Structural analysis of the extracellular matrix and urethral striated muscle in diabetic pregnant rats

Abstract

Diabetes mellitus during pregnancy is associated with high levels of urinary incontinence and pelvic floor muscle dysfunction. A previous study developed by our group found several serious effects of severe diabetes (blood glucose higher than 300mg/dL) on urethral striated muscle in pregnant rats, including: atrophy, thinning and change in the distribution and in the muscle fiber type. For the performance of this project, it was used the experimental model of pregnant rats with smaller blood glucose intensity diabetes (blood glucose between 120 and 300 mg /dL, mild diabetes), in order to establish a bigger correlation with blood glucose levels found at the clinic. So, after removing and processing the urethra, we intend to analyze the extracellular matrix and urethral striated muscle structure from theses rats, in an associated study with the "Case Western University". 92 Wistar rats were evaluated, divided into four groups: virgin, pregnant, diabetic and diabetic pregnant virgin. The first three groups were considered as control of the main group, the diabetic pregnant. Mild diabetes was induced at the first day of birth by subcutaneous administration of streptozotocin at a dose of 100 mg/kg body weight. The inclusion criteria was to have two or more glycemic points changed in the glucose tolerance test performed at the first pregnancy day. In the end of the experiment, the rats were anesthetized, euthanized, and the vagina and the urethra were removed in monobloc. Some samples were frozen in liquid nitrogen and kept at -80 ° C for future cuts in cryostat (6 µm thick), and others were fixed on paraformaldehyde, embbebed in paraplast, cutted in microtome (4 µm thick) and submitted to the to cytochemical staining for future analysis of the extracellular matrix and urethral striated muscle components. It will be performed morphometric analysis to measure the thickness of the muscle layer, of the propria slide layer and of the connective tissue area around the urethral striated muscle of the four groups. Our hypothesis is that, in the binomial diabetes and pregnancy, the joint analysis of the urethral striated muscle and extracellular matrix makes clear that the change in this muscle composition leads to tissue remodeling where the matrix has a fundamental function. Moreover, we expect to better understand the structural effects of diabetes and pregnancy interaction on the extracellular matrix and the urethral striated muscle, and verify if the moderate diabetes (blood glucose between 120-300mg/dL) leads to the muscle changes similar to the ones found in severe diabetes (blood glucose> 300mg/dL). (AU)