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The Role of Microglia and Peripheral Monocytes in Retinal Damage after Corneal Chemical Injury

  • Eleftherios I. Paschalis
    Correspondence
    Address correspondence to Eleftherios I. Paschalis, Ph.D., Department of Ophthalmology, Boston Keratoprosthesis Laboratory, Massachusetts Eye and Ear and Schepens Eye Research Institute, Harvard Medical School, 3rd Floor KPro, 20 Staniford Street, Boston, MA 02114.
    Affiliations
    Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts

    Massachusetts Eye and Ear/Schepens Eye Research Institute, Boston Keratoprosthesis Laboratory, Harvard Medical School, Boston, Massachusetts

    Disruptive Technology Laboratory, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
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  • Fengyang Lei
    Affiliations
    Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts

    Massachusetts Eye and Ear/Schepens Eye Research Institute, Boston Keratoprosthesis Laboratory, Harvard Medical School, Boston, Massachusetts

    Disruptive Technology Laboratory, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
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  • Chengxin Zhou
    Affiliations
    Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts

    Massachusetts Eye and Ear/Schepens Eye Research Institute, Boston Keratoprosthesis Laboratory, Harvard Medical School, Boston, Massachusetts

    Disruptive Technology Laboratory, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
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  • Vassiliki Kapoulea
    Affiliations
    Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts

    Massachusetts Eye and Ear/Schepens Eye Research Institute, Boston Keratoprosthesis Laboratory, Harvard Medical School, Boston, Massachusetts

    Disruptive Technology Laboratory, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
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  • Aristomenis Thanos
    Affiliations
    Angiogenesis Laboratory, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
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  • Reza Dana
    Affiliations
    Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
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  • Demetrios G. Vavvas
    Affiliations
    Angiogenesis Laboratory, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
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  • James Chodosh
    Affiliations
    Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts

    Disruptive Technology Laboratory, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts
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  • Claes H. Dohlman
    Affiliations
    Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts

