Retinoic Acid Induced MMC-associated NBD In Fetal Rats: Characterization By Histopathological And Immunohistochemical Analysis

IntroductionNurogenic bladder dysfunction(NBD) is one of common diseases in Pediatric urology. The natural history of NBD, however, remains controversial and has not been formally assessed.This lack of understanding hinders the development of more appropriate treatment strategies for MMC-associated NBD.The aim of this study was to create a reliable method of inducing an isolated MMC-like lesion in fetal rats by maternal administration of retinoic acid(RA) and to analysis its related histopathological and immunohisto-chemical characterizations.Materials and Methods1.Retinoic acid induced MMC-associated NBD in fetal ratsTime-dated primigravida Sprague-Dawley rats were randomly grouped. A) E9 group:dams were gavage fed different doses of RA (40-70 mg/kg body weight) dissolved in 2 ml of olive oil at embryonic day9 (E9).B) E10 group: dams were gavage fed different doses of RA (40-130 mg/kg)dissolved in 2 ml of olive oil at E10.C) Control group:animals were fed 2 ml of olive oil alone at E10.Fetuses were collected at E21 by cervical dislocation.The numbers of fetuses were recorded.Features with MMC were compared to those in their phenotypically normal littermates as well as to control fetuses.2.histopathological and immunohistochemical analysis Time-dated primigravida S-D rats were gavage fed 120 mg/kg RA dissolved in 2 ml of olive oil at E10.Control animals were fed 2 ml of olive oil alone.Fetuses which collected at E21 were divided into 3 groups:MMC (RA-exposed with MMC), RA (RA-exposed but with no malformation) and OIL (control fetuses).Fetuses of 3 groups were embedded in paraffin wax and sectioned at 5μ-m thickness. Sections were dewaxed, serially rehydrated and stained with hematoxylin and eosin (H-E).The expression patterns of GFAP,α-SMA, tubulin-β-Ⅲ, nNOS were analyzed by immunohistochemistry.ResultsOverall,we have examined 810 fetuses of 75 rats,in which MMC-like defects were present at term in 97(12.0%).99.2% fetuses of E9 group(16 rats) were dead at term,no MMC-like defects were present.637 fetuses of E10 group(56 rats), in which MMC-like defects were present in 97(15.2%).A dose-response relationship for the incidence of associated malformations and a decrease in number of live fetuses observed.A single administration of 120 mg/kg of RA resulted in the highest incidence of MMC-like defects (43.0%). All the fetuses in control group were normal.Similar to the findings in human fetuses with MMC, the severity of the lesion spanned the spectrum of severity observed in MMC fetuses, ranging from exposure of the cord with intact neural elements to a destroyed spinal cord with no intact neural elements.H-E staining of bladder from OIL, MMC, and RA fetuses demonstrates normal arrangement of the outer and inner layer of smooth muscle composing the bladder detrusor.Immunoreactivity for GFAP was observed within the spinal cord.The staining intensity of GFAP of MMC fetuses were increased.Immunoreactivity forα-SMA,tubulin-β-Ⅲand nNOS was observed within the bladder wall. Staining intensity for a-SMA did not vary between OIL, MMC, and RA bladders.Meanwhile, the density of tubulin-β-Ⅲ-positive nerve fibers and nNOS-positive nerve fibers within the detrusor muscle of MMC bladders were both reduced. ConclusionsRA has been shown to be highly effective in inducing MMC-associated NBD in fetal rats.The efficiency of RA for induction of MMC is dependent upon the timing and dosage of administration.RA induced MMC-associated NBD in rat is a reliable and useful model.It can help us to evaluate the biomolecular mechanisms of NBD in MMC and to improve our understanding of the athophysiological processes involved in the development of myelodysplastic bladders.