Model of diabetic embryopathy

We (3, 4, 7, 8) and others (59) have used a rodent model of streptozotocin (STZ)–induced diabetes in research on diabetic embryopathy. Briefly, 10-week-old WT or KO female mice were intravenously injected daily with STZ (75 mg/kg) for 2 days to induce diabetes. STZ from Sigma-Aldrich (St. Louis, MO) was dissolved in sterile 0.1 M citrate buffer (pH 4.5). We used a U-100 insulin syringe (Becton Dickinson) with 28G1/2 needles for the injections. Approximately 140 μl of STZ solution was injected per mouse. Diabetes was defined as a 12-hour fasting blood glucose level of ≥16.7 mM. Male and female mice were paired at 3:00 p.m., and E0.5 of pregnancy was established at noon of the day when a vaginal plug was present. Embryos were harvested for subsequent analysis at different developmental stages. Embryos were harvested at 2:00 p.m. at E8.5 for biochemical and molecular analyses. At E10.5, embryos were examined under a Leica MZ16F stereomicroscope (Bannockburn, IL) for NTDs. Images of embryos were captured by a DFC420 5 MPix digital camera with software (Leica, Bannockburn, IL) and processed with Adobe Photoshop CS2. Normal embryos were classified as having a completely closed neural tube with no evidence of other malformations. Malformed embryos were classified as showing evidence of failed closure of the anterior neural tubes resulting in exencephaly, a lethal type of NTD with the absence of a major portion of the brain, skull, and scalp. Because our model induces NTD at E10.5, we did not examine other major structural malformations, such as cardiovascular defects, which do not occur until later embryonic stages (E13.5).