engleski

Changes induced by heat shock in in vitro culture of gastrulating mammalian embryo- application of FTIR and RAMAN spectroscopy

An original in vitro organ culture model of the rat gastrulating embryo-proper was used to investigate the teratogenic effects of a severe heat shock. Additionally, FTIR and RAMAN spectroscopy were applied to detect possible changes in composition of the culture medium. Embryonic parts of 9, 5-days-old rat embryos were cultivated for two weeks at the air-liquid interface in serum-supplemented Eagle&#8217; s MEM (50%). Heat shock (430C) was applied for 24 hours and embryos were transferred to 370C. Controls have spent the 14-day culture period at 370C. Culture medium was changed every other day. Culture medium as well as culture conditioned medium was frozen at minus 200C. Two diameters of explants were measured several times by an ocular micrometer and compared by t-test. FTIR and FT-Raman spectroscopy were done on defrosted and dyalized culture media. The infrared spectra in transmission mode were recorded using Perkin Elmer GX spectrometer and Raman spectra with Raman modul of same spectrometer. The changes in spectra of heat shocked and control group were followed in region of 1800 cm-1 to 600 cm-1 which is the region of active vibrations of biological macromolecules. The diameters of heat shocked embryos were always significantly smaller than in controls (p< 0, 01) which showed that the growth of embryos was impaired by severe heat shock. FTIR and RAMAN spectroscopy showed comparable results. It seems that either of these complementary spectroscopical methods may serve as a suitable method for a quick evaluation of the impact of extraneous factors on a complex biological system.

Mammalian embryo culture; heat shock; FTIR; RAMAN

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