Post-mortem changes of prostanoid concentrations in tissues of mice: Impact of fast cervical dislocation and dissection delay

Abstract

Prostanoids are potent lipid mediators involved in a wide variety of physiological functions like blood pressure regulation or inflammation as well as cardiovascular and malign diseases. Elucidation of their modes of action is mainly carried out in pre-clinical animal models by quantifying prostanoids in tissues of interest. Unfortunately, prostanoids are prone to post-mortem artifact formation and de novo synthesis can already be caused by external stimuli during the euthanasia of animals like prolonged hypercapnia or ischemia. Therefore, this study investigates the suitability and impact of fast cervical dislocation for the determination of prostanoids (6-keto-PGF1α, TXB2, PGF2α, PGD2, PGE2) in seven tissues of mice (spinal cord, brain, sciatic nerve, kidney, liver, lung, and spleen) to minimize time-dependent effects and approximate physiological concentrations. Tissues were dissected in a standardized sequence directly or after 10 min to investigate the influence of dissection delays. The enzyme inhibitor indomethacin (10 µM) in combination with low processing temperatures was employed to preserve prostanoid concentrations during sample preparation. Quantification of prostanoids was performed via LC-MS/MS. This study shows, that prostanoids are differentially susceptible to post-mortem artifact formation which is closely connected to their physiological function and metabolic stability in the respective tissues. Prostanoids in the brain, spinal cord, and kidney that are not involved in the regulatory response post-mortem, i.e. blood flow regulation (6-keto-PGF1α, PGE2, PGF2α) showed high reproducibility even after dissection delay and could be assessed after fast cervical dislocation if prerequisites like standardized pre-analytical workflows with immediate dissection and inhibition of residual enzymatic activity are in place. However, in tissues with high metabolic activity (liver, lung) more stable prostanoid metabolites should be used. Moreover, prostanoids in the spleen were strongly affected by dissection delays and presumably the method of euthanasia itself.