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Angela L. Pool, Nicholas Brecha; Decreased Retinal Ganglion Cell Survival and Differential Immune Response in Thy1-CFP DBA/2J Mouse Line Following Ocular Injury. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5358.
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Our aim was to determine whether intravitreal injections of the glutamate receptor agonist N-methyl-D-aspartate (NMDA) caused disparate amounts of ganglion cell loss or differential immune response in the retina of Thy1-CFP C57BL/6 (Thy1-CFP) and Thy1-CFP DBA/2J (CFP-DBA) mice.
8 to 12-week old Thy1-CFP and CFP-DBA mice were anesthetized and subjected to scleral puncture (poke) or intravitreal injections of sterile saline (pH 7.2) or NMDA (20nmol). Tissue was collected after 72 hours as retinal wholemounts and labeled with antibodies to GFAP (astrocytes) and Iba1 (microglia). Retina were incubated in primary antibody solution for 3-5 days and secondary antibodies conjugated with Alexa 568 or Alexa 633 (1:1000) for 1-2 days at 4°C. Immune cells and CFP-expressing inner retinal cells were manually counted in projected confocal images and average densities for each cell type were determined. Scans were acquired every 500 µm from the optic nerve head along the dorsoventral and nasotemporal axes.
CFP-DBA retina showed a larger decrease in the number of CFP-expressing cells than Thy1-CFP retina following poke, saline injection, and NMDA injection. Average CFP-positive cell density was determined to be 1782 ± 260 cells/mm² in Thy1-CFP retina and 1485 ± 227 cells/mm² in CFP-DBA retina. At 72 hours post-excitotoxic insult, Thy1-CFP animals had lost an average of 36% of CFP-positive cells while CFP-DBA mice had lost over 60%. CFP-DBA retina also showed a larger increase in the number of GFAP-positive cells than Thy1-CFP retina in all experimental conditions. Average astrocytic density was determined to be 190 ± 29 cells/mm² in Thy1-CFP mice and 132 ± 12 cells/mm² in CFP-DBA mice. Following poke, saline, and NMDA injections, Thy1-CFP animals showed average increases of 6%, 38%, and 56% respectively while CFP-DBA mice showed increases of 26%, 50%, and 97% respectively. Average microglial density was determined to be 101 ± 10 cells/mm² in Thy1-CFP mice and 124 ± 17 cells/mm² in CFP-DBA mice. Following poke, saline, and NMDA injections, Thy1-CFP animals showed average increases of 219%, 273%, and 741% respectively while CFP-DBA mice showed increases of 229%, 540%, and 636% respectively.
Our results demonstrate that CFP-DBA mice show decreased ganglion cell survival and increased immune response following ocular injury when compared to Thy1-CFP animals.
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