    Massachusetts Eye and Ear/Schepens Eye Research Institute, Boston Keratoprosthesis Laboratory, Harvard Medical School, Boston, Massachusetts
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Open ArchivePublished:April 06, 2018DOI:https://doi.org/10.1016/j.ajpath.2018.03.005
      Eyes that have experienced alkali burn to the surface are excessively susceptible to subsequent severe glaucoma and retinal ganglion cell loss, despite maximal efforts to prevent or slow down the disease. Recently, we have shown, in mice and rabbits, that such retinal damage is neither mediated by the alkali itself reaching the retina nor by intraocular pressure elevation. Rather, it is caused by the up-regulation of tumor necrosis factor-α (TNF-α), which rapidly diffuses posteriorly, causing retinal ganglion cell apoptosis and CD45+ cell activation. Herein, we investigated the involvement of peripheral blood monocytes and microglia in retinal damage. Using CX3CR1+/EGFP::CCR2+/RFP reporter mice and bone marrow chimeras, we show that peripheral CX3CR1+CD45hiCD11b+ MHC-II+ monocytes infiltrate into the retina from the optic nerve at 24 hours after the burn and release further TNF-α. A secondary source of peripheral monocyte response originates from a rare population of patrolling myeloid CCR2+ cells of the retina that differentiate into CX3CR1+ macrophages within hours after the injury. As a result, CX3CR1+CD45loCD11b+ microglia become reactive at 7 days, causing further TNF-α release. Prompt TNF-α inhibition after corneal burn suppresses monocyte infiltration and microglia activation, and protects the retina. This study may prove relevant to other injuries of the central nervous system.
      Patients with ocular surface injuries (chemical, other traumas, surgery) often experience eventual vision loss from aggressive glaucoma, despite maximal antiglaucoma treatment.
      • Smith R.E.
      • Conway B.
      Alkali retinopathy.
      • Cade F.
      • Grosskreutz C.L.
      • Tauber A.
      • Dohlman C.H.
      Glaucoma in eyes with severe chemical burn, before and after keratoprosthesis.
      • Crnej A.
      • Paschalis E.I.
      • Salvador-Culla B.
      • Tauber A.
      • Drnovsek-Olup B.
      • Shen L.Q.
      • Dohlman C.H.
      Glaucoma progression and role of glaucoma surgery in patients with Boston keratoprosthesis.
      Our recent experimental work, in rabbits and mice, using alkali burn to the cornea as model, has demonstrated that substantial damage occurs not only to the anterior segment but also to the retina.
      • Paschalis E.I.
      • Zhou C.
      • Lei F.
      • Scott N.
      • Kapoulea V.
      • Robert M.C.
      • Vavvas D.
      • Dana R.
      • Chodosh J.
      • Dohlman C.H.
      Mechanisms of retinal damage after ocular alkali burns.
      The retinal ganglion cells (RGCs; the key cell layer for glaucoma) show apoptosis within 24 hours of the burn.
      • Cade F.
      • Paschalis E.I.
      • Regatieri C.V.
      • Vavvas D.G.
      • Dana R.
      • Dohlman C.H.
      Alkali burn to the eye: protection using TNF-α inhibition.
      This damage is neither because of a direct pH effect on the retina (the alkali is effectively buffered at the iris-lens level) nor is the damage secondary to intraocular pressure elevation. Rather, tumor necrosis factor (TNF)-α and other inflammatory cytokines, which become up-regulated in the anterior segment of the eye by the injury, diffuse posteriorly, and they rapidly cause activation of CD45+ cells and subsequent RGC apoptosis.
      • Paschalis E.I.
      • Zhou C.
      • Lei F.
      • Scott N.
      • Kapoulea V.
      • Robert M.C.
      • Vavvas D.
      • Dana R.
      • Chodosh J.
      • Dohlman C.H.
      Mechanisms of retinal damage after ocular alkali burns.
      The end result is a gradual decline in vision and eventual blindness, after months or years.
      • Smith R.E.
      • Conway B.
      Alkali retinopathy.
      • Cade F.
      • Grosskreutz C.L.
      • Tauber A.
      • Dohlman C.H.
      Glaucoma in eyes with severe chemical burn, before and after keratoprosthesis.
      • Crnej A.
      • Paschalis E.I.
      • Salvador-Culla B.
      • Tauber A.
      • Drnovsek-Olup B.
      • Shen L.Q.
      • Dohlman C.H.
      Glaucoma progression and role of glaucoma surgery in patients with Boston keratoprosthesis.
      The presumed critical role of TNF-α is supported by the strong neuroprotective effect on the retina of infliximab, a TNF-α inhibitor, when it is administered promptly after the burn.
      • Paschalis E.I.
      • Zhou C.
      • Lei F.
      • Scott N.
      • Kapoulea V.
      • Robert M.C.
      • Vavvas D.
      • Dana R.
      • Chodosh J.
      • Dohlman C.H.
      Mechanisms of retinal damage after ocular alkali burns.
      • Cade F.
      • Paschalis E.I.
      • Regatieri C.V.
      • Vavvas D.G.
      • Dana R.
      • Dohlman C.H.
      Alkali burn to the eye: protection using TNF-α inhibition.
      • Zhou C.
      • Robert M.-C.
      • Kapoulea V.
      • Lei F.
      • Stagner A.M.
      • Jakobiec F.A.
      • Dohlman C.H.
      • Paschalis E.I.
      Sustained subconjunctival delivery of infliximab protects the cornea and retina following alkali burn to the eye.
      Herein, the role of retinal CD45+ cells and, in particular, the retinal microglia and blood-derived monocytes/macrophages was explored in the pathogenesis of the retinal degeneration after alkali injury to the cornea. Microglia and macrophages are both implicated in most neurodegenerative disorders of the central nervous system (CNS) and brain injuries.
      • Kim S.U.
      • de Vellis J.
      Microglia in health and disease.
      • Block M.L.
      • Zecca L.
      • Hong J.-S.
      Microglia-mediated neurotoxicity: uncovering the molecular mechanisms.
      • Dheen S.T.
      • Kaur C.
      • Ling E.-A.
      Microglial activation and its implications in the brain diseases.
      • Prinz M.
      • Priller J.
      Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease.
      • Rice R.A.
      • Spangenberg E.E.
      • Yamate-Morgan H.
      • Lee R.J.
      • Arora R.P.S.
      • Hernandez M.X.
      • Tenner A.J.
      • West B.L.
      • Green K.N.
      Elimination of microglia improves functional outcomes following extensive neuronal loss in the hippocampus.
      Macrophage infiltration and microglia activation in the CNS are both signs of pathology,
      • Ajami B.
      • Bennett J.L.
      • Krieger C.
      • Tetzlaff W.
      • Rossi F.M.V.
      Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.
      • Mildner A.
      • Schmidt H.
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      • Hanisch U.-K.
      • Mack M.
      • Heikenwalder M.
      • Brück W.
      • Priller J.
      • Prinz M.
      Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.
      • Kettenmann H.
      • Hanisch U.-K.
      • Noda M.
      • Verkhratsky A.
      Physiology of microglia.
      • Ransohoff R.M.
      • Brown M.A.
      Innate immunity in the central nervous system.
      and microglia activation precedes glaucomatous damage in ocular hypertension models.
      • Inman D.M.
      • Horner P.J.
      Reactive nonproliferative gliosis predominates in a chronic mouse model of glaucoma.
      • Bosco A.
      • Steele M.R.
      • Vetter M.L.
      Early microglia activation in a mouse model of chronic glaucoma.
      Both microglia and macrophages can release TNF-α on activation
      • Hanisch U.-K.
      • Kettenmann H.
      Microglia: active sensor and versatile effector cells in the normal and pathologic brain.
      and mediate RGC apoptosis.
      • Block M.L.
      • Zecca L.
      • Hong J.-S.
      Microglia-mediated neurotoxicity: uncovering the molecular mechanisms.
      • Tezel G.
      • Li L.Y.
      • Patil R.V.
      • Wax M.B.
      TNF-alpha and TNF-alpha receptor-1 in the retina of normal and glaucomatous eyes.
      TNF inhibition has been shown to protect the retina in various ocular injury models.
      • Paschalis E.I.
      • Zhou C.
      • Lei F.
      • Scott N.
      • Kapoulea V.
      • Robert M.C.
      • Vavvas D.
      • Dana R.
      • Chodosh J.
      • Dohlman C.H.
      Mechanisms of retinal damage after ocular alkali burns.
      • Cade F.
      • Paschalis E.I.
      • Regatieri C.V.
      • Vavvas D.G.
      • Dana R.
      • Dohlman C.H.
      Alkali burn to the eye: protection using TNF-α inhibition.
      • Zhou C.
      • Robert M.-C.
      • Kapoulea V.
      • Lei F.
      • Stagner A.M.
      • Jakobiec F.A.
      • Dohlman C.H.
      • Paschalis E.I.
      Sustained subconjunctival delivery of infliximab protects the cornea and retina following alkali burn to the eye.
      • Roh M.
      • Zhang Y.
      • Murakami Y.
      • Thanos A.
      • Lee S.C.
      • Vavvas D.G.
      • Benowitz L.I.
      • Miller J.W.
      Etanercept, a widely used inhibitor of tumor necrosis factor-α (TNF-α), prevents retinal ganglion cell loss in a rat model of glaucoma.
      In this study, reporter mice and bone marrow chimeras were used to study the role of microglia and peripheral monocytes in retinal degeneration after corneal alkali burn. The therapeutic role of TNF-α inhibition, as a prelude to potential human clinical application, was further explored.
      • Dohlman C.H.
      • Cade F.
      • Regatieri C.V.
      • Zhou C.
      • Lei F.
      • Crnej A.
      • Harissi-Dagher M.
      • Robert M.-C.
      • Papaliodis G.N.
      • Chen D.
      • Aquavella J.V.
      • Akpek E.K.
      • Aldave A.J.
      • Sippel K.C.
      • D'Amico D.J.
      • Dohlman J.G.
      • Fagerholm P.
      • Wang L.
      • Shen L.Q.
      • Gonzalez-Andrades M.
      • Chodosh J.
      • Kenyon K.R.
      • Foster C.S.
      • Pineda R.
      • Melki S.
      • Colby K.A.
      • Ciolino J.B.
      • Vavvas D.G.
      • Kinoshita S.
      • Dana R.
      • Paschalis E.I.
      Chemical burns of the eye: the role of retinal injury and new therapeutic possibilities.

      Materials and Methods

      Study Approval

      All animal experiments were reviewed and approved by the Animal Care Committee of the Massachusetts Eye and Ear Infirmary and were performed in accordance with the Association for Research in Vision and Ophthalmology.

      Mouse Model of Alkali Burn

      All animal-based 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 the NIH’s Guide for the Care and Use of Laboratory Animals.
      Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research Council
      Guide for the Care and Use of Laboratory Animals: Eighth Edition.
      This study was approved by the Animal Care Committee of the Massachusetts Eye and Ear Infirmary (Boston, MA). C57BL/6J, B6.129P-Cx3cr1tm1Litt/J (CX3CR1EGFP/EGFP Stock 005582), B6.129(Cg)-Ccr2tm2.1lfc/J (CCR2RFP/RFP Stock 017586), B6.129-Tnfrsf1atm1Mak/J (TNFR1−/− Stock 002620), B6.129S2-Tnfrsf1btm1Mwm/J (TNFR2−/− Stock 002818), and B6.129S-Tnfrsf1atm1Imx Tnfrsf1btm1Imx/J (TNFR1/2−/− Stock 003243) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice between the ages of 6 and 12 weeks were used for this study. This model of alkali chemical burn is based on our previously published study.
      • Paschalis E.I.
      • Zhou C.
      • Lei F.
      • Scott N.
      • Kapoulea V.
      • Robert M.C.
      • Vavvas D.
      • Dana R.
      • Chodosh J.
      • Dohlman C.H.
      Mechanisms of retinal damage after ocular alkali burns.
      In brief, mice were anesthetized using 60 mg/kg ketamine and 6 mg/kg xylazine, and deep anesthesia was confirmed by a toe pinch test. Proparacaine hydrochloride USP 0.5% (Bausch and Lomb, Tampa, FL) eye drop was applied to the cornea, and after 1 minute, was carefully dried with a Weck-Cel (Beaver Visitec International, Inc., Waltham, MA). A 2-mm diameter filter paper was soaked into 1 mol/L NaOH solution for 10 seconds, dried of excess NaOH by a single touch on a paper towel, and applied onto the mouse cornea for 20 seconds. Complete adherence of the filter paper on the corneal surface was confirmed by gentle pushing of the perimeter using forceps. After the filter paper was removed, prompt irrigation with sterile saline for 10 seconds was applied using a 50-mL syringe with a 25 gauge needle. The mouse was then placed on a heating pad, positioned laterally, and the eye irrigated for another 15 minutes at low pressure using sterile saline. Buprenorphine hydrochloride (0.05 mg/kg; Buprenex Injectable; Reckitt Benckiser Healthcare Ltd, Slough, UK) was administered subcutaneously for pain management. A single drop of topical Polytrim antibiotic was administered after the irrigation (polymyxin B/trimethoprim; Bausch & Lomb Inc., Bridgewater, NJ). Mice were kept on the heating pad until fully awake.

