Effects of Hemorrhagic Shock and Fraction of Inspired Oxygen on Hydrogen Peroxide and Apoptosis in Rat Lung and Diaphragm

Abstract Hemorrhagic shock (HS) is the single most common cause of death in civilian and military personnel experiencing trauma (Alam & Rhee, 2007). Immediate resuscitation for HS can involve the administration of supplemental oxygen (O2) above ambient levels (0.21) using a non-rebreather face mask and high flow O2 of 10-15 L/min (Trauma, 2008). With supplementary O2, reactive oxygen species (ROS) may be increased leading to oxidative damage to deoxyribonucleic acid (DNA), proteins and lipid membranes (Rushing & Britt, 2008). During hyperoxia, ROS can cause a secondary oxidative injury to cells and tissues especially in the lungs and diaphragm. The optimal amount of O2 to be administered following HS is not clearly defined. The purpose of this dissertation was to investigate the optimal fraction of inspired oxygen (FIO2) to be administered following HS by determining the amount of hydrogen peroxide (H2O2) and apoptosis in the lungs and diaphragm. Previous animal studies have demonstrated that dopamine (DA) can scavenge free radicals or decrease ROS by increasing blood flow and can decrease apoptosis by activating β-2 adrenoreceptors (Communal, Singh, Sawyer, & Colucci, 1999; Patterson et al., 2004; J. D. Pierce, Goodyear-Bruch, Hall, & Clancy, 2006; J. D. Pierce, Goodyear-Bruch, Hall, Reed, & Clancy, 2008) Therefore, we conducted additional experiments to determine if DA with various FIO2s following HS reduces apoptosis in these tissues. Adult male Sprague-Dawley rats (n = 112) were anesthetized; a tracheostomy was performed and catheters were inserted in the carotid and femoral arteries. HS was elicited by withdrawing 40% of the rat's blood volume over 30 minutes. This was followed by the rat breathing one of the following FIO2 concentrations (0.21, 0.40, 0.60, and 1.00) without and with concurrent DA (10 mcg/kg/min) for 60 minutes. The animal was euthanized and the lungs and diaphragm excised and prepared for measurement of H2O2 and nuclear DNA damage (apoptosis). Hydrogen peroxide was quantified using dihydrofluorescein diacetate (Hfluor-DA) and laser scanning cytometry. Percent apoptosis was determined using differential dye up-take and fluorescent microscopy. The amount of lung and diaphragm H2O2 and percent apoptosis were greatest in the 0.21 and 1.00 FIO2 concentrations and the least amounts were observed in rats when using 0.40. Infusing DA significantly decreased H2O2 and apoptosis in both tissues at all FIO2's except 0.40. The lack of difference in rats receiving 0.40 with DA was because of the already reduced H2O2 and apoptosis values. In conclusion, an FIO2 of 0.40 was optimal for attenuation of lung and diaphragm H2O2 and apoptosis following HS. When greater FIO2s are necessary, adding DA to the resuscitation regimen may diminish ROS-induced cellular injury