Comparative Histomorphological and Histostereological Teratogenic Effects of Prenatal Exposure to Phenytoin and Phenobarbital on the Differentiation of Epiphyseal Growth Plate in Albino Rats (Rattus norvegicus)

Abstract

The prenatal exposure to phenytoin and phenobarbital in the management of maternal neurological disorders like epilepsy has been associated with a wide range of fetal neurological and musculoskeletal malformations. What is yet to be established is their comparative histostereological and histomorphological teratogenic effects on the fetal skeletogenesis of the long bones, hence the basis of this study. In carrying out this study, a post-test-only experimental study design was adopted. All the animal experimentation was carried out in the animal research facility situated at the University of Nairobi (UON), while the bone tissue processing for histology and histostereological was carried out in the Department of Human Anatomy of JKUAT. A Sample size of 30 albino rat dams weighing between 220±30 grams were used, for each of the study medicine. This sample size was determined by the use of the resource equation for the one-way Analysis of Variance method (ANOVA). The 30 Albino rats in each of these two study categories of phenobarbital and phenytoin were first broadly divided into two study groups of 3 rats for the control group and 27 rats for the experimental group. To evaluate whether the teratogenic effects of both medicines are dose-dependent, the 27 rats in their experimental groups were further subdivided into three study subgroups of 9 rats as follows; (i) 9 rats for low doses of phenobarbital and phenytoin group (31mg/kg/3.1mg/kg, respectively), (ii) 9 rats for medium doses of phenobarbital and phenytoin group (medium 62mg/kg and 19mg/kg, respectively), (iii) 9 rats for the high phenobarbital and phenytoin group (124mg/kg and 41.35mg/kg, respectively). To further evaluate whether the observed teratogenic effects are time-dependent, the 9 rats in each of the three-dose categories were further sub-divided into three subgroups of 3 rats each according to the trimesters of exposure as follows; (i) 3 rats for trimester one (TM1); (ii) 3rats for trimester two (TM2) and (iii) 3 rats for trimester three (TM3). All rats in both control and experimental groups were fed on rodent pellets and water ad-libitum and sacrificed on the 20th gestational day. The fetal bones were harvested for both histomorphological and stereological analysis. Histomorphological data information of photomicrographs was taken using a Swift 3.0 microscope digital camera (20mega pixels), and uploaded to Swift 3.0 software for labelling. The maternal and fetal pregnancy outcomes and stereological data (parametric) were collected using structured checklists, stored in Excel spreadsheets Windows 10, version 2016, and then exported for analysis into the SPSS program for Windows version 25 for analysis (Chicago Illinois). Data was expressed as mean ± SD for all values. The analysis applied both one-way ANOVA for intragroup comparisons, MANOVA for interaction effects and pairwise comparisons. All results whose P<0.05 were considered significant. The findings of this study established that both phenobarbital and phenytoin had teratogenic inhibitory effects on the developing epiphyseal growth plates of the long bones in a dose and time-dependent manner particularly at TM1 and TM2, with phenobarbital having more detrimental effects than phenytoin. It was therefore recommended that high dosages of the two medicines where possible should be avoided at TM1 and TM2. Further studies with non-human primates were recommended to help come up with findings that will apply to humans.