      Treatment of Burns

      Anti–TNF-α treatment was performed 15 minutes after the burn with an i.p. injection (6.25 mg/kg reconstituted in normal saline) of infliximab (Janssen Biotech, Horsham, PA). Wild-type naive mice were used as controls. The contralateral nonburned eye was used as internal control. Human IgG1 κ isotype antibody (GeneTex, Irvine, CA) was used as vehicle control (6.25 mg/kg; i.p.), when appropriate, and no treatment as no-treatment control to simulate the clinical outcomes of patients who do not receive treatment after burn.

      Tissue Preparation for Immunohistochemically Frozen Sections

      After the alkali burn, eyes were enucleated at predetermined time points and processed using OCT (Tissue Tek, 4583; VWR, Randor, PA) compound. Multiple sagittal sections of approximately 10 μm in thickness were obtained from the center and the periphery of the globe. Sectioned tissues were transferred to positive charged glass slides (Superfrost Plus, 75 × 25 mm, 1-mm thickness; VWR) and stored at −80°C for further processing.

      Tissue Preparation for Flat Mount Imaging

      After the alkali burn, eyes were enucleated at predetermined time points and fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) solution for 1 hour at 4°C. The cornea and retina tissue were carefully isolated using microsurgical techniques and washed three times for 5 minutes in phosphate-buffered saline (PBS) solution (Sigma-Aldrich) at 4°C. The tissues were then blocked using 5% bovine serum albumin (Sigma-Aldrich) and permeabilized using 0.3% Triton-X (Sigma-Aldrich) for 1 hour at 4°C. The specific antibody was added in blocking solution, incubated overnight at 4°C, and then washed three times for 10 minutes with 0.1% Triton-X in PBS. Tissues were transferred from the tube to positive charged glass slides using a wide piper tip with the concave face upwards. Four relaxing incisions from the center to the periphery were made to generate four flat tissue quadrants. VECTRASHIELD mounting medium (H-1000; Vector Laboratories, Burlingame, CA) was placed over the tissue, followed by a coverslip.

      TUNEL Labeling and Quantitation of DNA Fragmentation

      After the alkali burn, eyes were enucleated and cryosectioned. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) labeling was performed using a Roche TUNEL kit (12156792910; Roche, Indianapolis, IN), as previously published.
      • Cade F.
      • Paschalis E.I.
      • Regatieri C.V.
      • Vavvas D.G.
      • Dana R.
      • Dohlman C.H.
      Alkali burn to the eye: protection using TNF-α inhibition.
      Mounting medium with DAPI (UltraCruz; sc-24941; Santa Cruz Biotechnology, Dallas, TX) was placed over the tissue, followed by a coverslip. Images were taken with an epifluorescent microscope (Zeiss Axio Imager M2; Zeiss, Oberkochen, Germany), using the tile technique. DAPI signal (blue) was overlayed with Texas red (TUNEL+ cells) and quantified using ImageJ software version 1.5s (http://imagej.nih.gov/ij; NIH, Bethesda, MD) to measure the number of TUNEL+ cells overlapping with DAPI in the areas of interest. All experiments were performed in triplicate.

      Immunolocalization of TNF-α and β3-Tubulin Protein Expression

      After the alkali burn, eyes were enucleated at predetermined time points and processed for frozen section, as described above. Sectioned tissues were fixated onto the glass slides using 4% paraformaldehyde for 15 minutes at room temperature. The slides were then washed three times for 5 minutes in PBS and dried for paraffin boundary marking using paraffin pen.

      TNF-α

      Tissues were blocked with 5% bovine serum albumin and incubated at 4°C overnight using 1:100 dilution of monoclonal anti-mouse fluorescein isothiocyanate conjugated antibody (NBP1-51502; Novus Biologicals, Littleton, CO). Tissues were washed three times for 5 minutes in PBS at room temperature and mounted using either VECTASHIELD or mounting medium with DAPI, and covered with a coverslip.

      β3-Tubulin

      Tissues were blocked with 5% bovine serum albumin in PBS + 0.1% Tween-20 for 30 minutes at room temperature. Overnight incubation at 4°C was performed using conjugated mouse monoclonal antibody (NL557; R&D Systems, Minneapolis, MN) at 1:100 dilution. Tissues were washed three times for 5 minutes in PBS at room temperature and mounted using either VECTASHIELD or mounting medium with DAPI, and covered with a coverslip.

      Retinal Flat Mount Preparation for Microglia/Macrophage and Nerve Fiber Layer Assessment

      Alkali burned eyes from CX3CR1+/EGFP mice were enucleated at different time points, and whole retinas and corneas were processed with anti–neuron-specific β-III tubulin (NL557; R&D Systems) conjugated antibody in 1:80 dilution. The tissues were prepared for flat mount, as described above, and imaged using a confocal microscope (Leica SP-5; Leica Microsystems Inc., Buffalo Grove, IL). Images were taken at 10×, 20×, 40×, and 63× objective lenses using Z-stack of 0.7-, 0.6-, 0.4-, and 0.3-μm step, respectively. Image processing with ImageJ version 1.5s was used to obtain maximum and average projections of the z axis, color depth maps, and three-dimensional volumetric images. Retinal microglia/macrophage cells were quantified by layer-by-layer technique, total Z-stack projection technique, and volumetric three-dimensional analysis. Retinal nerve fiber layer (RNFL) was quantified by using Z-stack projection and binary image conversion or by orthogonal transverse two-dimensional cuts of the nerves and area measurements.

      PCR Array for Measuring Proinflammatory Cytokine Expression

      After the alkali burn, eyes were harvested at predetermined time points. Corneal and retinal tissues were surgically dissected, placed in cryovials, and rapidly frozen in liquid nitrogen. RNA isolation was performed using RNeasy Mini Kit (74106; Qiagen, Valencia, CA) for retinas. RNA quantification was performed using a nanodrop spectrophotometer (Nanodrop 2000; Thermo Fisher Scientific, Waltham, MA), and each sample was normalized before cDNA synthesis. cDNA synthesis was performed using superscript III (18080-044; Invitrogen, Carlsbad, CA). One microliter of cDNA was used in each RT-PCR. RT2 Profiler PCR Array Mouse Inflammatory Cytokines & Receptors Kit (PAMM_011Z; Qiagen, San Diego, CA) was used to evaluate retinal inflammation after the ocular alkaline injury. The assay was performed per manufacturer's instruction, and data were analyzed by using Qiagen's data analysis web portal.

