engleski

Differentiation of neural tissue in rat embryos cultivated in vitro

In the organ culture of gastrulating rat embryos without extraembryonic parts it is possible to investigate various signals required for normal or impaired development (Bulić-Jakuš et al., 1990 ; Škreb et al., 1993 ; Vlahović et al., 1993 ; Strahinić et al., 1996). The aim of the study presented here was to evaluate the degree of neural tissue differentiation in cultivated rat embryos with electron microscopy and immunohistochemistry. Fisher rat embryos 9, 5 days old were microsurgically isolated and cultivated for 14 days on a metal grid in Eagle's Minimal Essential Medium with 50% rat serum. Resulting teratoma-like eksplants were fixed in 4% glutaraldehyde, postfixed in 1% OsO4, embedded in Durcopan, sectioned for transmission electron microscopy (TEM) and analysed with Zeiss 902 A microscope. Some of the explants were fixed in St. Marie's solution, embedded in paraffin, serially sectioned and processed for immunohistochemistry. Primary antibody against Proliferating Cell Nuclear Antigen ( anti-PCNA, DAKO) and LSAB2 peroxidase kit (DAKO) for detection of the antigene-antibody complex were used. After two-weeks of culture, among differentiated tissues in embryonic explants, features typical for neural tissue were discovered with TEM : neuropil and myelinization in the brain. A strong intranuclear PCNA signal was also discovered in some cells which indicated that they were still proliferating. It was concluded that after cultivation of gastrulating rat embryos, terminal differentiation of brain cells has occurred although some cells were still retained in the cycling compartement.

neural tissue; rat embryo culture

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