H.Seda VATANSEVER, Mehmet SELÇUKİ, V.Sevinç İNAN, A.Şükrü UMUR

The effects of methotrexate on the development of neural tube defects in the chick $embryo^sharp$

Tavuk gelişiminde en erken farklılanan doku nöral tüptür. Tavuk yumurtasının 24 saatlik inkübasyonundan sonra, farklılaşma başlar ve 30-48 saatlik inkübasyondan sonra nöral plak nöral tüpü oluşturmak üzere baştan kuyruğa doğru kapanır. Bu dönemde, eğer nöral tüp kapanmasını kontrol eden faktörler etkilendiğinde, nöral tüp kapanma kusurlarına neden olur. Bu çalışmada, erken tavuk embriyosu gelişimi sırasında metotreksat'ın nöral tüp gelişimi üzerindeki etkilerinin incelenmesi amaçlanmıştır. Bu nedenle, 40 patojen içermeyen (SPF) beyaz Leghorn tipi tavuk embriyosu kullanıldı. Embriyolar 37,8 ± 2 °C'de 30 saat inkübe edildi. Kompetitif mekanizma ile dihidrofolat redüktaz enzimini inhibe eden metotreksat, terapötik dozlarda (10 mg/m2, 20 mg/m2, 40 mg/m2) in ovo olarak enjekte edildi. Yumurtaların 10 tanesine ise kontrol grubu olarak % 0,9 NaCl enjekte edildi. Tüm gruplar, enjeksiyondan sonra 48 ve 72 saat inkübe edildi. Daha sonra yumurtalar açıldı ve embriyolar % 10'luk formalin solüsyonu içinde 2 saat tespit edildi. Embriyolar parafin bloklar içinde gömüldü ve 5 u seri kesitler alındı. Kesitler hematoksilen ile boyandıktan sonra ışık mikroskobu altında değerlendirildi. 20 mg/m2 veya 40 mg/m2 metotreksat verilen embriyolar 48 saatlik inkübasyon sonrası açıldığında yaşamaz iken, 10 mg/m2 metotreksat verilen embriyoların 48 ve 72 saatlik inkübasyondan sonra normal gelişimlerine devam ettikleri gözlendi. Bununla beraber, kontrol grubu ile karşılaştırıldığında, metotreksat enjekte edilen embriyolarda gelişme geriliği olduğu, aynı zamanda beyinin gelişmesinde de gerileme olduğu ve beyin veziküllerinin hacimlerinin, kontrol grubuna oranla azaldığı görüldü. Araştırma sonuçları, bir folik asit antimetaboliti olan metotreksatın, terapötik dozlarda enjeksiyonu sonucunda nöral tüp kapanma defektine neden olduğunu desteklemektedir. Folik asit sinir sistemi gelişimi için esastır, bu yüzden folat antagonistleri de santral sinir sistemi üzerinde diğer gelişen organlara oranla daha fazla zararlı etkilere sahip olabilir.

Tavuk embiryosunda gelişen nöral tüp defekti üzerine metotreksat'ın etkileri

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 $circ$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 m 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.

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