      Bone Marrow Chimera Model

      C57BL/6J mice were myelodepleted with three i.p. injections of busulfan (Sigma-Aldrich; 35 mg/kg) 7, 5, and 3 days before bone marrow transfer. CX3CR1+/EGFP::CCR2+/RFP or only CX3CR1+/EGFP bone marrow cells (5 × 106 total bone marrow cells) were transferred to the myelodepleted C57BL/6J mice through tail vein injection 1 month before corneal alkali burn. Bactrim (trimethoprim/sulfamethoxazole, 80/400 mg, respectively) was resuspended in 400-mL drinking water and given ad libitum for 15 days after busulfan treatment.

      Assessment of Immune Cell Activation/Infiltration in the Retina by Flow Cytometry

      Eyes were harvested at various, predetermined, time points. Retinas were isolated surgically and processed for flow cytometry using Collagenase Type I and Papain Dissociation System (Worthington Biochemical Corp., Lakewood, NJ), respectively. Thy1.1-phosphatidylethanolamine antibody (BioLegend, San Diego, CA) was used to identify RGCs, whereas retinal microglia/macrophage cells were detected as enhanced green fluorescent protein (EGFP)+ population in CX3CR1+/EGFP mice. Different fluorescent CD45 (clone 104), CD11b (clone M1/70), I-A/I-E [major histocompatibility complex (MHC)-II; clone M5/114.15.2], CX3CR1 (clone SA011F11), Ly6C (clone HK1.4), and Ly6G (clone 1A8) antibodies (BioLegend) were used to perform the flow cytometry. Cells were analyzed on a BD LSR II cytometer (BD Biosciences, San Jose, CA) using FlowJo software version 10.4.2 (Tree Star, Ashland, OR).

      Quantification of Immunohistochemistry

      Macroglia were quantified using ImageJ version 1.5s, on whole eye tissue cross sections stained with DAPI and glial fibrillary acidic protein monoclonal antibody, at indicated times after injury. Different time points were selected for analysis. For each time point, three different tissue sections from three different mice (n = 3) were used. The area of interest was marked using the freehand selection tool, according to DAPI boundaries. The enclosed area was measured (mm2), and the image was decomposed to red and blue channels. The red and blue channels that corresponded to glial fibrillary acidic protein and DAPI expression, respectively, were selected and converted to binary information. The positive pixels (bright pixels) were quantified, the outcome was normalized according to the total sampled area, and the resulting number was plotted. Microglia quantification was also performed in retinal flat mounts of CX3CR1+/EGFP mice. The three distinct retinal layers known to be occupied by tissue resident microglia [ganglion cell layer (GCL), inner plexiform layer, and outer plexiform layer] were analyzed. The longest cell process length, which is a marker of cell quiescence, was used as a morphometric descriptor to analyze microglia activation. RNFL quantification was performed in flat mount retinas. The red channel that corresponded to β3-tubulin expression was selected and converted to binary information. The positive pixels (bright pixels) were quantified, the outcome was normalized according to the total sampled area, and the resulting number was plotted.

      Statistical Analysis

      Results were analyzed with the Statistical Package of Social Sciences Version 17.0 (SPSS Inc., Chicago, IL). The normality of continuous variables was assessed using the Kolmogorov-Smirnov test. Quantitative variables were expressed as means ± SEM, and qualitative variables were expressed as frequencies and percentages. The Mann-Whitney test was used to assess differences between the groups. All tests were two-tailed, and statistical significance was determined at P < 0.05. The independent t-test was used to compare between two groups, and pairwise t-test was used to compare changes within the same group. Analysis of variance was used for comparison of multiple groups. α Level correction was applied, as appropriate, for multiple comparisons.

