The Effects of Methotrexate on the Development of Neural Tube Defects in the Chick Embryo^#


Abstract: During chick development, one of the earliest differentiated tissues is the neural tube. After 24 h of incubation, a chick egg starts to differentiate and 30-48 h after incubation the neural plate is closed from head to tail to form the neural tube. If factors controlling the neural tube's closing are disrupted, this consequently causes neural tube closure defects during this time. In this study, the effect of methotrexate on the developing neural tube was investigated during early chick development. For this research, 40 specific pathogen free (SPF) white Leghorn type chick embryos were used. They were incubated for 30 h at 37.8 ± 2 °C. Methotrexate, which inhibits the dihydrofolate reductase enzyme by a competitive mechanism, was injected within therapeutic dosage limits (10 mg/m^2, 20 mg/m^2, 40 mg/m^2) in ovo. Ten eggs were injected with 0.9% NaCl and used as a control group. All groups, after the injection, were incubated for 48 and 72 h. They were then dissected and the embryos were fixed in 10% (v/v) formalin for 2 h. The embryos were embedded in paraffin wax and 5 \mu serial sections were taken. Sections were stained with haematoxylin and then observed under light microscopy. While 20 mg/m^2 or 40 mg/m^2 methotrexate embryos were not alive when they were opened at 48 h incubation, 10 mg/m^2 methotrexate embryos maintained normal development after 48 and 72 h incubation. However, there was developmental retardation in the methotrexate injected group when compared with the control group with development of the brain being retarded; the volume of brain vesicles was lower than in the control group. Our results suggested that methotrexate, an antimetabolite of folic acid, caused neural tube closure defects when injected at therapeutic dosage levels. Folic acid is essential for normal development of the nervous system; therefore, folate antagonists might be more harmful to the central nervous system than to other parts of the developing body.

Keywords: Methotrexate, early chick embryo, nervous system

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