Histochemical determination of glycosaminoglycans (GAGs)during normal and alochol-induced neural tube development

Neural tube defects (NTD) are a group of malformations that result from the failure ofthe neural tube to close early in embryonic development and among the most commoncongenital malformations in humans. NTD are associated with a number of other centralnervous system (CNS) and non-neural malformations. Proteoglycans (PG) that are common inthe extracelular matrix (ECM), are formed a core protein and related to carbohydrate chains,which that is termed glycosaminoglycan (GAG). GAG formation is important for the NTdevelopment. Prenatal ethanol exposure produces NTD and growth retardation in experimentalanimals. Alcohol is known to induce folate deficiency and produce oxidative stress. Therefore,we investigated the effect of moderate alcohol exposure on the GAGs during development NTby critical electrolyte concentration (CEC) principle which allows GAG to be properly preservedand visualized.In this study, SPF chicken eggs that were obtained from Manisa Chicken Diseases andvaccine Production Center (Veterinary Control and Research Institute Administration) wereused. Eggs were divided into two groups, control and expose, and incubated. 33. hours ofincubation (HH-stage-9-10), eggs were injected with 100 µL % 10 ethanol. The control goupwas injected serum physiologic. Ethanol injected eggs were opened after exposition 24 and 48hours later and embryos were fixed in Saint-Marie?s solution. The control group was openedand fixed at the same time with injected embryos. A part of total embryos were photographedto determine difference between control and exposed group. Haematoxylin- Eosine (HE)stained slides were studied to observe general structure of NT. Entity, distribution andalterations of GAGs were examined histochemically by stainig Alcian-Blue 8 GX within CriticalElectrolyte Concentrations and Periodic acid- Schiff (PAS) staining with light microscope.Ectoderm cells in the midline became columnar epithelia by elongation at induction ofnotocord. The nuclei of the cells were located in the basal. After neural plate formation,neuroepithelia formed neural folds by performing elevator-like movement. The NT formed byconjuction of neural folds.Staining of KEK showed that basal layers were densely stained and the dominant GAGalteration was hyaluronic acid (HA). With the progress of development, amount of GAGs inalcohol group were reduced compared to those of control group. In addition, all sulphated GAGs(sGAG) including chondroitin sulphate (Ch-S) and dermatan sulphate (DS) particularly in theneural basal layer, Ch-S in the basal layer of notocord and Ch-S with DS in the lumen face ofNT including migrating cells getting away from NT were increased compared to those of at otherregions. Application of %10 alcohol produced more HA in the basal layer of NT and moreglycoprotein (GP) in the lumen face of NT. Increasing alcohol by application of %15 producedless HA and more DS and Ch-S in the basal layer of NT and more DS and Ch-S in the lumenface of NT, more heparin (HP) and heparan sulphate (HS) in the migrating cells getting awayfrom NT. Application of %20 alcohol produced more HP and HS, and siginificantly less Ch-S inthe basal layer of notochord, more HA in the basal layer of NT and clearly less even none GAGin the lumen face of NT.Alterations of GAG during development of NT play important role for normal productionof brain and medulla spinalis. Moderate exposure of alcohol during NT formation which mimicsthe effect of alcohol on the embryo in pregnant women, caused decrease and topographicalalterations of GAG. These results require further investigation of these effects on the laterstages of embryos which may be of significance in the etiology of NTD.Key words: Neural tube development, ethanol, glycosaminoglycans, chick.