      Results

      Corneal Alkali Burn Leads to Retinal Damage in Humans and Animals

      Corneal alkali injury causes severe corneal scarring and neovascularization in patients (Figure 1A) and advanced cupping and pallor of the optic nerve (ON) (Figure 1B). The latter cannot be explained alone by typical intraocular pressure–induced glaucomatous damage.
      • Cade F.
      • Grosskreutz C.L.
      • Tauber A.
      • Dohlman C.H.
      Glaucoma in eyes with severe chemical burn, before and after keratoprosthesis.
      • Crnej A.
      • Paschalis E.I.
      • Salvador-Culla B.
      • Tauber A.
      • Drnovsek-Olup B.
      • Shen L.Q.
      • Dohlman C.H.
      Glaucoma progression and role of glaucoma surgery in patients with Boston keratoprosthesis.
      In vivo retinal analysis using optical coherence tomography typically reveals significant loss of neuroretinal tissue and thinning of the RNFL in all retinal quadrants, similar to that seen in end-stage glaucoma (Figure 1C). Corneal alkali burns in animals, such as rabbits or mice, result in phenotypical changes similar to human burns, including severe corneal scarring and neovascularization (Figure 1, D and H), progressive loss of β3 tubulin+ neuroretinal tissue, thinning of the RNFL (Figure 1, E and I), and ON axon degeneration (Figure 1, F and J), without intraocular pressure elevation or chemical diffusion posteriorly.
      • Paschalis E.I.
      • Zhou C.
      • Lei F.
      • Scott N.
      • Kapoulea V.
      • Robert M.C.
      • Vavvas D.
      • Dana R.
      • Chodosh J.
      • Dohlman C.H.
      Mechanisms of retinal damage after ocular alkali burns.
      • Cade F.
      • Paschalis E.I.
      • Regatieri C.V.
      • Vavvas D.G.
      • Dana R.
      • Dohlman C.H.
      Alkali burn to the eye: protection using TNF-α inhibition.
      • Zhou C.
      • Robert M.-C.
      • Kapoulea V.
      • Lei F.
      • Stagner A.M.
      • Jakobiec F.A.
      • Dohlman C.H.
      • Paschalis E.I.
      Sustained subconjunctival delivery of infliximab protects the cornea and retina following alkali burn to the eye.
      Within 3 months of the injury, the retinas of rabbits and mice exhibit a significant 40% RGC loss (Figure 1, G and K). Microarray analysis in retinal tissues of burned mice showed marked up-regulation of a range of inflammatory genes within 24 hours of the burn (Figure 1L). In particular, significant up-regulation (more than twofold change log2) was found in chemokine (C-C motif) ligands 2, 6, 7, and 12 and receptors CCR 1, 2, 3, and 5, which are responsible for monocyte recruitment and signal transduction.
      • Le Y.
      • Zhou Y.
      • Iribarren P.
      • Wang J.
      Chemokines and chemokine receptors: their manifold roles in homeostasis and disease.
      Up-regulation of colony stimulating factor 1 gene, which is essential for microglia survival, was also noted.
      • Erblich B.
      • Zhu L.
      • Etgen A.M.
      • Dobrenis K.
      • Pollard J.W.
      Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits.
      Chemokine (C-X-C motif) ligands 1, 5, and 10 and receptor Cxcr2 genes
      • Tsai H.H.
      • Frost E.
      • To V.
      • Robinson S.
      • Ffrench-Constant C.
      • Geertman R.
      • Ransohoff R.M.
      • Miller R.H.
      The chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration.
      • Pelus L.M.
      • Fukuda S.
      Peripheral blood stem cell mobilization: the CXCR2 ligand GRObeta rapidly mobilizes hematopoietic stem cells with enhanced engraftment properties.
      • Boivin N.
      • Menasria R.
      • Gosselin D.
      • Rivest S.
      • Boivin G.
      Impact of deficiency in CCR2 and CX3CR1 receptors on monocytes trafficking in herpes simplex virus encephalitis.
      • Arnoux I.
      • Audinat E.
      Fractalkine signaling and microglia functions in the developing brain.
      were also up-regulated, as well as inflammatory mediators of the IL and tumor necrosis factor-α families (Figure 1L).
      Figure thumbnail gr1
      Figure 1Corneal alkali burn causes retinal damage in humans and animals. A: Digital image of a human cornea after alkali burn presented with extensive neovascularization and conjunctivalization. B: Fundus digital imaging of the optic nerve (ON) reveals end-stage glaucoma associated with advanced ON cupping and parlor. C: In vivo optical coherence tomography confirms significant loss of retinal nerve fiber layer (RNFL) thickness (dotted black line) compared with normal age-matched controls (green section). D: Digital image of a rabbit cornea after alkali burn with extensive neovascularization and conjunctivalization. E: Immunohistochemical analysis of rabbit retina using βIII-tubulin after corneal alkali burn (top panel) and of control eye (bottom panel) shows significant reduction in RNFL thickness in the burned eye compared with control retina (white arrowheads denote ganglion layer cells). F and J: P-phenylenediamine staining of rabbit and mouse ONs after corneal alkali burn (top panel) and of control eye (bottom panel) shows significant axonal degeneration in burned eyes (black arrows). G: Corresponding 40% of retinal ganglion cell (RGC) loss. H: Digital image of a mouse cornea after alkali burn with extensive neovascularization and conjunctivalization. I: Immunohistochemical analysis of flat mount mouse retinas using βIII-tubulin marker after corneal alkali burn (top panel) and of control eye (bottom panel) shows significant loss of RNFL density (white arrowheads). K: Corresponding 40% of RGC loss assessed by Brn3a marker. L: Microarray RNA analysis in mouse retinas 24 hours after the burn shows significant up-regulation (more than twofold log2) of C-C and C-X-C motif genes involved in monocyte recruitment, colony stimulating factor 1 gene required for microglia survival, and inflammatory genes of the IL and tumor necrosis factor (TNF)-α families. G and K: Independent t-test was used. n = 3 (G, K, and L). ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bars: 20 μm (E and I); 10 μm (F and J). Cnt., control; GCL, ganglion cell layer; INF, inferior (refers to different retinal segments); NA, not applicable; NAS, nasal; SUP, superior; TEMP, temporal.

