Despite adequate and presumably successful hemodynamic resuscitation, morbidity and mortality from hemorrhagic shock remains high due to the high incidence of multi system organ failure (MSOF) , , , . Common complications in such patients include Acute Respiratory Distress Syndrome (ARDS), acute renal failure (ARF), myocardial depression, and liver dysfunction. The delayed onset of these complications points to an additional mechanism of injury besides ischemia alone. We hypothesize that the production of toxic free radicals in the post-ischemic reperfusion phase may contribute to this organ damage.
We will attempt to demonstrate this in two steps. First, we will directly measure and quantitate the production of free radicals during ischemia and reperfusion in the lungs, kidneys, heart, and liver in animals being resuscitated following hemorrhagic shock. Second, we will attempt to determine if the administration of free radical scavengers during the period of resuscitation can reduce mortality in animals recovering from profound shock , . Positive data should provide both direct and indirect evidence that the production of free radicals plays a significant role in the delayed morbidity and mortality seen in hemorrhagic shock.
Free radicals, especially those derived from molecular oxygen, have been implicated in a variety of disease processes. In 1983, Schoenberg and colleagues demonstrated that the administration of free radical scavengers could reduce the amount of damage to the small bowel undergoing ischemia and reperfusion . Jolly and co-workers have demonstrated a comparable effect in the myocardium . Other studies carried out on the effect of antioxidant therapy following hemorrhagic shock on the liver ,  and overall survival  have shown similar results. There is also a growing body of experimental evidence that implicates free radical dependent lipid peroxidation in injury to the central nervous system (CNS), as well as the myocardium , . Overall, there have been many investigators who have indirectly demonstrated that free radicals play an important role in the pathogenesis of reperfusion injury in multiple organ systems. Some investigators (including ourselves) have been able to quantitate free radical production by electron paramagnetic spectroscopy (EPR) , , . Further, a novel fluorescence probe, 2-[6-(4-hydroxyl)phenoxy-3H-Xanthen-3-on-9-yl]benzoic acid (HPF) has been used by some workers to investigate the generation of highly reactive oxygen species (hROS) under ischemia, both in vivo and in vitro. These techniques have directly demonstrated the production of free radicals during reperfusion after a given period of ischemia to the heart. It has also been demonstrated that there is a significant rise in free radicals in ischemic lung tissue, and are probably involved in the pathogenesis of ischemia reperfusion injury .
Due to the ubiquitous nature of this molecular species and the growing evidence of its production during ischemia-reperfusion sequences, we suspect that free radicals may play some role in the pathogenesis of delayed morbidity following shock. It is possible that reperfusion of ischemic organ systems during the period of resuscitation results in the production of free radicals that create additional tissue damage through membrane destabilization. Furthermore, free radicals catalyze lipid peroxidation, a chain reaction mediated by polymorphonuclear leukocytes (PMN), and a phenomenon extremely toxic to cellular membranes.
Our experiments will directly measure the production of free radicals during shock and resuscitation in organ systems that have been clinically shown to be the most vulnerable to delayed morbidity. Confirmation of the generation of significant quantities of free radicals may be of great clinical importance, since it represents a potentially treatable regional molecular event that could otherwise be responsible for a serious clinical problem. Further information regarding increased survival following the administration of free radical scavengers or iron chelators that could interrupt lipid peroxidation would have an obvious clinical significance. The second part of the experiment would potentially provide such data, thus opening the possibility for future clinical trials using scavengers.
Literature is abound with evidence that the administration of Superoxide Dismutase (SOD – a superoxide scavenger) or Mannitol (a hydroxyl free radical scavenger) can reduce reperfusion injury. This phenomenon has been documented in the intestines , , liver, kidneys, pancreas, and in the heart both in vivo,  and in the global ischemic model . Our laboratory has reported that the administration of either Superoxide Dismutase/Catalase or Mannitol will significantly reduce the mortality of Escherichia coli/Hemoglobin-induced peritonitis in the rat . In this study, the suspected mechanism of action is that free radical scavengers prevent overwhelming production of hydroxyl free radicals generated by the Fenton reaction (driven by PMN in the setting of bacteria and hemoglobin within the peritoneal cavity).
