In vivo Bromodeoxyuridine Incorporation in Normal Rat Liver: Immunohistochemical Detection of Streaming Cells and Measurement of Labelling Indices.

Abstract

The aim of the present study was to determine if normal liver cell kinetics can be evaluated using bromodeoxyuridene (BrdU) as the label. BrdU was injected intraperitoneally into 10 male adult rats, 5 were sacrified after 1 hr, and the remainder after 30 days. The livers were fixed in 70% ethanol, embedded in paraffin and histological slides were prepared.The labelled nuclei were revealed using indirect immunoperoxidase staining and easily counted by light microscopy.One hr after labelling, labelled hepatocytes and littoral cells were detected within 164 ± 12.3 ~tm and 161 ± 13 pin respectively from the portal tract rim. After 30 days, both cell types reached the respective distances of 290 ± 14.6 pm and 294 ± 17.7 pm. Their respective displacement velocities were 4.2 pm/d and 4.3 pm/d. These results were identical to those we obtained using tritiated thymidine.This laboratory method provides a useful and simpler alternative method to tritiated thymidine.Cell kinetics have been traditionally studied by measuring tritiated thymidine (TdR) incorporation into DNA in the S-phase of the cell cycle. The validity of BrdU labelling for cell kinetic studies has been well established in comparison with other methods and it is now regarded as a useful alternative to TdR (13). Most studies using BrdU involve rapidly proliferating tissues. However, BrdU incorporation has also been used successfully in systems exhibiting low levels of proliferation, although such studies are sparse (5,11,12,15) heir results were compatible with measurements made using TdR. On the other hand, DeFazio et al. (5) claimed that normal liver remained unstained after three days of exposure to BrdU.The aim of the present study was to demonstrate that BrdU can serve as a reliable marker for determination of liver cell kinetics in normal mature rats not exposed to any liver insult. The histochemical method used is quick and relatively simple (Darmon et al.,(4) ), and the results are comparable to those we obtained using TdR (1-3, 17). MATERIALS AND METHODSTen male adult rats, random-bred Tel Aviv University strain weighing 250-300 g, were injected intraperitoneally (IP) with 50 mg/kg body weight BrdU (Sigma Chemical Company Ltd, Poole, Dorset) at a concentration of 6 mg/ml in 0.05 M phosphate buffered saline, pH 7.4 (PBS). Five rats were sacrified after one hr, the remaining five rats were fed and watered, ad libitum, for 30 days and then sacrified. The livers, and a short section of the small intestine were immediately removed and fixed in 70% ethanol