      Microglia Activation and Blood-Derived Monocyte Infiltration after Ocular Injury

      To investigate the inflammatory cellular components of retinal damage, two major immune cell populations involved in acute inflammatory response (the microglia and the blood-derived monocytes) were studied. Microglia are the resident tissue phagocytes of the retina, expressing CX3C chemokine receptor 1 (CX3CR1).
      • Salter M.W.
      • Stevens B.
      Microglia emerge as central players in brain disease.
      On activation, they undergo morphologic and biochemical changes, characterized by shortening of their dendritic processes and assuming a more amoeboid shape.
      Corneal injury led to morphometric CX3CR1+ cell activation in the GCL at 24 hours and in all retinal layers (GCL, inner nuclear layer, outer plexiform layer) at 7 days (Figure 2, A and B ). Cell activation was associated with significant shortening of the cell process length and uniaxial elongation of cell body in the direction of the nerve fiber layer (Figure 2A). An increased number of amoeboid CX3CR1+ cells were found around the outer segment of the ON head (ONH) and the RNFL (Figure 2C and Supplemental Video S1). Treatment with TNF-α inhibitor prevented CX3CR1+ cell activation and improved morphometric ramification in the GCL and inner nuclear layer compared with vehicle control (Figure 2, A and B). In addition, anti–TNF-α treatment reduced the accumulation of CX3CR1+ cells around the outer segment of the ONH (Figure 2C and Supplemental Figure S1).
      Figure thumbnail gr2
      Figure 2Corneal alkali burn causes CX3CR1+ cell activation in the retina. A: Confocal images of flat-mounted retinas of CX3CR1+/EGFP reporter mice 24 hours and 7 days after corneal alkali burn. Cells are color-depth coded on the basis of their position within the retina layers. Blue indicates cells in the ganglion cell layer (GCL; 0 μm), red indicates cells in the outer plexiform layer (OPL), and intermediate colors indicate cells between the GCL and OPL. Color bar translates color to μm. B: Morphometric analysis of CX3CR1+ cells was performed in three distinct retina layers: the GCL, inner nuclear layer (INL), and OPL. The longest process length measured from the edge of the cell body (in μm) was used as a morphometric descriptor of CX3CR1+ cell activation. A and B: At 24 hours after the burn, CX3CR1+ cells in the GCL start to become amoeboid, exhibiting significant reduction in processes length. CX3CR1+ cells in the INL and OPL are not affected. Anti–tumor necrosis factor (TNF)-α treatment preserves cell ramification in the GCL. C: At 7 days, CX3CR1+ cells accumulate around the outer segment of the optic nerve head (ONH) and align to the direction of the retina nerve fiber layer. Also, CX3CR1+ cells in all retinal layers became more amoeboid, with significant shortening of the cell processes. Anti–TNF-α treatment significantly reduces CX3CR1+ cell amoeboid transformation in all retinal layers and reduces cell accumulation around the ONH. B: Multiple comparisons using Holm-Sidak method. n = 3 per group (B). P < 0.05, ∗∗P < 0.01. Scale bars = 100 μm (A). Original magnification, ×40 (C).
      Because CX3CR1 is a marker of both resident microglia and peripheral monocyte/macrophages, flow cytometry was performed to differentiate and analyze the two cell populations. MHC-II marker was used to assess activation. CX3CR1+ CD45hi CD11b+ cells were used to define blood-borne macrophages, and CX3CR1+ CD45lo CD11b+ cells were used for microglia.
      • Kettenmann H.
      • Hanisch U.-K.
      • Noda M.
      • Verkhratsky A.
      Physiology of microglia.
      • Mittal R.
      • Gonzalez-Gomez I.
      • Panigrahy A.
      • Goth K.
      • Bonnet R.
      • Prasadarao N.V.
      IL-10 administration reduces PGE-2 levels and promotes CR3-mediated clearance of Escherichia coli K1 by phagocytes in meningitis.
      • Mizutani M.
      • Pino P.A.
      • Saederup N.
      • Charo I.F.
      • Ransohoff R.M.
      • Cardona A.E.
      The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.
      • Hambardzumyan D.
      • Gutmann D.H.
      • Kettenmann H.
      The role of microglia and macrophages in glioma maintenance and progression.
      MHC-II marker was used to assess cell activation.
      • Berman N.E.
      • Marcario J.K.
      • Yong C.
      • Raghavan R.
      • Raymond L.A.
      • Joag S.V.
      • Narayan O.
      • Cheney P.D.
      Microglial activation and neurological symptoms in the SIV model of NeuroAIDS: association of MHC-II and MMP-9 expression with behavioral deficits and evoked potential changes.
      Retinal cells from CX3CR1+/EGFP mice were analyzed with flow cytometry. Two distinct CX3CR1+ cell populations were identified: CX3CR1+ CD11b+ CD45hi (macrophages) and CX3CR1+ CD11b+ CD45lo (microglia) (Figure 3). In naive eyes, most CX3CR1+ cells were CD45lo, with no MHC-II expression. A negligible (<0.1%) fraction of CX3CR1+ cells were CD45hi but did not express MHC-II (Figure 3). At 24 hours after the injury, the percentage of CX3CR1+ CD11b+ CD45hi cells increased with concomitant increase in the number of MHC-II+ cells from 1500 to 100,000 MHC-II+ cells/retina (P = 0.002) (Figure 3). At the same time, the percentage of CX3CR1+ CD11b+ CD45lo cells reduced slightly, without significant change in the number of MHC-II+ cells (P > 0.999) (Figure 3).
      Figure thumbnail gr3
      Figure 3Activated retinal CX3CR1+ cells have microglia and macrophage signatures. A, B, and D: Biochemical assessment of CX3CR1+ cells using flow cytometry. CX3CR1+ CD11b+ cells divided into macrophages (CD45hi) and microglia (CD45lo). Major histocompatibility complex (MHC)-II expression was used as a metric of cell activation. Naive eyes express predominantly CD45lo (90%), with only 4% of the CX3CR1+ cells expressing CD45hi. None of the CD45 cell populations express MHC-II. Twenty-four hours after the burn, the percentage of CX3CR1+ CD11b+ CD45hi cells in the retina increases with concomitant increase in MHC-II expression. Seven days after the burn, CX3CR1+ CD11b+ CD45hi population decreases with concomitant reduction in MHC-II expression. At the same time, the percentage of CX3CR1+ CD11b+ CD45lo remains unchanged, but the number of MHC-II+ cells increases. Treatment with monoclonal anti–tumor necrosis factor (TNF)-α antibody significantly reduces the number of CX3CR1+ CD11b+ CD45hi MHC-II+ cells in the retina, but it has no effect on the number of CX3CR1+ CD11b+ CD45lo MHC-II+ cells. At 7 days, anti–TNF-α treatment increases the percentage of CX3CR1+ CD45lo CD11b+ and their corresponding MHC-II expression, but it has no effect on the number of CX3CR1+ CD45hi CD11b+ MHC-II+ cells. Overall, anti–TNF-α treatment reduces the infiltration of CX3CR1+ CD11b+ CD45hi cells in the retina and prevents MHC-II expression by these cells. C: Statistical comparisons of the number of CX3CR1+ CD11b+ CD45hi MHC-II+ and CX3CR1+ CD11b+ CD45lo MHC-II+ cells between the treatment groups using analysis of variance with Holm-Sidak method. Statistically significant values (≤0.05) are shown in bold. n = 3 in all groups. ANOVA, analysis of variance; FSC, forward scatter.
      Seven days after the burn, the percentage of CX3CR1+ CD11b+ CD45hi cells was reduced, with concomitant reduction in the number of MHC-II+ cells from 100,000 to 20,000 MHC-II+ cells/retina (P < 0.005). At the same time, the percentage of CX3CR1+ CD11b+ CD45lo cells remained unchanged, but the number of MHC-II+ cells increased from 900 to 15,000 (P < 0.0001) (Figure 3).
      Anti–TNF-α treatment significantly reduced the number of activated CX3CR1+ CD11b+ CD45hi MHC-II+ cells at 24 hours from 100,000 to 38,000 cells/retina (P < 0.01) but did not affect the number of CX3CR1+ CD45lo CD11b+ MHC-II+ cells (P = 0.807) (Figure 3). At 7 days, anti–TNF-α treatment increased the expression of CX3CR1+ CD45lo CD11b+ cells and their corresponding MHC-II expression (P = 0.014), but had no effect on the number of CX3CR1+ CD45hi CD11b+ MHC-II+ cells (P > 0.999) (Figure 3). Overall, anti–TNF-α treatment reduced early infiltration of blood-derived monocytes and subsequent MHC-II activation of these cells.

      Blood-Derived CX3CR1+ CCR2+ Monocytes Rapidly Infiltrate into the Retina after Corneal Alkali Injury

