Effect Of Caffeine On The Neural Development Of Chicken Embryo

Objectives:Clinical research had proved that high caffeine intake during gestation could affect normal development of fetus, but its mechanism had not yet been confirmed. The current experiment used developing chicken embryo model to investigate the effect of caffeine on neural development, and to reveal the mechanism of caffeine interference during fetal development. The purpose is to provide reasonable scientific theory and reduce the blemished new-borns and increase population quality.Methods:(1) Morphological changes of neural tube development after exposure of 0.5 mg,1.0 mg,1.5 mg caffeine on 1.5 d chicken embryo were observed using carmine staining and immunohistochemical staining. (2) Contents of caffeine in brains of 8 d chicken embryos during different developing stages were measured using HPLC after injection of 0.5 mg,1.0 mg,1.5 mg caffeine respectively. (3) Single or multiple injections of caffeine were given to 8d chicken embryo and monoamine neurotransmitters content in brain was measured on 17 d using HPLC. (4) Gene expressions of neurotransmitters interferer related enzymes were measured by RT-PCR.Results:(1) Result of carmine staining showed that neural tube closure defection and irregular cell arrangement were found in all caffeine groups. Result of IHC showed that 1.0 mg caffeine induced uneven distribution of neural crest cell, and 1.5 mg caffeine could inhibit the immigration of neural crest cell. (2) After injesting different doeses of caffeine to 8d embryo, the content of caffeine in embryo brain were increasing gradually, and existed mainly in the form of prototype. (3) Caffeine (1.0 mg and 1.5 mg) significantly increased contents of 5-HT related monoamine neurotransmitters (P＜0.05). (4) Result of RT-PCR revealed that caffeine (1.0 mg and 1.5 mg) significantly enhanced the expression of neurotransmitters interferer related gene, such is TPH2 gene (P＜0.05).Conclution:(1) Caffeine (0.5 mg) could induce neural development defection in early chicken embryo, and high dose of caffeine could even affect the distribution and immigration of neural crest cell. (2) Reason for embryotoxicity by caffeine maybe due to the high concentration and its accumulation of caffeine in the brain of embryo. (3) Medium and high dose of caffeine could significantly elevate contents of monoamine neurotransmitters, and was related to the frequency of interference. (4) The mechanism of the effect of caffeine on monoamine transmitters should be related to the enhanced the level of TPH2 mRNA expression.