Platelets Accumulate in the Diabetic Retinal Vasculature Following Endothelial Death and Suppress Blood-Retinal Barrier Breakdown Kenji Yamashiro, Akitaka.

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Platelets Accumulate in the Diabetic Retinal Vasculature Following Endothelial Death and Suppress Blood-Retinal Barrier Breakdown Kenji Yamashiro, Akitaka Tsujikawa, Susumu Ishida, Tomohiko Usui, Yuichi Kaji, Yoshihito Honda, Yuichiro Ogura, Anthony P. Adamis The American Journal of Pathology Volume 163, Issue 1, Pages 253-259 (July 2003) DOI: 10.1016/S0002-9440(10)63648-6 Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

Figure 1 Platelet accumulation in the diabetic retinal circulation. Labeled platelets were visualized as white dots (arrows) in the retinas of non-diabetic rats (A) and 2-week diabetic rats (B). C: Time course of platelet adhesion in the retinas of diabetic rats. Values represent the mean ± SEM *P < 0.01 versus non-diabetic rats. The American Journal of Pathology 2003 163, 253-259DOI: (10.1016/S0002-9440(10)63648-6) Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

Figure 2 Immunofluorescence for Factor XIIIA in the retinas of rats 24 hours after platelet infusion. Green fluorescence from the CFSE-labeled adhering platelets (arrowheads in A) and red fluorescence from the anti-Factor XIIIA antibody (arrows in B). Co-localization was confirmed when the images were superimposed (C). The American Journal of Pathology 2003 163, 253-259DOI: (10.1016/S0002-9440(10)63648-6) Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

Figure 3 Effect of diabetic platelets and endothelial cells on platelet adhesion. N to N, platelets from non-diabetic rats infused into non-diabetic rats; D to D, platelets from 2-week diabetic rats infused into 2-week diabetic rats; N to D, platelets from non-diabetic rats infused into 2-week diabetic rats; D to N, platelets from 2-week diabetic rats infused into non-diabetic rats. Values represent the mean ± SEM *P < 0.01 versus N to N. The American Journal of Pathology 2003 163, 253-259DOI: (10.1016/S0002-9440(10)63648-6) Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

Figure 4 Western blotting for Fas (top) and FasL (bottom) showed that retinal Fas and FasL protein levels in 2-week diabetic rats (DM2w) were elevated compared to non-diabetic rats (non-DM). Similar results were obtained in three separate experiments. The American Journal of Pathology 2003 163, 253-259DOI: (10.1016/S0002-9440(10)63648-6) Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

Figure 5 Effect of an anti-FasL neutralizing antibody on platelet trapping. non-DM, platelets from non-diabetic rats were infused into non-diabetic rats; DM2w, platelets from 2-week diabetic rats were infused into 2-week diabetic rats; DM2w/control Ab, platelets from 2-week diabetic rats were infused into 2-week diabetic rats treated with the control antibody; DM2w/anti-FasL Ab, platelets from 2-week diabetic rats were infused into 2-week diabetic rats treated with the anti-FasL antibody. Values represent the mean ± SEM *P < 0.01 versus DM2w. The American Journal of Pathology 2003 163, 253-259DOI: (10.1016/S0002-9440(10)63648-6) Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

Figure 6 Dying and/or injured retinal endothelial cells were labeled in vivo with propidium iodide (PI) 24 hours after platelet infusion into 2-week diabetic rats. Co-localization of green fluorescence from the CFSE-labeled trapped platelets (arrowheads in A) and red fluorescence from the PI-labeled endothelial cells (arrows in B) was confirmed when the images were superimposed (C). The American Journal of Pathology 2003 163, 253-259DOI: (10.1016/S0002-9440(10)63648-6) Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

Figure 7 Time course of platelet counts in the peripheral blood of normal rats following the intraperitoneal administration of the anti-platelet serum (APS). Values represent the mean ± SEM (n = 6 at each time point). The American Journal of Pathology 2003 163, 253-259DOI: (10.1016/S0002-9440(10)63648-6) Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

Figure 8 The effect of platelet depletion on blood-retinal barrier breakdown. non-DM, non-diabetic rats; non-DM + APS, non-diabetic rats treated with intraperitoneal anti-platelet serum; DM2w, 2-week diabetic rats; DM2w + APS, 2-week diabetic rats treated with the anti-platelet serum. Values represent the mean ± SEM *P, < 0.01 versus non-DM. †P, < 0.01 versus DM2w. The American Journal of Pathology 2003 163, 253-259DOI: (10.1016/S0002-9440(10)63648-6) Copyright © 2003 American Society for Investigative Pathology Terms and Conditions