      To understand the role of blood-derived monocyte infiltration in retinal damage, a bone marrow chimera model with fluorescent labeled CCR2+/RFP and CX3CR1+/EGFP cells was used. Busulfan myelodepletion was performed instead of γ radiation, to prevent radiation-induced blood-retina barrier disruption and subsequent infiltration of monocytes. Briefly, bone marrow cells from CCR2+/RFP::CX3CR1+/EGFP mice were harvested and transferred to busulfan myelodepleted C57BL/6 wild-type mice. At 4 weeks, bone marrow engraftment reached 95% efficiency, as determined by flow cytometry (data not shown).
      In naive mice, a small number of dendritiform CCR2 CX3CR1+ cells were found around the ONH 10 weeks after bone marrow transfer. However, these cells did not appear to migrate beyond the ONH boundary (Figure 4 A, F, and G ). A few amoeboid CCR2+ CX3CR1 cells were found across the entire GCL (Figure 4A and Supplemental Figure S1). Twenty-four hours after the injury, CCR2+ CX3CR1+ cells had accumulated into the retina and around the ONH. Cell infiltration into the retina appeared to occur through the ONH, and infiltrated cells appeared to align along the retinal vessels (Figure 4, B and F–H). Infiltrated CCR2hi CX3CR1lo cells were highly amoeboid in shape (Figure 2, B and C) and scattered homogeneously across the GCL (Figure 4B and Supplemental Figure S1). At 7 days, the number of infiltrated CCR2+ CX3CR1+ cells increased within the GCL and inner plexiform layer (Figure 4B and Supplemental Figure S1), and appeared to adopt a more ramified morphology, expressing CCR2hi CX3CR1hi (Figure 4, D, F, and G).
      Figure thumbnail gr4
      Figure 4Monocyte infiltration into the retina after corneal burn. Image quantification of CX3CR1+ and CCR2+ cell infiltration in the retina after corneal alkali burn using CX3CR1::CCR2EGFP/RFP bone marrow (BM) chimera. A, F, and G: In naive mice, only few ramified CX3CR1+ CCR2 cells are present around the optic nerve head (ONH). These cells do not migrate beyond its boundary. However, scant CX3CR1 CCR2+ cells are found across the retina, located in the ganglion cell layer (GCL). B: Twenty-four hours after corneal surface injury, amoeboid CX3CR1+ CCR2+ cells from the blood infiltrate the retina and locate themselves primarily in the GCL. An increased number of amoeboid CX3CR1+ CCR2+ cells is present around the ONH (white arrows) and along the retinal vessels (red arrows). Only a few cells are CX3CR1+ CCR2. C, F, and G: At 24 hours, anti–tumor necrosis factor (TNF)-α treatment (Tx) reduces the infiltration of blood-derived CX3CR1+ CCR2+ cells in the retina. D, F, and G: At day 7, infiltrated blood-derived CX3CR1+ CCR2+ cells accumulate between the GCL and outer nuclear layer (ONL) and exhibit a phenotypic differentiation from amoeboid to dendritiform. The number of blood-derived CX3CR1+ CCR2+ cells in the GCL and inner plexiform layer remains elevated, whereas a significant number of CCR2+ cells are located around the outer segment of the ONH (white arrow). EG: TNF-α inhibition reduces the number of peripheral CX3CR1+ CCR2+ cell infiltrates and promotes differentiation from amoeboid to dendritiform morphology and concomitant abolishment of CCR2 marker. CCR2 remains expressed by amoeboid cells. H: Flow cytometry in CX3CR1+/EGFP BM chimeras confirms the reduction in CX3CR1+ cell infiltration after anti–TNF-α treatment. F and G: Independent group comparisons with naive mouse as reference using Holm-Sidak method. n = 6 per group (F and G); n = 3 per group (H). P < 0.05, ∗∗∗∗P < 0.0001 versus naive. Scale bars = 100 μm (AE). BMT, BM transfer; EGFP, enhanced green fluorescent protein.
      Using flow cytometry, the inhibitory effect of anti–TNF-α treatment on CX3CR1+ cell infiltration into the retina 24 hours and 7 days after the burn was confirmed (Figure 4H). In addition to reducing CCR2+ CX3CR1+ cell infiltration 24 hours and 7 days after the burn, anti–TNF-α treatment facilitated cell differentiation from amoeboid to dendritiform, and shifted their expression from CCR2hi CX3CR1−/lo to CCR2−/lo CX3CR1hi at 7 days (Figure 4, C and E–G).
      Further analysis of monocyte infiltration in the ON was performed with confocal microscopy. Peripheral CCR2+ CX3CR1+ cells were shown to physiologically populate the ON within 4 weeks after bone marrow transfer. Most of the cell infiltrates were either CCR2hi CX3CR1lo or CCR2lo CX3CR1hi (Figure 5). Fewer cells simultaneously expressed CCR2 and CX3CR1 markers. CCR2hi CX3CR1lo cells were mostly amoeboid, whereas CCR2lo CX3CR1hi cells were ramified, with some rare exceptions (Figure 5). Corneal alkali burn appeared to increase CX3CR1 and CCR2 expression in the ON 24 hours after the injury, and anti–TNF-α treatment appeared to inhibit this increase (Figure 5). Collectively, these results suggest that both the retinal and ON CCR2+ CX3CR1+ cells respond to the corneal injury within 24 hours, and that TNF-α blockade appears to affect both tissues.
      Figure thumbnail gr5
      Figure 5Monocyte infiltration into the retina through the optic nerve head. Image quantification of CX3CR1+ CCR2+ cell infiltration in the optic nerve (ON) after corneal alkali burn using CX3CR1+/EGFP::CCR2+/RFP bone marrow (BM) chimera model. AE: Confocal microscopy of ONs of BM-transferred (BMT) mice. A, F, and G: Blood-derived CX3CR1+ and CCR2+ cells physiologically populate the ON within 10 weeks after BMT. Most cells are either CX3CR1+CCR2 or CX3CR1CCR2+, with only few cells expressing both markers. CX3CR1+CCR2 and CX3CR1+CCR2+ acquire a semiramified morphology, whereas CX3CR1CCR2+ are mainly amoeboid in appearance. BD, F, and G: Twenty-four hours after corneal alkali burn, CX3CR1/CCR2 cell numbers increase in the optic nerves, followed by gradual reduction at 7 days (P < 0.005). C and EG: Anti–tumor necrosis factor (TNF)-α treatment (Tx) significantly reduces the number of CX3CR1/CCR2+ cells in the ON 24 hours and 7 days after the burn, and leads to reduction in the number of migrating cells into the retina. F and G: Independent group comparisons with naive mouse as reference using Holm-Sidak method. n = 3 per group (F and G). ∗∗P < 0.01, ∗∗∗∗P < 0.0001 versus naive. Scale bars = 75 μm (ΑΕ).

      Activated Neuroglia and Blood Monocytes Promote Neuroinflammation and Degeneration

      Using immunofluorescence, it was found that 24 hours after the burn, CX3CR1+ cells in the GCL express TNF-α (Figure 6, A and B ). Flow cytometry of CX3CR::CCR2EGFP/RFP mice confirmed TNF-α expression from CD45lo CCR2 (microglia)
      • Mizutani M.
      • Pino P.A.
      • Saederup N.
      • Charo I.F.
      • Ransohoff R.M.
      • Cardona A.E.
      The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.
      and CD45hi CCR2+ (peripheral monocytes)
      • Mizutani M.
      • Pino P.A.
      • Saederup N.
      • Charo I.F.
      • Ransohoff R.M.
      • Cardona A.E.
      The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.
      (Figure 7, A and B ). Furthermore, peripheral CX3CR1+ CCR2+ monocytes and CX3CR1+ CCR2 microglia expressed activation marker MHC-II, with monocytes expressing higher levels of MHC-II than microglia (Figure 7, C–E). These data suggest that monocytes respond more vigorously to the injury than microglia.
      Figure thumbnail gr6
      Figure 6Activated neuroglia and blood monocytes promote neuroinflammation and degeneration. A and B: Tumor necrosis factor (TNF)-α staining in flat-mounted CX3CR1+/EGFP mouse retinas. CX3CR1 and TNF-α markers are coexpressed in the ganglion cell layer 24 hours after the burn. B: Inset: Magnification of the dotted boxed area. C and D: Four weeks after the burn, CX3CR1+ cells (green) remain activated (amoeboid), and the retinal nerve fiber layer (βIII-tubulin; red) exhibits 40% tissue density loss (P < 0.05) compared with treatment with anti–TNF-α antibody that abrogates CX3CR1+ cell activation (less amoeboid) and inhibits retinal nerve fiber layer (RNFL) loss. Dotted boxed areas represent the section of the retina nerve fiber layer that was used for quantification. E and F: Quantification of RNFL thickness using vertical projection analysis of flat-mounted retinas 20 weeks after the burn shows 40% RNFL loss (P < 0.0001), compared with anti–TNF-α treated mice. The dotted line represents the location of the cross-sectional cut for quantification. G and H: Three-dimensional rendering of a retina flat mount shows amoeboid CX3CR1+ cells (semitransparent green) appearing to ensheathe (white arrows) β3-tubulin+ neuronal tissue (red) 4 weeks after corneal alkali burn. D and F: Independent group comparisons using Holm-Sidak method. n = 3 per group (D and F). P < 0.05, ∗∗∗∗P < 0.0001. Scale bars = 50 μm (C and E). Original magnification: ×63 (A, B, and H); ×40 (G).
      Figure thumbnail gr7
      Figure 7CX3CR1+ CCR2+ monocyte activation and tumor necrosis factor (TNF)-α expression in the retina. Flow cytometry of CX3CR1::CCR2EGFP/RFP mice. A and B: Ocular injury increases the percentage of CD45hi CCR2+ cells in the retina at 24 hours. TNF-α is expressed by CD45lo CCR2 (microglia) and CD45hi CCR2+ (peripheral monocytes). CE: At 24 hours after ocular surface injury, peripheral CX3CR1+ CCR2+ monocytes are shown to express high levels of activation marker major histocompatibility complex (MHC)-IIhi. The ocular burn has no significant effect on MHC-II expression by CX3CR1+ CCR2 microglia, which remain at low levels (MHC-IIint). Independent group comparisons between naive and burn 24 hours using analysis of variance and Holm-Sidak method. n = 3 in all groups. P < 0.05 verus naive (unpaired t-test). FSC, forward scatter; GFP, green fluorescent protein; RFP, red fluorescent protein.
      Inflammation after burn was associated with significant neuronal tissue loss at both 4 and 20 weeks after the injury. Neuronal dropout was abrogated by TNF-α inhibition (Figure 6, C–F).
      Interestingly, activated CX3CR1+ cells were spatially juxtaposed with β3-tubulin, and they appeared to phagocytose neuronal tissue (Figure 6, G and H, and Supplemental Video S2).

