Experimental Study on the Effects of Cabarmazepine on the Neurulation in Early Chick Embryos and Immunohistochemical Staining for Fibronectin

Lee, Mou Seop; Kim, Dong Ho; Han, Dae Hee

Abstract

Teratogenic effects of carbamazepine, an anticonvulsant, on the neurulation of the explanted early chick embryos were studied utilizing the punched-out filter paper explantation and culture technique. Fresh fertilized white leghorn hen eggs were incubated for 20-30 hours in an egg incubator. The Hamburger and Hamilton stage 4-11 chick embryos were explanted using the punched-out filter paper explantation technique and cultured in the CO2 cell culture incubator for 6-10 hours. They were randomly divided into a control group and an experimental group. The experimental group was divided into five subgroups according to the carbamazepine concentrations of 20micrometer 40micrometer 100micrometer 200micrometer 400micrometer with which the Ham's F-10 culture media were treated. The morphological characteristics and the incidences of teratogenic effects on the neurulation of early chick embryos in the control and experimental groups were compared with each other using the stereomicroscope and the electron microscope. The chick embryos of the same developmental stage were selected from the control and experimental groups, and immunohistochemical staining for fibronectin was done by the double-bridge PAP method. The results were as follows. 1) Of the 41 chick embryos cultured in the Ham's F-10 media without carbamazepine, 38 embryos(92.7%) developed normally, and 3 embryos(7.3%) developed abnormally. In contrast, among the 98 embryos cultured in the carbamazepine-treated media, 54 embryos(55.1%) developed abnormally. The frequent anomalous features were deformities of the neural folds, failure of neural tube closure, derangement of somites, and developmental arrest. 2) The frequency and severity of abnormal embryos increased in dose-dependent fashion. The embryos cultured in the media treated each with 20micrometer 40micrometer 100micrometer 200micrometer 400micrometer of carbamazepine developed abnormally in 12.5%, 21.1%, 60.0%, 81.0%, 86.4% respectively. 3) The scanning electron microscopic findings in neuroepithelial cells of abnormally developed embryos were flattened and smooth cellular surface with diminished surface blebs and microvilli, and size irregularity of the cells. On transmission electron microscope, underdevelopment of intracellular microfilaments was seen, but there was no significant change in the intracellular organelle. 4) The immunohistochemical stainability of the extracellular fibronectin at the basal side of the neuroepithelium was decreased in the carbamazepine-treated embryos.