Date of Award

7-22-2011

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Integrative Physiology

Department

Graduate School of Biomedical Sciences

First Advisor

Robert T. Mallet

Second Advisor

Albert Olivencia-Yurvati

Third Advisor

Peter Raven

Abstract

Gurji, HA. Pyruvate-Enriched Ringer’s Solution Protects Hindlimb and Myocardial Tissue During Hemorrhagic Shock and Hindlimb Ischemia. Doctor of Philosophy (Integrative Physiology), July 22, 2011, 111 pp, 1 table, 23 figures, 209 references, 142 titles. Copious blood loss is the leading cause of death in military combat. Extreme exsanguination following traumatic injury causes hypotension which may culminate in hemorrhagic shock, multiple open organ failure, and death. Currently, the only available strategy to treat hemorrhage is to apply tourniquets and administer resuscitative fluids. Although necessary to limit blood loss, protracted tourniquet application imposes ischemia on distal tissues. Revascularization of the injured limb reintroduces oxygenated blood into the ischemic zone, forming toxic reactive oxygen species. These highly reactive compounds can inactivate key metabolic enzymes, hamper ATP production, and cause end organ dysfunction. Fluid resuscitation provides crucial hemodynamic support, and affords an opportunity to treat the deleterious effects of hemorrhagic shock and ischemia-reperfusion. In order to mitigate the harmful effects of hemorrhagic shock and ischemia-reperfusion of tourniqueted extremities, a fluid resuscitant should contain agents capable of suppressing the formation of reactive oxygen and nitrogen species, thus protecting cellular metabolic function; stabilizing tissue energetics; and safeguarding end organic function. Pyruvate, an endogenous energy substrate, possesses strong antioxidative properties. This study tested whether substituting pyruvate for lactate in a Ringer’s solution would be effective at mitigating reactive oxygen species formation, protect key ATP-generating and ATP-shuttling enzymes from inactivation, bolster skeletal and cardiac muscle phosphorylation potentials, and stabilize cardiac electrical function in goats subjected to hemorrhagic shock and hindlimb ischemia-reperfusion. Isoflurane-anesthetize goats were subjected to a controlled hemorrhaged to reduce the mean arterial pressure to c. 50 mmHg. After reaching this target pressure, hindlimb ischemia (HLI) was imposed for a total of 90 min by femoral artery crossclamp and tourniquet application around the hindlimb. After 30 min of hindlimb ischemia, pyruvate- (PR) or lactate- enriched (LR) Ringer’s solution was infused intravenously (10mL/min) for 90 min. Time control (TC) goats were neither hemorrhaged nor subjected to hindlimb ischemia. At the conclusion of- and 3.5 h after- fluid resuscitation, the left ventricle and the right gastrocnemius were biopsied and flash-frozen for biochemical analysis of metabolites, enzymes, and markers of oxidative stress. In addition, custom-written software was developed to analyze QT interval variability- a marker of electrical instability- from the lead II electrocardiogram. The first phase of this project tested the hypothesis that resuscitation with PR vs. LR effectively protects cardiac metabolism and preserves cardiac electrical performance during hemorrhagic shock and hindlimb ischemia. Resuscitation with PR effectively suppressed the formation of myocardial tissue 8-isoprostane vs. goats resuscitated with LR during the acute and subacute phases of the protocol. In addition, myocardial creatine kinase (CK) activity fell after LR administration vs. TC; however, PR preserved CK activity better than LR during fluid resuscitation and 4 h after hindlimb ischemia reperfusion. PR administration augmented myocardial phosphocreatine phosphorylation potential during fluid administration and 3.5 h later to values significantly higher than those in LR-resuscitated goats. Pro-arrhythmic QTc variability was markedly increased in LR vs. PR and TC during both phases of the protocol. The second phase of this project tested the hypothesis that resuscitation with PR preserves tissue energetics in the reperfused gastrocnemius during hemorrhagic shock and hindlimb ischemia. Resuscitation with PR vs. LR effectively protected the gastrocnemius from oxidative stress in both protocols, as evidenced by the suppression of 8-isoprostane formation. PR prevented CK and aconitase inactivation vs. LR during the acute phase of reperfusion, and this enzyme protection persisted at least 3.5 h after completing fluid resuscitation. Additionally, PR augmented muscle phosphocreatine phosphorylation potential vs. TC and LR during the acute phase of reperfusion, and, like CK and aconitase activities, this augmented energy state persisted 3.5 h after the end of fluid resuscitation. We conclude that 1) Pyruvate Ringer’s resuscitation during hemorrhagic shock and hindlimb ischemia provides antioxidative protection in skeletal and cardiac muscle during fluid resuscitation; 2) Pyruvate-fortified fluid resuscitation prevents inactivation of enzymes involved in production and shuttling of ATP; 3) PR augments cardiac and muscle phosphorylation potentials during fluid resuscitation; and 4) Resuscitation with PR effectively protects cardiac electrical rhythm in the face of hemorrhagic shock and hindlimb ischemia. These investigations demonstrate the powerful antioxidative protection imposed by pyruvate, its positive effects on muscle and cardiac metabolism and energy state and its role in stabilizing cardiac electrical function during hemorrhagic shock and hindlimb ischemia.

Comments

Gurji, HA. Pyruvate-Enriched Ringer’s Solution Protects Hindlimb and Myocardial Tissue During Hemorrhagic Shock and Hindlimb Ischemia. Doctor of Philosophy (Integrative Physiology), July 22, 2011, 111 pp, 1 table, 23 figures, 209 references, 142 titles.

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