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Crystal Koralis Colon Ortiz, Maria Avrutsky, Jacqueline Lawson, Fatima Morales, Claire Chen, Scott Snipas, Guy S Salvesen, Carol M Troy; Immunohistochemical profiling of caspases in a mouse model of Retinal Vein Occlusion (RVO). Invest. Ophthalmol. Vis. Sci. 2019;60(9):1636.
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Apoptosis and inflammation are main contributors to the development of ischemic neurovascular injury after Retinal Vein Occlusion (RVO). Caspases drive these two major events in mammals. To elucidate the caspase signaling pathways in RVO, we tested the hypothesis that caspases are differentially expressed post-RVO in a spatiotemporal manner.
We induced RVO by tail vein injection of rose bengal, and retinal veins were occluded by laser photocoagulation using the Micron IV (Phoenix Research Labs) in C57/BI6 adult male mice (Jackson Laboratories). Caspase-9 was inhibited by administering a cell permeant caspase-9 inhibitor using topical eye-drops. Immunohistochemistry against cleaved-caspase-9, caspase-8, cleaved-caspase-1, cleaved-caspase-7, and cleaved-caspase-3 was performed to assess the expression of caspases 24hrs post-RVO. Endothelial and immune cells were stained with Isolectin or CD31. Expression of caspases was evaluated in RVO injured retinas (n=4), RVO injured retinas treated with caspase-9 inhibitor (n=3), and compared to uninjured controls; vehicle (n=6) and caspase-9 inhibitor (n=5). Confocal images were blindly quantified for caspase positive endothelial cells, microglia, and retinal neurons. One-Way ANOVA and Dunnett’s multiple comparisons test were used for statistical analysis.
Caspase-9 (p=0.0002), caspase-8 (p=0.007), caspase-1 (p=0.01), and caspase-7 (p=0.002) were significantly increased in the retina post RVO, when compared to uninjured controls. On the contrary, expression of caspase-3 was maintained in basal levels, as its expression did not change in injured retinas and was not modulated by caspase-9. Caspase-9 had the highest levels of induction, followed by caspase-7. Inhibition of caspase-9 restricted the expression of caspase-7 and significantly reduced the levels of caspase-9 in endothelial cells, leukocytes, and neurons. Caspase-8 was significantly activated in microglia and caspase-1 in neurons.
We found that caspase-9 is the main caspase activated by RVO and caspase-9 cleaves caspase-7, but not caspase-3 in endothelial cells. Caspase-8 and caspase-1 may play an important role in microglial activation and neuronal inflammatory response. Evaluation of the implication of caspases in inflammation and apoptosis post-RVO will allow us to clarify the pathophysiology of ischemic neurovascular injury.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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