Other experiments with iron chelators have suggested that free radical production continues long after the initial burst due to the lipid peroxidation chain reaction that is mediated by PMN. Deferoxamine was found to attenuate shock-induced oxidative stress in the hepatocytes . Some investigators have found a decrease in CNS damage following ischemia and reperfusion in animals treated with Deferoxamine, an iron chelator . A study done by Liachenko et al. showed a significant increase in cerebral perfusion following the administration of deferoxamine . This is thought to be secondary to the interruption of lipid peroxidation, a process that generates intracellular free radicals and requires iron as a catalyst. A similar study has been carried out in cardiocytes with comparable results .
Free radicals in the ischemia-reperfusion setting may be derived from a neutrophil oxygen burst or from the metabolism of Hypoxanthine to Urea with the consequent generation of oxygen. Our group ,  and a group at Johns Hopkins University  have been successful in measuring free radical production in peri-ischemic conditions. Our work revealed that in the isolated rabbit heart model, global ischemia resulted in a decrease of free radicals, but that free radical production more than doubled upon reperfusion of the heart . The free radicals were measured by freezing the tissue in liquid nitrogen (77 K) and measuring signal size on the electron paramagnetic resonance spectrometer. This work provided direct evidence that free radicals are indeed produced in abundance during reperfusion, and may mediate reperfusion injury. Similarly, in a study carried out on a rat model of focal cerebral ischemia, transient occlusion of the middle cerebral artery generated more reactive oxygen species in comparison to permanent occlusion .
Carrying out the experiment we propose requires experience in the use of free radical scavengers, the measuring of free radicals, and the development of a suitable hemorrhagic shock model; all of which are well established procedures in our laboratory. We have also used Superoxide Dismutase/Catalase , Mannitol , Deferoxamine , Hyaluronidase , and perfluorocarbons  in an attempt to reduce myocardial reperfusion injury in the baboon.
Our work with the isolated rabbit heart represented one of the first successful attempts to demonstrate a decrease in free radical production during ischemia, followed by a dramatic increase after reperfusion. We have extensive hands-on experience with the use of spin traps for measurement of free radicals using EPR spectroscopy. Our laboratory has also developed a suitable hemorrhagic shock model in rabbits that affords an 80% mortality rate if treated only with resuscitation . This model provides an excellent vehicle through which to test the hypothesis.
To our knowledge, there is no study that has attempted to measure the production of free radicals in organ systems vulnerable to reperfusion injury following massive hemorrhage. There have been few attempts to study survival curves in animals resuscitated from shock, additionally treated with various free radical scavengers (including iron chelators). When one takes into account the recent experimental observations in the field of free radicals and reperfusion injury (summarized below), these tasks are the next logical steps.
The experimental protocol is described in two parts. The first (A) is designed to measure free radical production in various organs in rabbits subjected to profound hemorrhagic shock, followed by resuscitation (reperfusion). The second part (B) will attempt to study the effects that free radical scavengers and iron chelators have on survival when used as an adjunct to resuscitation.
In this part of the experiment, four groups of rabbits will be used. All groups will have their lungs, heart, liver, intestines, and kidneys sampled at different stages of shock. These tissues will be frozen with liquid nitrogen (77 K) and powdered. This powder will then be analyzed by EPR spectroscopy for the presence of free radicals. The first group will consist of animals that are anesthetized, then sacrificed before the initiation of shock. The second will be sampled during the period of shock itself. The third group will be sampled immediately after reperfusion (resuscitation). The fourth will be sacrificed and sampled, 12 h post-shock or upon expiration, whichever comes first. In this manner, a comparison can be made as to the quantity of free radicals produced during each phase by comparing the size of the EPR signals.
Initially, forty New Zealand white rabbits will be used (ten per group in four groups). They will be anesthetized with subcutaneous injections of Ketamine Hydrochloride (10 mg/kg). Sedation will be maintained with Ketamine and Diazepam (Valium, 5 mg/ml) in combined doses of 0.2 ml through an ear vein catheter. The rabbits will be secured in a supine position on a heating pad to maintain normothermia. Femoral cutdowns will be performed and all four vessels cannulated to monitor femoral arterial pressure, central venous pressure (CVP), body temperature, and cardiac output (CO). Routine arterial samples will be drawn to monitor pH and blood gases. All lines will be periodically flushed with heparinized saline (4 NIH units of Heparin/ml).
