A method of culturing coronary artery explants for measuring vascular smooth muscle proliferation in rainbow trout

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Abstract
A standardized method of in vitro [3H]thymidine incorporation during the S phase of cell division was developed to study vascular smooth muscle proliferation in the coronary artery of rainbow trout (Oncorhynchus mykiss). We established a reliable medium system, an optimum [3H]thymidine dose, and optimum incubation conditions. Incorporation of the radiolabel into vascular smooth muscle nuclei was confirmed with autoradiography. To test the sensitivity of the assay system, the coronary artery was surgically exposed and gently rubbed in anesthetized rainbow trout. Following either a 1-, 2-, or 3-day recovery period, fish were sacrificed and the coronary artery explants were cultured with [3H]thymidine. Gentle rubbing of the coronary artery in vivo resulted in a significant increase in [3H]thymidine incorporation into the coronary artery explant compared with sham-operated and untreated control groups of fish. Peak incorporation of [3H]thymidine occurred at day 2 in the treated group, when incorporation was three times that in the sham-operated group. This technique has potential application in the study of coronary arteriosclerosis in salmonids, where vascular smooth muscle proliferation is a primary event.

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