      Discussion

      This study aims to improve our understanding of the enigmatic retinal damage that follows acute corneal chemical burns. Clinical findings in patients experiencing ocular chemical injuries often show not only corneal and anterior chamber changes, but also glaucoma, with frequent relentless progression to end-stage blindness, despite efforts to prevent or slow down the process.
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      Glaucoma in eyes with severe chemical burn, before and after keratoprosthesis.
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      Glaucoma progression and role of glaucoma surgery in patients with Boston keratoprosthesis.
      This early retinal damage is not mediated by intraocular pressure elevation or posterior diffusion of the alkali itself in mice and rabbits; the preretinal vitreous pH, O2, and oxidation-reduction levels remain normal 24 hours after the burn,
      • Paschalis E.I.
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      • Chodosh J.
      • Dohlman C.H.
      Mechanisms of retinal damage after ocular alkali burns.
      suggesting that the iris and the lens act as barriers to further chemical diffusion posteriorly through the vitreous body. The alkali is unable to diffuse posteriorly through the suprachoroidal space 24 hours after the burn.
      • Paschalis E.I.
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      • Dohlman C.H.
      Mechanisms of retinal damage after ocular alkali burns.
      Rather, rapid changes in pH, oxidation-reduction, and O2 levels are restricted to the cornea, anterior chamber, and iris. These changes lead to a massive up-regulation of inflammatory cytokines in the anterior chamber, such as TNF-α and IL-1β, that rapidly diffuse posteriorly to the retina.
      • Paschalis E.I.
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      Mechanisms of retinal damage after ocular alkali burns.
      Within 24 hours of the burn, retinal CD45+ immune cells become activated and promote additional TNF-α synthesis.
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      Mechanisms of retinal damage after ocular alkali burns.
      The inflammatory response in the retina is sustained for weeks and months after the injury, triggering further RGC loss.
      • Paschalis E.I.
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      • Dohlman C.H.
      Mechanisms of retinal damage after ocular alkali burns.
      This hypothesis is further supported by the strong neuroprotective effect of infliximab, a TNF-α inhibitor, which prevents RGC loss and retinal damage, if administered promptly after the burn.
      • Paschalis E.I.
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      • Dohlman C.H.
      Mechanisms of retinal damage after ocular alkali burns.
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      • Dana R.
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      Alkali burn to the eye: protection using TNF-α inhibition.
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      Chemical burns of the eye: the role of retinal injury and new therapeutic possibilities.
      Building on the results of our prior studies,
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      Mechanisms of retinal damage after ocular alkali burns.
      • Cade F.
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      • Dana R.
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      Alkali burn to the eye: protection using TNF-α inhibition.
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      Chemical burns of the eye: the role of retinal injury and new therapeutic possibilities.
      the role of two major CD45+ immune cell populations (the retinal microglia and peripheral monocytes/macrophages) was investigated. Microglia have become the focus of intense scientific investigation because of their specialized functions.
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      Microglia-mediated neurotoxicity: uncovering the molecular mechanisms.
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      Macrophage biology in development, homeostasis and disease.
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      In conclusion, this study provides a plausible mechanism to explain the immunological processes that lead to retinal damage after acute corneal alkali injury. A link between the anterior and posterior segments that includes the retina and the ON is demonstrated. Furthermore, the involvement of microglia and peripheral monocytes in the inflammatory process is highlighted. Also, it was determined that ON and retina differ in peripheral monocyte regulation, where infiltrating monocytes/macrophages play a key role in early-phase retinal damage, whereas microglia play an important role in the late phase. In summary, ocular surface injury leads to recruitment of peripheral monocytes, up-regulation of inflammatory mediators, subsequent activation of microglia, and irreversible retinal damage (Figure 8). These findings may also prove relevant to other neurodegenerative diseases of the retina and the central nervous system.
      Figure thumbnail gr8
      Figure 8Proposed mechanism of retinal damage after corneal alkali burn. The effect of blood-derived monocytes and neuroglia in retinal degeneration after acute ocular surface trauma with alkali. A: Corneal alkali injury causes acute retinal inflammation and infiltration of CX3CR1+ and CCR2+ cells through the optic nerve head that align along the retinal vessels. B: Increased monocyte/macrophage trafficking causes neuroglial cell activation and subsequent elevation of inflammation. Activated CX3CR1+ cells enswathe retinal ganglion cells (RGCs) and nerve axons, a gliotic process that leads to retinal tissue damage. RNFL, retinal nerve fiber layer; TNF-α, tumor necrosis factor-α.

      Acknowledgments

      E.I.P. designed the study, conducted experiments, acquired and analyzed data, and wrote the manuscript; F.L., C.Z., V.K., and A.T. conducted experiments, acquired and analyzed data, and reviewed the manuscript; D.G.V., R.D., J.C., and C.H.D. critically reviewed the manuscript.

      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1Color-depth coding of CX3CR1+ and CCR2+ peripheral monocytes in the retina. Color-depth–coded confocal images of CX3CR1+/EGFP::CCR2+/RFP bone marrow–transferred (BMT) mice. Color analysis shows the position of the CX3CR1+ and CCR2+ cells within the retina tissue. Blue cells are located in the ganglion cell layer (GCL), and red in the outer plexiform layer (OPL). All intermediate colors are cells located between the GCL and OPL. Original magnification, ×63 (all images). TNF-α, tumor necrosis factor-α; Tx, treatment.

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