Baseline measurements will be taken, including arterial blood gases (ABG), mean arterial pressure (MAP), CVP, heart rate (HR), and CO. A control group of ten animals will be sacrificed at this point of the experiment. Biopsies of the lungs, heart, liver, intestines, and kidneys will be taken with a Van Doren clamp. The tissues will be immediately frozen in liquid nitrogen (77 K) and maintained in that state. The remaining animals will be slowly bled over 15 min to a MAP of 44 mm Hg. This blood will then be stored with 2 ml Heparin/50 ml blood. Aliquots of blood will either be withdrawn or infused to maintain the MAP at 44 mg Hg for 2½ h. At the end of this shock period, the withdrawn blood will be re-infused along with lactated Ringer’s solution until a MAP is obtained that is within 20% of the baseline or until a maximum of 100 ml of fluid is delivered. The animals will then be given maintenance IV fluid for one hour and then a bolus of three times the normal rate. The catheters will then be removed from the femoral vessels, and the vessels will be ligated. The wounds will be closed in layers and the animals returned to the cages for observation.
Of the three groups of animals undergoing shock, one group will be biopsied during the period of shock itself (Group 2). Group 3 will be biopsied immediately after reperfusion (5 min after resuscitation). The final group will be biopsied 12 h after the shock period, or the instant the animal dies, whichever occurs first. The tissue from these biopsies will be treated in an identical fashion to the control group.
The frozen, powdered tissue will then be placed in EPR tubes with a diameter of 3 mm and filled to a height of 7 mm. The EPR spectra of the tissues will be recorded on a Varian Model E-9 spectrometer (frequency = 9.19 GHz, power = 1.0 mW, modulation amplitude = 2.5 G). The amplitude of all signals generated will be measured and averaged for all rabbits in each group. The mean signal size for each of the groups will then be compared. Using calculated g values, the types of free radicals generated can be identified.
This part of the experiment will also consist of 4 groups of rabbits. All groups will undergo hemorrhagic shock to a MAP of 44 mm Hg for 2½ h. This degree of shock has been shown in our laboratory to be associated with a 20–30% survival rate, with the mean time of death for rabbits being approximately 20 h post-shock. The control group will be resuscitated with blood and lactated Ringer’s solution alone. The first treated group will receive superoxide Dismutase/Catalase along with the infusion. The second will receive the iron chelator, Deferoxamine. The third group will receive both drug regimens along with the standard reperfusion. Survival curves will be compared.
Sixty New Zealand white rabbits (15 per group) will be surgically prepared in an identical fashion as was proposed in Part A of the experiment. Shock will be induced by slowly bleeding the rabbits over a 15-min period to a MAP of 44 mm Hg. This blood will be stored with 2 ml Heparin/50 ml blood. Aliquots of blood will either be withdrawn or infused to maintain the MAP at 44 mm Hg for 2½ h.
The control group will be resuscitated at the end of the 2½ h period with the shed blood and lactated Ringer’s solution until the MAP achieved is within 20% of baseline or until a maximum of 100 ml of fluid is given. Group 2 will be resuscitated in the same manner, but will receive in addition a 50 mg/kg bolus of Superoxide Dismutase and a 5 mg/kg bolus of Catalase at the start of resuscitation, followed by a 1 mg/kg/min drip of each of Superoxide Dismutase and Catalase during the one hour maintenance period. The second treated group (Group 3) will receive Deferoxamine with the fluid resuscitation, at a dose of 50 mg/kg given as a continuous infusion, at the start of resuscitation and ending after the one hour maintenance period. The final group will receive all treatment modalities (Fluids, Superoxide Dismutase/Catalase, and Deferoxamine).
Once the resuscitation phase is complete, all animals will undergo a one hour period of maintenance IV fluid administration followed by a fluid bolus of three times the maintenance prior to closing. They will then be returned to their cages and allowed food and water. Survival will be assessed for each group up to a period of five days. At five days, all surviving animals will be sacrificed and autopsied. The lungs, heart, liver, intestines, and kidneys will be biopsied and examined grossly and microscopically for evidence of necrosis. Survival curves will be constructed for all four groups and statistical significance will be determined by use of the Kaplan–Meier methods.
