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Thomas F. Sabljic, Alexander K. Ball; Slit2 Delays The Death Of Retinal Ganglion Cells After Optic Nerve Crush Injury. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6601.
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Retinal ganglion cells (RGC) die over 4 weeks after optic nerve (ON) crush injury. ON crush activates retinal microglia and Robo-1 expressing blood-borne immune cells that localize to the site of injury, and the retina. The interaction between the systemic immune system and the retina is poorly understood, yet may play an important role in regulating RGC survival mechanisms after injury. Slit-2, the ligand for Robo-1 has been shown to be a potent immunomodulator, exerting its effects by inhibiting leukocyte chemotaxis. The purpose of this experiment was to determine the effect of Slit2 on RGC survival after ON crush injury in the rat.
Adult Sprague-Dawley rats were anaesthetized and RGCs were retrogradely labeled with Fluorogold (FG) injected into the superior colliculi. 7 days later animals received tail vein injections of either 1mL phosphate buffered saline (PBS) or 1mL Slit2 (2.5 μg/mL). The left intraoribital ONs were then exposed and crushed. Animals were fixed by transcardial perfusion with 4% buffered formaldehyde 7 and 14 days after injury. Cryostat sections of retinas and ONs were examined by epifluorescence microscopy. RGC survival was determined after injury by counting FG labelled RGCs in flat mounted retinas of injured and uninjured animals. Expression of markers for glial reactivity (GFAP; 1:200), and leukocytes (Robo-1; 1:200) in the retina and ON were examined by immunohistochemistry.
There was a 29% and 63% decrease in RGCs 7 and 14 days after ON crush, respectively, compared to uninjured retinas. In animals receiving Slit2 injections, there was no loss of RGCs after 7 days, and a 73% loss after 14 days compared to uninjured animals. GFAP was upregulated in retinal astrocytes of injured animals injected with PBS compared to uninjured animals. GFAP expression was not upregulated in animals injected with Slit2. Leukocytes expressing Robo-1 were observed on the vitreal surface of the retina, as well as within the retinal vasculature of injured animals injected with PBS. Robo-1 expression was absent in uninjured retinas, as well as animals injected with Slit2. Robo-1 expression by leukocytes was absent in the uninjured ON. At the crush sites of all injured animals, Robo-1 expressing cells were found on the surface of the ON.
Slit2 injection before ON crush reduced glial reactivity and delayed the death of RGCs 7 days after injury. The lack of Robo-1 labeled immune cells in the injured retinas suggests that Slit2 reduced pro-inflammatory signaling by the systemic immune system. These findings suggest that Robo-1 leukocytes are involved in the retinal response to intraorbital ON crush and that systemic administration of Slit2 only delays the death of RGCs by inhibiting leukocyte chemotaxis.
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