Circadian influence on the metaphase arrest technique in mouse cornea (light microscopy study) and rectum (flow cytometry study)

Abstract

Vincristine (VC) was used to arrest metaphases in the epithelium of the cornea and rectum. The cornea was studied by light microscopy (mitotic index). The rectum was analyzed by flow cytometry (%G2 + M). The results of a dose range finding study showed that 3.0 mg/kg body weight VC did not increase the number of metaphases in the cornea or the %G2 + M for the rectum over that recorded for a dose of 1.0 mg/kg VC. Therefore 1.0 mg/kg VC was selected as the dose to use. Mice were kept on a 12:12 light-dark cycle with light from 0600 to 1800. Mice were treated with 1.0 mg/kg VC at either 0200 or 1400. 0200 is a time when the native mitotic index is increasing toward a peak level, which is attained at 0800-1000 under these conditions. 1400 is a time when the mitotic index is decreasing to trough level, which is attained at 2000 to 2200. In one experiment, subgroups of mice were killed at +3 (0500 or 1700) and +6 (0800 or 2000) hours after VC. In a replicate experiment subgroups were killed at +2 (0400 or 1600), +4 (0600 or 1800), and +6 (0800 or 2000) hours after VC. The data from the cornea (mitotic indices) and the rectum (%G2 + M) were graphed against time. For both organs the slope of the metaphase accumulation line was different when VC at 0200 data were compared to VC at 1400 data. The conclusion reached was that by changing the biological time of the experiment one can significantly change the slope of the line obtained with the stathmokinetic technique.