J.V. Forrester

Role of inflammation in blood-retinal barrier breakdown: animal models

Purpose Animal models are valuable tools to study the pathogenic mechanisms of many diseases. Diabetic retinopathy is a low-grade chronic inflammatory disease. Nitric oxide (NO) is involved in leukostasis and blood-retinal barrier (BRB) breakdown in the early stages of the disease. However, the role of the different NO synthase (NOS) isoforms was not elucidated. We aimed to clarify the involvement of constitutive (eNOS, nNOS) and inducible NOS (iNOS) isoforms and the mechanisms underlying NO-mediated leukostasis and BRB breakdown, by using an experimental model of diabetes and iNOS KO mice. Methods Diabetes was induced by streptozotocin in normal and KO mice (2 weeks of diabetes). Normal mice were treated with L-NAME (NOS inhibitor). Vessel leakage was assessed with Evans blue. Leukostasis was quantified in flat-mounted retinas and in vivo. ICAM-1, occludin, ZO-1 and nitrotyrosine levels were assessed by Western blotting or immunohistochemistry. Results Diabetes increased the permeability of BRB and leukostasis, which were reduced by L-NAME. Similar effects were observed in diabetic iNOS KO mice. In diabetic mice, the immunoreactivity of tight junction proteins, occludin and ZO-1, decreased, whereas ICAM-1 protein levels increased. Those effects were prevented by L-NAME and in diabetic iNOS KO mice. Diabetes also upregulated all NOS isoforms and increased nitrotyrosine levels in normal mice, but did not significantly increase eNOS and nNOS, and nitrotyrosine levels, in iNOS KO mice. Conclusion These data demonstrate that iNOS plays a predominant role in leukostasis and BRB breakdown. The mechanism involves ICAM-1 upregulation and tight junction proteins downregulation. Support: FCT, Portugal