Materials and Methods
Induction of TON
Generation of Chimeric Mice
Retinal Imaging Using Confocal SLO
- Seeliger M.W.
- Beck S.C.
- Pereyra-Munoz N.
- Dangel S.
- Tsai J.Y.
- Luhmann U.F.
- van de Pavert S.A.
- Wijnholds J.
- Samardzija M.
- Wenzel A.
- Zrenner E.
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- Al Saati T.
- Johnson M.G.
- Sullivan T.J.
- Medina J.C.
- Collins T.L.
- Schmid-Alliana A.
- Schmid-Antomarchi H.
Real-Time Quantitative RT-PCR Analysis
Enzyme-Linked Immunosorbent Assay
Fluorescence in Situ Hybridization
Dark-Adapted ERG Analysis
Immunostaining of Retinal Whole Mounts
Co-Culture of Primary RGCs and Leukocytes
Leukocytes Are Recruited to the Retina after Optic Nerve Injury
Leukocyte Recruitment Occurs before Neuronal Cell Dysfunction and Death
- Galindo-Romero C.
- Aviles-Trigueros M.
- Jimenez-Lopez M.
- Valiente-Soriano F.J.
- Salinas-Navarro M.
- Nadal-Nicolas F.
- Villegas-Perez M.P.
- Vidal-Sanz M.
- Agudo-Barriuso M.
CXCL10/CXCR3 Pathway Is Up-Regulated in TON
CXCL10/CXCR3 Pathway Is Involved in Leukocyte Recruitment and RGC Injury in TON
Pharmacological Blockade of CXCR3 Partially Prevents RGC Dysfunction and Cell Loss after TON
- Johnson M.
- Li A.R.
- Liu J.
- Fu Z.
- Zhu L.
- Miao S.
- Wang X.
- Xu Q.
- Huang A.
- Marcus A.
- Xu F.
- Ebsworth K.
- Sablan E.
- Danao J.
- Kumer J.
- Dairaghi D.
- Lawrence C.
- Sullivan T.
- Tonn G.
- Schall T.
- Collins T.
- Medina J.
Up-Regulation of CXCL10 in TON Is Mediated by STAT
- Joussen A.M.
- Poulaki V.
- Mitsiades N.
- Cai W.Y.
- Suzuma I.
- Pak J.
- Ju S.T.
- Rook S.L.
- Esser P.
- Mitsiades C.S.
- Kirchhof B.
- Adamis A.P.
- Aiello L.P.
- Supplemental Figure 1
Genotype and phenotype analysis of representative offspring mice. A: Representative genotyping results of PCR analysis for wild type (WT; 1, only with 337-bp WT allele) and CXCR3 knockout (KO; 2, only with 485-bp KO allele) mice. B: Representative phenotyping results of GFP+ transgenic mice using blue light-emitting diode laser point.
- Supplemental Figure 2
Analysis of green fluorescent protein (GFP)–positive leukocytes in the blood of GFP bone marrow transplanted (BMT) mice. C57BL/6J wild-type (WT) mice were transplanted with bone marrow from GFP transgenic mice and 4 weeks later, blood was collected for flow cytometry. Representative histogram shows the percentage of GFP+ leukocytes in the blood of WT and BMT mice. n = 3 mice. Max, maximum.
- Supplemental Figure 3
Distribution of leukocytes and microglia in retinas after axonal injury. Wild-type mice received bone marrow transplant from green fluorescent protein (GFP) mice and 4 weeks later they were subjected to TON. At 24 hours after TON, retinas were fixed and labeled with Iba1 antibody (red) to identify microglia/monocytes. Green dots indicate GFP-positive cells. n = 3 mice. Scale bars = 50 μm. CON, control.
- Supplemental Figure 4
CD19 staining in the spleen. Spleen frozen section was used as a positive control for antibody against CD19 (red). DAPI (blue) stains nuclei. Scale bars = 50 μm.
- Supplemental Figure 5
Characterization of leukocytes in the TON retinas. Retinas were collected at 24 hours after TON and enzymatically dissociated to produce single cells for flow cytometric analysis. Cells were stained with antibodies against markers for leukocyte subtypes: CD45, CD3, CD11b, Ly6G, Ly6C, F4/80, and NK1.1. Plots of flow cytometric analyses are shown. Percentage indicates the proportion of CD45+ leukocytes, CD3+ T cells, CD11b+Ly6Ghigh neutrophils, CD11b+Ly6G− nonneutrophil myeloid cells, and NK1.1+ natural killer cells in total retinal cells from Control (Ctrl) and TON retinas. n = 14 mice.
- Supplemental Figure 6
Confirmation of purity of isolated primary RGCs. RGCs were isolated from wild-type pups at postnatal day 3 and stained with retinal ganglion cell marker Tuj1 antibody (green). DAPI (blue) stains nuclei. Scale bar = 50 μm.
- Supplemental Figure 7
Characterization of leukocytes isolated from bone marrow and blood. Leukocytes were isolated from bone marrow (A) and blood (B), and the populations and proportions of individual leukocyte subtypes were analyzed by flow cytometry. Plots for the percentage of CD4+ T cells, CD8+ T cells, γδ T cells, natural killer (NK) cells, NK T cells (CD3+NK1.1+), B cells (CD19+), neutrophils (CD11b+Ly6Ghigh), and nonneutrophil myeloid cells [CD11b+Ly6G− (eg, monocytes and dendritic cells)] are shown. n = 8 mice. SSC, side scatter.
- Supplemental Figure 8
Confirmation of nontoxicity of vehicle used for AMG487 treatment. Wild-type mice were injected with vehicle (Veh, i.p.) twice a day for 7 days. A: Body weight of naive and vehicle-treated mice. B: Representative images of retinas labeled with Tuj1 antibody (green) at 7 days after vehicle injection. Graph represents the number of Tuj1-positive cells per field. C: Electroretinographic analysis for retinal neuronal function under scotopic conditions between naive and vehicle-treated groups after 7 days of treatment. Graphs represent average amplitudes of a- and b-waves and positive scotopic threshold response over a range of stimulus strengths. n = 4 mice (B and C). Scale bars = 50 μm.
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Supported by NIH grant EY022694 , American Heart Association grant 11SDG4960005 , the John Sealy Memorial Endowment Fund for Biomedical Research, Retina Research Foundation (W.Z.), the University of Texas System Neuroscience and Neurotechnology Research Institute (R.K. and W.Z.), BrightFocus Foundation grant G2015044 (Y.H.), and American Heart Association grant 15POST22450025 (H.L.).
Disclosures: None declared.
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