4.1. Streptozotocin (STZ)-Induced Diabetic Mice

All experimental procedures were approved by CWRU IACUC (2016-0025 first approved on 2/13/2016) and LSCVAMC ACORP (16-012-MS-19-002-C first approved on 3/1/2016). Animals were housed under SPF conditions at Case Western Reserve University Animal Resource Center Health Sciences Animal Facility according to NIH guidelines. Male C57BL/6 mice aged 8 to 10 weeks were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Diabetes was induced over five consecutive days with intraperitoneal injections of streptozotocin (STZ) at 60 mg/kg body weight. Diabetes was defined by 6-h, fasted-blood-glucose concentrations greater than 275 mg/dL, which was verified using glucose-dehydrogenase-based strips 17 days after the last STZ injection (day 22). Hyperglycemia was quantified by hemoglobin A1c levels using the Crystal Chem Mouse A1c kit at six and 29 weeks after diabetes was confirmed. Subcutaneous insulin (Eli Lilly NPH) was administered as needed (0 to 0.2 U, one to three times per week) to maintain body weight and prevent catabolism. Retinal inflammation, oxidative stress, and leukostasis analyses were performed at a two-month diabetic time point, while capillary degeneration analyses were performed at an eight-month diabetic time point. As previously described, these durations are optimal for these analyses in this murine model [23,26].

Briefly, the research design of this study was as follows: 30 mice per group (non-diabetic, VAF347-treated diabetic, and untreated diabetic mice), and 10 non-diabetic VAF347-treated (controls for VAF347 treatment toxicity) totaling 100 mice were used in this study; all HbA_1c scores are shown in Table 1. All 10 non-diabetic VAF347-treated mice were euthanized eight months after diabetes was confirmed for toxicity examination. Non-lethal blood collections of five mice per group were performed two and eight months after diabetes was confirmed for sera analysis of IL-6 and IL-17A. Two months after diabetes was confirmed, 20 mice/group (n = 60 mice total) were euthanized and used to analyze: the number of T helper cells in the spleen (n = 3 mice/group), the number of adherent leukocytes in the retinal vasculature (n = 3 mice/group), the level of oxidative stress in the retina (n = 5 mice/group), and the level of VEGF, IL-17A, IL-6, and TNF-α in the retina (n = 9 mice/group). Eight months after diabetes was confirmed, 10 mice/group (n = 30 mice total) were euthanized and used to analyze: the number of T helper cells in the spleen (n = 3 mice/group), the number of acellular capillaries in the retinal vasculature (n = 5 mice/group), and toxicity comparisons (n = 2 mice/group).