks after exposure, the retina showed a "scar" in the area of the hot spot with loss of photoreceptors and remaining fluorescent cells within the vasculature of the posterior eye (data not shown). Around the optic nerve head, distinct green cell populations were seen throughout the entire observation period up to 10 weeks (data not shown). CX3CR1−/− knockout mice express GFP instead of CX3CR1 and specifically label microglia.8Jung S Aliberti J Graemmel P Sunshine MJ Kreutzberg GW Sher A Littman DR Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.Mol Cell Biol. 2000; 20: 4106-4114Crossref PubMed Scopus (1788) Google Scholar GFP-fluorescent microglial cells of unexposed control mice showed an even distribution throughout the entire retina (Figure 3A, left panel). Three days after light exposure, retinas displayed massive accumulation of green cells in the optic nerve and in the hot spot region similar to BMC mice (Figure 3A, right panel, arrow). Compared with the exposed BMC retina where green cells were absent in the contralateral side of the light-exposed eye, CX3CR1−/− retinas showed microglia in the outer plexiform layer and in the inner plexiform layer that are likely resident (Figure 3A, left panel, arrowhead). Immunostaining with the lectin GSA-IB4 and the anti-Iba1 antibody revealed that microglia was strongly activated 3 and 6 days after light exposure in the hot spot region and showed macrophage morphology (Figure 3B). Similarly to BMC mice, GFP-microglia stained with anti-CD68, a specific marker for activated macrophages, confirmed that retinal glia-derived macrophages populated the hot spot 6 days after exposure (Figure 3C, arrows). Interestingly, 6 days after exposure, GFP-fluorescent microglial cells were located at the level of damage, ie, in the photoreceptor outer segment layer (Figure 3D). The colocalization of microglia-derived macrophages with an anti-rhodopsin antibody (anti-Rho4D2, red) indicated that microglial cells phagocytosed photoreceptor debris (Figure 3D, arrowheads). On the contralateral side of the light-exposed retina, photoreceptor outer segments were still preserved and no invasion of green cells was noticed in the photoreceptor layer (Figure 3D; compare also Figure 1, B and C). To analyze specifically the retinal localization and activation pattern of microglial cells, CX3CR1−/− mice were analyzed by confocal laser scanning microscopy. In control non-exposed retinas, the resting microglia showed a dense network of fine arborizations of their processes in the nerve fiber layer, the inner plexiform layer and in the outer plexiform layer (Figure 4, A–C). One day after light exposure, a remarkable migration of microglial cells from the nerve fiber layer and the inner plexiform layer into the outer plexiform layer, close to the region of damage occurred (Figure 4, D–G). Microglial cells within the outer plexiform layer above the hot spot became rounded and amoeboid with fewer processes, thereby displaying signs of phagocytosis (Figure 4F, arrow). Within the hot spot, the majority of microglial cells were indistinguishable from macrophages (Figure 4G). Notably, an activation pattern of microglia in the entire retina occurred as seen in the contralateral undamaged retina of the light-exposed eye where increased and thickened microglial cell processes and somata were observed (Figure 4, H–J). These activated microglial cells in an unaffected retinal area possibly indicated a similar surveillance function as was shown for microglia in the normal and diseased CNS.25Nimmerjahn A Kirchhoff F Helmchen F Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.Science. 2005; 308: 1314-1318Crossref PubMed Scopus (3720) Google Scholar
Referência(s)