New Zealand white rabbits, all young adults, will be used for this experiment. A total of 100 (40 for Part A and 60 for Part B) will be necessary for the two parts of the experiment. These animals will weigh approximately 2–3 kg. They are relatively inexpensive and freely available from local sources.
A computer and statistical consultant is required to be on staff or available for data analysis.
Such agreements may be necessary, especially in relation to equipment which may not be available in the principal investigator’s laboratory, where the work will be conducted.
There are no author disclosures. This is a protocol and does not have any financial issues.
This was a protocol and sources of funding are not applicable.
Shyamal Premaratne: planning of experiment, protocol development, literature search, manuscript preparation.
Dhanushya Amaratunga: protocol development, literature search.
Francis Mensa: protocol development, statistical analysis.
Late J. Judson McNamara: principal investigator on the project, chairman of the department.
Dr. Shyamal Premaratne and Dr. J. Judson McNamara were the initial guarantors. However, with the passing of Dr. McNamara, Dr. Premaratne will assume that role.
McNamara, J.J., Suehiro, G.T., Suehiro, A. and Jewett, B. (1983). Resuscitation from hemorrhagic shock. J Trauma 23(7): 552. [PubMed]
Bulkley, G.B. (1983). The role of oxygen free radicals in human disease processes. Surgery 94(3): 407. [PubMed]
McGhan, L.J. and Jaroszewski, D.E. (2012). The role of toll-like receptor-4 in the development of multi-organ failure following traumatic hemorrhagic shock and resuscitation. Injury 43(2): 129–136. [PubMed]
Casale, A.S., Bulkley, G.B., Bulkley, B.H., Flaherty, J.T., Gott, V.L. and Gardner, T.J. (1983). Oxygen free radical scavengers protect the arrested globally ischemic heart upon reperfusion. Surg Forum 34: 313.
Schoenberg, M.H., Muhl, E., Sellin, D., Younes, M., Schildberg, F.W. and Haglund, U. (1984). Posthypotensive Generation of superoxide free radicals – possible role in the pathogenesis of the intestinal mucosal damage. Acta Chir Scand 150(4): 301–309. [PubMed]
Jolly, S.R., Kane, W.J., Bailie, M.B., Abrams, G.D. and Lucchesi, B.R. (1984). Canine myocardial reperfusion injury: its reduction by the combined administration of superoxide dismutase and catalase. Circ Res 54(3): 277–285. [PubMed]
Orfanos, N.F., Mylonas, A.I., Karmaniolou, I.I., Stergiou, I.P., Lolis, E.D., Dimas, C., Papalois, A.E., Kondi-Pafiti, A.I., Smyrniotis, V.E. and Arkadopoulos, N.F. (2016). The effects of antioxidants on a porcine model of liver hemorrhage. J Trauma Acute Care Surg 80(6): 964–971. [PubMed]
Bauer, C., Walcher, F., Holanda, M., Mertzlufft, F., Larsen, R. and Marzi, I. (1999). Antioxidative resuscitation solution prevents leukocyte adhesion in the liver after hemorrhagic shock. J Trauma 46(5): 886–893. [PubMed]
Kentner, R., Safar, P., Behringer, W., Wu, X., Kagan, V.E., Tyurina, Y.Y., Henchir, J., Ma, L., Hsia, C.J. and Tisherman, S.A. (2002). Early antioxidant therapy with Tempol during hemorrhagic shock increases survival in rats. J Trauma 53(5): 968–977. [PubMed]
Demopoulos, H.B., Flamm, E.S., Pietronigro, D.D. and Seligman, M.L. (1980). The free radical pathology and the microcirculation in the major central nervous system disorders. Acta Physiol Scand Suppl 492: 91–119. [PubMed]
Premaratne, S., Limm, W., Mugiishi, M.M., Piette (Late), L.H. and McNamara, J.J. (1993). Quantitative assessment of free radical generation during ischemia and reperfusion in the isolated rabbit heart. Coron Artery Dis 4(12): 1093–1096. [PubMed]
