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L. Zhao, W. Ma, R. Fariss, W. T. Wong; Cellular Mechanisms Associated With Photoreceptor Cell Loss in a Mouse Model of Subretinal Hemorrhage. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6197.
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Hemorrhage in the subretinal space, such as that occurring in exudative age-related macular degeneration (AMD), often results in retinal atrophy and vision loss. The cellular mechanisms through which subretinal hemorrhage causes retinal atrophy as well as the potential treatment approaches targeting these pathological factors remain poorly defined. The aim of this study was to create a mouse model for subretinal hemorrhage in order to examine cellular and molecular alterations occurring specifically in response to subretinal blood and to evaluate mechanisms driving retinal cell loss.
Adult C57BL/6 mice (2-4 months old) were anesthetized with intraperitoneal ketamine and xylazine, and submitted to a blood draw via the tail vein. A small volume (1.6 µl) autologous blood was then injected slowly into the subretinal space in the temporal quadrant in one eye. An equal volume of viscoelastic gel (Provisc, Alcon) was similarly delivered to the contralateral eye served as control. Eyes were harvested at 6 hours, 12 hours, 1 day, 2 days, 3 days, and 5 days after subretinal injection, and then sectioned and analyzed by immunohistochemistry and TUNEL assay. Molecular analyses were also performed on treated retinas using ELISA.
Photoreceptor apoptosis, as marked by TUNEL positivity in the outer nuclear layer (ONL), was rapidly induced within12 hours after subretinal hemorrhage, increasing to a maximum by 3 days. Thinning of the outer nuclear layer was evident by 2-3 days, decreasing to a layer 0-4 cells thick by 5 days. In control eyes the number of TUNEL + were significantly lower with only minimal ONL thinning by 5 days. Microglial infiltration into the ONL was also prominent in eyes with subretinal blood, consisting largely of activated amoeboid microglia that were MAC2+. ELISA analyses also revealed a greater elevation of chemotactic cytokines (CCL2, CCL5, SDF-1) and adhesion molecules (VCAM, ICAM-1) in retinas with subretinal blood compared to controls.
Our murine model of subretinal hemorrhage demonstrated cellular features of photoreceptor apoptosis and outer retinal atrophy that recapitulates features seen in human disease. Microglial infiltration and activation, as well as increased retinal expression of chemokines and adhesion molecules that attract and retain microglia, were also key features present. This model may be useful in elucidating cellular and molecular mechanisms and for testing treatment strategies in subretinal hemorrhage.
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