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Thomas F. Sabljic, Alexander K. Ball; Robo-1 Immunoreactive Leukocytes are Present in the Eye Following Optic Nerve Crush. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3103.
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© ARVO (1962-2015); The Authors (2016-present)
Optic nerve (ON) crush results in the gradual death of retinal ganglion cells (RGCs) over 4 weeks. Resident immune cells, retinal microglia, become activated in response to ON crush. Understanding the immune response following ON crush is essential for the development of interventions to prevent RGC death after injury. The purpose of this experiment was to characterize immune cells in the retina and ON following intraorbital ON crush in the rat.
Adult Sprague-Dawley rats were anaesthetized and RGCs were retrogradely labelled with Fluorogold (FG) (4% FG) injected into the superior colliculi. Seven days later, the left intraorbital ONs were exposed and crushed by closing fine flat tipped jeweller's forceps completely around the ON approximately 3 mms from the optic disc for 3 seconds. Animals were fixed by transcardial perfusion with 4% buffered formaldehyde 7 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 both injured and uninjured animals. Expression of markers for glial reactivity (GFAP; 1:200), microglia (Iba-1; 1:200), and leukocytes (Robo-1; 1:200) in the retina and ON were examined by immunohistochemistry.
Seven days after ON crush there was a 28% decrease in RGCs compared to uninjured retinas. GFAP and Iba-1 were upregulated in retinal astrocytes and microglia, respectively, of injured animals compared to uninjured animals. Robo-1 expression was absent in uninjured retinas. Expression of Robo-1 by leukocytes was observed on the vitreal surface of the retina, as well as within blood vessels of injured animals. ON astrocytes and microglia expressed GFAP and Iba-1, respectively. In the ONs of injured animals, GFAP expression was found to be absent at the crush site but persisted in the areas adjacent to the site of injury. The number of Iba-1 expressing microglial cells was dramatically increased at the ON crush site but did not differ from uninjured animals a short distance on either side of the crush site. Robo-1 expression by leukocytes was absent in the uninjured ON. At the crush site of injured animals, Robo-1 expressing cells were found on the surface of the ON.
Microglial Iba-1 expression increases in both the retina and ON in response to ON crush. GFAP is upregulated in the retina but is decreased at the ON injury site. Robo-1 expression is absent in the retina and ON of uninjured animals but found in both the retina and ON after ON crush. Robo-1 expressing leukocytes are involved in the retinal response to intraorbital ON crush.
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