Streptozotocin (stz)

All animal experiments in the study were approved by the Institutional Animal Care and Use Committee at the Soochow University, and all methods in the study were performed in accordance with the relevant guidelines and regulations. Adult female Sprague-Dawley (SD) rats (weighing 160~180 g) were housed four per cage in a 12 hour-12 hour light-dark cycle and temperature-controlled (25 ± 1 °C) room. All rats were fed with tap water and standard laboratory chow, ad libitum. Streptozotocin (STZ; Sigma Chemicals, St. Louis, MO, USA) was used to induce diabetic models by a single intraperitoneal injection in 65 mg/kg dose, which was freshly dissolved in citrate buffer (10 mmol/L, Na citrate, pH 4.3~4.4), as described previously^{35 (link), 44 (link)}. The control (CON) rats only received citrate buffer in the same volume. Fasting blood glucose concentration was measured from the tail vein by glucometer (Johnson & Johnson, New Brunswick, NJ, USA) 1 week after STZ injection. Only rats with blood glucose concentration higher than 15.0 mmol/L (270 mg/dL) were further used in the study.

Severe diabetes was induced at adult life of female rats (approximately at 90 days of age) by beta cytotoxic drug (Streptozotocin, STZ; Sigma Chem. Company, USA). STZ was dissolved in a citrate buffer (0.1 mol/L, pH 4.5) and intravenously (i.v.) administered at a dose of 40 mg/kg body weight [18 (link)–20 (link)]. Control rats received only citrate buffer using similar route and administration period. Seven days after STZ injection, the diabetic state was confirmed by blood glucose levels ≥ 300 mg/dL using a conventional glucometer (OneTouch Ultra—Johnson & Johnson). For nondiabetic rats, the inclusion criteria used was blood glucose levels ≤ 120 mg/dL. Glycemic values were expressed in milligrams per deciliter (mg/dL). After one week of diabetes confirmation or buffer administration (control), all adult female rats were mated overnight with nondiabetic adult male rats. The morning on which spermatozoa were found in the vaginal smear was designated pregnancy day 0 [19 (link)]. The offspring was born by spontaneous delivery.

STZ (WAKO, Tokyo, Japan) was injected into 6-week-old male mice for five days (50 mg/kg/day via intraperitoneal injection). Blood glucose was measured by OneTouch® UltraVue (Johnson and Johnson, New Brunswick, NJ, USA) three days after the STZ injection, and was defined as true diabetes, not prediabetes, if the serum glucose levels were higher than 250 mg/dl.

Eight‐week‐old male C57BL/6 mice were starved for 4 h before streptozotocin (STZ, Sigma, St. Louis, MO) injection. They then received an intraperitoneal injection of STZ (50 mg/kg) or citrate buffer (vehicle control) for 5 days consecutively. The fasting blood glucose of the mice was evaluated at 7 days after the last STZ injection by a glucometer (ONETOUCH, Johnson & Johnson, USA). Mice with glucose levels of 16.7 mM or above were deemed diabetic. The fasting blood glucose was continuously monitored per month to ensure the models were successful (those failed mice were excluded). For antibodies, mice were received intravitreal injections of anti‐Sema4D neutralizing antibody (2 μg per eye, BMA‐12), anti‐VEGF neutralizing antibody (2 μg per eye, R&D Systems) alone or combinedly in the indicated time. For adenoviruses in STZ model, adenoviruses (1 μl) containing double‐floxed Cre‐inducible GFP and mir30‐shRNA were intravitreally injected into Tie2‐Cre and Pdgfrβ‐Cre mice at the beginning of diabetes induction and were repeated once a month, and the retinas were harvested at 2 months of diabetes. The animals were randomized into different treatments. The wild‐type or knockout mice were randomly allocated to experimental groups. For intravitreal injections, the mice were treated with antibiotics to prevent infection. Those with eyeball infections after injection were excluded.