Date of Award

5-2009

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Integrative Physiology

Department

Graduate School of Biomedical Sciences

First Advisor

Dr. Robert T. Mallet

Abstract

Traumatic blood loss often necessitates the use of resuscitative fluids to replenish blood volume and stabilize blood pressure. The use of tourniquets to achieve hemostasis imposes ischemia-reperfusion on wounded limbs after release. Resuscitation with the physiological antioxidant and natural intermediary metabolite pyruvate may abrogate reperfusion injury of muscle by scavenging oxyradicals and stabilizing cytoprotective proteins. This study was designed to determine the effects of pyruvate in the setting of hemorrhagic shock with resuscitation and hindlimb ischemia-reperfusion. All experiments were conducted on isoflurane-anesthetized male goats. A controlled hemorrhage was performed to lower mean arterial pressure (MAP) to c. 50 mmHg, then the right femoral artery and vein were occluded for 90 min. Lactate Ringer’s (LR) or pyruvate Ringer’s (PR) was infused intravenously (10 ml/min) for 90 min, from 30 min occlusion until 30 min after reperfusion. At 4 h reperfusion, the right gastrocnemius muscle and left ventricular myocardium were biopsied and flash-frozen for analyses of metabolites, enzymes, pro- and anti-apoptotic proteins and markers of oxidative and inflammatory stress.

During the first phase of experimentation we hypothesized that controlled resuscitation with PR vs. LR more effectively stabilizes MAP and attenuates myocardial inflammation post-resuscitation. MAP (mmHg) was increased in PR (59 ± 4) vs. LR (47 ± 3) resuscitated goats (p<0.05) at 4 h post-occlusion. In addition, PR more effectively augmented circulating HCO3- and total base excess, thus counteracting metabolic acidosis caused by systemic hypoperfusion. Marked tyrosine nitration, a footprint of nitrosative stress, was detected in myocardium 4 h after LR resuscitation, but was suppressed by PR. Finally, PR prevented the increase in circulating neutrophils that occurred during and following LR resuscitation.

During the second phase of experimentation, we hypothesized that PR resuscitation would protect ischemic hindlimb muscle in the setting of hemorrhagic shock and limb reperfusion. Lactate dehydrogenase and creatine kinase activities fell by 36 and 20%, respectively in LR-resuscitated vs. sham muscle (p<0.05). PR infusion preserved lactate dehydrogenase activity and more than doubled activity of the antioxidative enzyme glutathione reductase vs. the sham value. NADPH oxidase activity in LRresuscitated muscle had an increased activity compared with PR-treated and sham muscle (p = 0.056). Poly(ADP-ribose) polymerase-2 (PARP-2) cleavage, a measure of apoptosis, was increased in the LR-resuscitated muscle but PR resuscitation prevented this pro-apoptotic effects. Moreover, LR-treated muscle had decreased content of the antiapoptotic protein Bcl-xL vs. the PR-treated and sham muscle. Nitrotyrosine content, a measure of nitrosative stress, more than doubled in LR-treated vs. sham muscle, but PR prevented the increase in nitrotyrosine. Finally, muscle water content (ml/100g) increased from 74.7 ± 1.2 in sham to 81.4 ± 2.2 4 h after LR resuscitation, indicating tissue edema; PR attenuated the increase in water content (78.1 ± 1.0).

We conclude that 1) Systemic hypotension and hindlimb ischemia-reperfusion with conventional LR treatment imposed pro-oxidative and pro-inflammatory stress both systemically and locally, thus preventing stabilization of MAP during recovery and initiating apoptotic mechanisms in the hindlimb musculature; 2) Pyruvate-fortified Ringer’s effectively stabilized hemodynamics and dampened systemic inflammation after hemorrhagic shock with resuscitation and hindlimb ischemia-reperfusion; 3) PR-fortified resuscitation blunted oxidative and inflammatory stress within the ischemic hindlimb and suppressed pro-apoptotic signaling. These investigations demonstrate the anti-oxidative and anti-inflammatory effects of pyruvate in a system exposed to hemorrhagic shock with fluid resuscitation, as well as identify the cytoprotection pyruvate affords tissue experiencing ischemia-reperfusion.

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