Methods for study of rat adrenal function were adapted and optimized from several previously published reports.26 (link)–28 (link) Immediately following weighing and IV catheter placement, dexamethasone (0.2 mg/kg IV; American Regent, Shirley, NY) was administered to each rat to inhibit endogenous adrenocorticotropic hormone (ACTH) release, to suppress baseline corticosterone production, and to inhibit the variable stress response to restraint and handling. The IV tail vein catheter, used for both drug administration and blood draws, was heparin-locked after each use with 10 U/ml heparin to maintain patency; the heparin locking solution was “wicked” out of the catheter prior to drug administration and blood draws to minimize rat and sample heparinization. All blood draws were approximately 0.3 mls in volume. All drugs administrations were followed by a 1 ml normal saline flush to assure complete drug delivery.
Two hours following dexamethasone treatment, blood was drawn (for baseline measurement of serum corticosterone concentration) and a second dose of dexamethasome (0.2 mg/kg) was administered along with either IV MOC-etomidate, etomidate, propofol, or vehicle (35% propylene glycol v/v in water) as a control. Fifteen minutes later, ACTH1–24 (25 μg/kg; Sigma-Aldrich Chemical Co, St. Louis, MO) was given intravenously to stimulate corticosterone production. Fifteen minutes after ACTH1–24 administration (i.e., 30 min after drug or vehicle administration), a second blood sample was drawn to measure the ACTH1–24-stimulated serum corticosterone concentration. ACTH1–24 was dissolved in 1 mg/ml in deoxygenated water as stock, aliquoted, and frozen (−20 °C); a fresh aliquot was thawed just prior to each use. Rats in all three groups (vehicle, etomidate, and MOC-etomidate) received the same volume of propylene glycol.
Blood samples were allowed to clot at room temperature (10 to 60 min) before centrifugation at 3500g for 5 min. Serum was carefully expressed from any resulting superficial fibrin clot using a clean pipette tip prior to a second centrifugation at 3500g for 5 min. Following the second centrifugation, the resultant straw colored, clot-free serum layer was transferred to a fresh vial for a final, high-speed centrifugation (16000g, for 5 min) to pellet any contaminating red blood cells or particulates. The serum was transferred to a clean vial and promptly frozen (−20 °C) pending corticosterone measurement within 1 to 2 days. Following thawing and heat inactivation of corticosterone binding globulins (65 °C for 20 min), serum baseline and ACTH1–24 stimulated corticosterone concentrations were quantified using an Enzyme-Linked ImmunoSorbent Assay (ELISA) (Diagnostic Systems Laboratories, Webster, TX) and a 96-well plate reader (Molecular Devices, Sunnyvale, CA).
Two hours following dexamethasone treatment, blood was drawn (for baseline measurement of serum corticosterone concentration) and a second dose of dexamethasome (0.2 mg/kg) was administered along with either IV MOC-etomidate, etomidate, propofol, or vehicle (35% propylene glycol v/v in water) as a control. Fifteen minutes later, ACTH1–24 (25 μg/kg; Sigma-Aldrich Chemical Co, St. Louis, MO) was given intravenously to stimulate corticosterone production. Fifteen minutes after ACTH1–24 administration (i.e., 30 min after drug or vehicle administration), a second blood sample was drawn to measure the ACTH1–24-stimulated serum corticosterone concentration. ACTH1–24 was dissolved in 1 mg/ml in deoxygenated water as stock, aliquoted, and frozen (−20 °C); a fresh aliquot was thawed just prior to each use. Rats in all three groups (vehicle, etomidate, and MOC-etomidate) received the same volume of propylene glycol.
Blood samples were allowed to clot at room temperature (10 to 60 min) before centrifugation at 3500g for 5 min. Serum was carefully expressed from any resulting superficial fibrin clot using a clean pipette tip prior to a second centrifugation at 3500g for 5 min. Following the second centrifugation, the resultant straw colored, clot-free serum layer was transferred to a fresh vial for a final, high-speed centrifugation (16000g, for 5 min) to pellet any contaminating red blood cells or particulates. The serum was transferred to a clean vial and promptly frozen (−20 °C) pending corticosterone measurement within 1 to 2 days. Following thawing and heat inactivation of corticosterone binding globulins (65 °C for 20 min), serum baseline and ACTH1–24 stimulated corticosterone concentrations were quantified using an Enzyme-Linked ImmunoSorbent Assay (ELISA) (Diagnostic Systems Laboratories, Webster, TX) and a 96-well plate reader (Molecular Devices, Sunnyvale, CA).
