Influence of morphine on cytokine production in horses stimulated with LPS in vitro and in vivo

Abstract

Systemic inflammatory response syndrome (SIRS) is an exaggerated inflammatory condition, triggered, among other causes, by the release of endotoxins into the circulation and is related to organ failure and death. Causes of endotoxemia that result in SIRS in horses include pleuropneumonia, acute abdomen, endometritis, and neonatal sepsis. In order to avoid the worst outcome, besides treating the causal factor, it is necessary to control the production of cytokines and inflammatory mediators. Due to its immunomodulatory properties observed in humans, mice and equine synoviocytes, morphine is a drug with therapeutic potential for SIRS in horses affected by endotoxemia. The aim of this study is to verify the immunomodulatory effects of morphine on the production of cytokines by immune cells of horses stimulated with lipopolysaccharide (LPS) in vitro and in vivo. For this, the study will be divided into two stages. The first will be conducted in vitro, with the selection of eight animals, aged between 2 and 4 years, healthy and carrying no history of diseases caused by gram-negative bacteria. From each of these animals, 40 mL of blood will be collected through jugular vein puncture. The leukocyte portion will be separated and deposited in culture plates containing the following treatments: medium (negative control); LPS 200 ng/mL (positive control); RPMI medium + 300 µM morphine; LPS 200 ng/mL + morphine 300 µM + medium; LPS 200 ng/mL + morphine 100 ¼M + medium; LPS 200 ng/mL + morphine 30 ¼M + medium. The samples will be kept in a 5% CO2/37ºC incubator and the supernatant collected at 60, 90 and 180 minutes for analysis of cytokines TNF-±, IL-1², IL-6 and IL-10 by ELISA. The second stage will be conducted in vivo, in which 16 healthy horses carrying no history of diseases by gram-negative bacteria will be selected. Each subject will receive 100 ng/kg of LPS diluted in 250 mL of 0.9% NaCl solution, administered intravenously (IV) in 15 minutes. After the induction of endotoxemia with LPS, the animals will be randomly divided into two groups: Morphine (M), which will receive 0.1 mg/kg of IV morphine 15 minutes after the infusion of LPS, and Saline (C), receiving the equivalent volume of 0.9% NaCl at the same time. For analysis of blood count, fibrinogen and measurement of cytokines by the ELISA method, samples will be collected before T0 treatments (baseline) and after treatments at 15 minutes, 1, 2, 3, 4, 5 and 6 hours. We hope that this study will clarify whether the immunomodulatory properties of morphine are present in the equine species and whether the drug may be useful as an adjuvant in the treatment of animals suffering from endotoxemia.