Studies of the kinetics of in vivo labelling of proliferating wool follicle bulb cells with 5-Bromo-2′-deoxyuridine (BrdU): Intracutaneous labelling with BrdU and pharmacokinetics of free BrdU in the skin tissue of sheep

Abstract

After establishing an immunocytochemical technique for the identification of BrdU labeled S-phase cells in the follicle bulb of wool fibres, the usefulness of a facilitated intracutaneous injection method for BrdU was investigated. This method enables easy and quick administration of the labelling agent and thus permits in vivo examination of cellular processes in the wool follicle bulb under field conditions. Because of very low dose rates, the local i.c. injection decreases the risk of toxic side effects of BrdU in the skin and more generally in the whole body, that accompany intravenous infusion. The accumulation of BrdU labelled cells over time in the follicle bulb was measured with immunocytochemical detection techniques. Intracutaneously injected BrdU was incorporated immediately into nuclear DNA of proliferating cells, and the labelling effect was confined to an area within 20 mm from the injection site. This allows sequential injections to be made on the same midside area of the sheep without interference of adjacent BrdU pools. Labelled S-phase nuclei accumulated in a linear fashion from 5 min to 24 h. Intradermally administered BrdU has a slow tissue clearance rate, with levels up to 10% of the initial peak still present after 24 h. The BrdU labelling effect showed detectable immunoreactivity at total injection loads as low as 5 8g. The free pool clearance rate in association with strong immunoreactivity suggests that a single intradermal injection can be regarded as comparable to a continuous labelling technique, if cell cycle parameters and growth fractions are to be determined. The method of administering BrdU intradermally opens a new perspective for cell population studies of wool follicles. It allows serial as well as concurrent estimates of cell activity to be made in the same animal, while eliminating difficulties and potentially toxic side effects associated with whole body infusion techniques.