Premaratne, S., Suehiro, A., Suehiro, G.T., Arakaki, H.Y. and McNamara, J.J. (1994). Detection of hydroxyl free radicals in the reperfused primate heart. Free Radical Res 21(1): 19–25. [PubMed]
Zweier, J.L., Flaherty, J.T. and Willerson, M.L. (1987). Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci USA 84(5): 1404–1407. [PubMed]
Tomizawa, S., Imai, H., Tsukada, S., Simizu, T., Honda, F., Nakamura, M., Nagano, T., Urano, Y., Matsuoka, Y., Fukasaku, N. and Saito, N. (2005). The detection and quantification of highly reactive oxygen species using the novel HPF fluorescence probe in a rat model of focal cerebral ischemia. Neurosci Res 53(3): 304–313. [PubMed]
Gielis, J.F., Boulet, G.A., Briedé, J.J., Horemans, T., Debergh, T., Kussé, M., Cos, P. and Van Schil, P.E. (2015). Longitudinal quantification of radical bursts during pulmonary ischaemia and reperfusion. Eur J Cardiothorac Surg. 48(4): 622–629. [PubMed]
Izumi, M., McDonald, M.C., Sharpe, M.A., Chatterjee, P.K. and Thiemermann, C. (2002). Superoxide dismutase mimetics with catalase activity reduce the organ injury in hemorrhagic shock. Shock 18(3): 230–235. [PubMed]
Kloner, R.A., Reimer, K.A., Wilkerson, J.T. and Jennings, R.B. (1976). Reduction of experimental myocardial infarct size with hyperosmolar mannitol. Proc Soc Exp Biol Med 151(4): 677–683. [PubMed]
Otani, H., Umemoto, M., Kagawa, K., Nakamura, Y., Omoto, K., Tanaka, K., Sato, T., Nonoyama, A. and Kagawa, T. (1986). Protection against oxygen induced reperfusion injury of the isolated canine heart by superoxide dismutase and catalase. J Surg Res 41(2): 126–133. [PubMed]
Limm, W., Mugiishi, M.M., Piette, L. and McNamara, J.J. (1987). The use of oxygen free radical scavengers in the rat peritonitis model In: Presented at the 4th International Congress on Oxygen Radicals. University of California at San Diego.
Kompala, S.D., Babbs, C.F. and Blaho, K.E. (1986). Effect of deferoxamine on late deaths following CPR in rats. Ann Emerg Med 15(4): 405–407. [PubMed]
Liachenko, S., Tang, P. and Xu, Y. (2003). Deferoxamine improves early postresuscitation reperfusion after prolonged cardiac arrest in rats. J Cereb Blood Flow Metab. 23(5): 574–581. [PubMed]
Watanabe, B.I., Premaratne, S., Limm, W., Mugiishi, M.M. and McNamara, J.J. (1993). High and low dose super oxide dismutase plus catalase does not reduce myocardial infarct size in a subhuman primate model. Am Heart J 126(4): 840–846. [PubMed]
Watanabe, B.I., Limm, W., Suehiro, A., Suehiro, G., Premaratne, S. and McNamara, J.J. (1993). Failure of deferroxamine to reduce myocardial infarct size in a primate model of ischemia-reperfusion injury. J Surg Res 55(5): 537–542. [PubMed]
Premaratne, S., Watanabe, B.I., LaPenna, W. and McNamara, J.J. (1995). Effects of hyaluronidase on reducing myocardial infarct size in a baboon model of ischemia-reperfusion injury. J Surg Res 58(2): 205–210. [PubMed]
Premaratne, S., Harada, R.N., Chun, P., Suehiro, A. and McNamara, J.J. (1995). Effects of perfluorocarbon exchange transfusion on reducing myocardial infarct size in a primate model of ischemia-reperfusion injury: a prospective study. Surgery 117(6): 670–676. [PubMed]
Pohlson, E.C., Ziegler, E.J. and McNamara, J.J. (1985). Antiserum to endotoxin: a new approach to investigate the role of endotoxin in hemorrhagic shock in rabbits In: Baltimore, Maryland: Presented at the Shock Society Meeting. June 9-12.
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