Effect of cannulation surgery and restraint stress on the plasma corticosterone concentration in the rat: application of an improved corticosterone HPLC assay.

Purpose: Stress increases plasma corticosterone concentration in rats. We studied the time-course of plasma corticosterone post jugular vein cannulation, a potential stress producing process, and after 1 h restraint stress using an improved assay. Current HPLC methods for corticosterone analysis require long extraction times with large volumes of solvent.

Methods: Adult male, Sprague-Dawley rats were used. Tail vein blood samples were collected from uncannulated rats (n=4). Other rats were cannulated in the right jugular vein, allowed to recover overnight and assigned to days 1, 2, 3 or 4 groups (n=4-6/group). Blood was sampled before and after exposure to 1 h of restraint stress. Plasma was separated and extracted with 5 mL ethyl acetate (betamethasone as internal standard), and then the extract was washed with sodium hydroxide (0.1 M) and water. After evaporation of ethyl acetate, the residue was dissolved in mobile phase (acetonitrile-water-acetic acid-TEA, 22:78:0.1:0.03, v/v) and injected into an isocratic HPLC consisting of a 10 cm C18 column, and UV detector at 254 nm.

Results: The minimum quantifiable concentration was 10 ng/mL of corticosterone based on 0.5 mL plasma. The standard curve was linear over the concentration range of 10-500 ng/mL. The extraction efficiency was 84-87%. The coefficient of variation for intra-day and inter-day precision was <10% with <8% error. Plasma corticosterone before cannulation (74 +/- 17 ng/mL) and those measured daily before restraint stress were not significantly different from one day to another (day 1, 60 +/- 21; day 2, 59 +/- 39, day 3, 45 +/- 23, and day 4, 41 +/- 8 ng/mL). Exposure to restraint stress elevated corticosterone (day 1, 122 +/- 33; day 2, 82 +/- 23; day 3, 148 +/- 33; and day 4, 134 +/- 20 ng/mL). Except on day 2, all concentrations were significantly elevated as compared to the pre-stress levels.

Conclusions: Corticosterone concentrations were stable after jugular vein cannulation, and were increased by restraint stress. The present assay is a more convenient method as compared to those previously reported.