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INDUCED HYPOTHERMIA DURING RESUSCITATION FROM HEMORRHAGIC SHOCK ATTENUATES MICROVASCULAR INFLAMMATION IN THE RAT MESENTERIC MICROCIRCULATION Garrett N. Coyan,*† Michael Moncure,* James H. Thomas,* and John G. Wood*‡ Departments of *Surgery, † Preventive Medicine and Public Health, and ‡ Molecular & Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas Received 4 May 2014; first review completed 21 May 2014; accepted in final form 16 Jul 2014 ABSTRACT— Microvascular inflammation occurs during resuscitation following hemorrhagic shock, causing multiple organ dysfunction and mortality. Preclinical evidence suggests that hypothermia may have some benefit in selected patients by decreasing this inflammation, but this effect has not been extensively studied. Intravital microscopy was used to visualize mesenteric venules of anesthetized rats in real time to evaluate leukocyte adherence and mast cell degranulation. Animals were randomly allocated to normotensive or hypotensive groups and further subdivided into hypothermic and normothermic resuscitation (n = 6 per group). Animals in the shock groups underwent mean arterial blood pressure reduction to 40 to 45 mmHg for 1 h via blood withdrawal. During the first 2 h following resuscitation by infusion of shed blood plus double that volume of normal saline, rectal temperature of the hypothermic groups was maintained at 32-C to 34-C, whereas the normothermic groups were maintained between 36-C to 38-C. The hypothermic group was then rewarmed for the final 2 h of resuscitation. Leukocyte adherence was significantly lower after 2 h of hypothermic resuscitation compared with normothermic resuscitation: (2.8 T 0.8 vs. 8.3 T 1.3 adherent leukocytes, P = 0.004). Following rewarming, leukocyte adherence remained significantly different between hypothermic and normothermic shock groups: (4.7 T 1.2 vs. 9.5 T 1.6 adherent leukocytes, P = 0.038). Mast cell degranulation index (MDI) was significantly decreased in the hypothermic (1.02 T 0.04 MDI) versus normothermic (1.22 T 0.07 MDI) shock groups (P = 0.038) after the experiment. Induced hypothermia during resuscitation following hemorrhagic shock attenuates microvascular inflammation in rat mesentery. Furthermore, this decrease in inflammation is carried over after rewarming takes place. KEYWORDS—Hemorrhagic shock, resuscitation, hypothermia, leukocyte adherence, mast cell degranulation, mesenteric microcirculation
INTRODUCTION
related systemic hypoxia (3, 5). The generation of reactive oxygen species (ROS) initiates cytokine formation through mediators such as tumor necrosis factor ! (TNF-!) and interleukin 1" (IL-1"), followed by endothelial activation (6). This increases leukocyte adherence and emigration. In addition, mast cells located in the perivascular tissue are activated to release granules via immunoglobulin E (7). These granules contain histamine, additional inflammatory cytokines, and even some anti-inflammatory markers that modulate immune function. This complex pathway leads to localized tissue death due to edema and apoptosis, ending with multiple organ dysfunction and death of the patient. Hypothermia has seen a recent increase in usage as a therapeutic modality (8). A recent case series has shown that inducedhypothermia protocols may be used after traumatic cardiac arrest with positive outcomes (9). Several laboratory studies have sought to clarify the role of hypothermia during resuscitation from hemorrhagic shock and trauma (10Y17). In one study directly examining microvascular inflammation Childs et al. induced mild-moderate hypothermia in rats prior to hemorrhagic shock and resuscitation, and noted a decrease in ROS formation and vascular permeability (18). In this study, however, the effects of hypothermia on shock-induced mast cell degranulation or of rewarming to normal body temperature were not examined.
Trauma continues to be a major cause of morbidity and mortality in the general population (1). Along with traumatic brain injury, hemorrhagic shock shares a large responsibility for these poor outcomes (2). Even with optimal resuscitation, delayed morbidity and mortality occur in this population secondary to microvascular inflammation. Although major efforts have been made to overcome this inflammatory response to injury and resuscitation, little improvement in standardized care has been realized (3). Hemorrhagic shock has been shown to induce microvascular inflammatory responses that are not attenuated with volume resuscitation (4). Various mechanisms of this injury have been hypothesized, most notably ischemia-reperfusion injury with Address reprint requests to John G. Wood PhD, 3901 Rainbow Blvd, Mailstop 3043, Kansas City, KS 66160. E-mail: [email protected]. Co-correspondence: Garrett N. Coyan, MS, 3901 Rainbow Blvd, Mailstop 3043, Kansas City, KS 66160. E-mail: [email protected]. Funding support provided by the Department of Surgery Research Fund and the Department of Molecular & Integrative Physiology, University of Kansas Medical Center. G.C. is supported in part by a CTSA grant from National Center for Research Resources and National Center for Advancing Translational Sciences awarded to the University of Kansas Medical Center for Frontiers: The Heartland Institute for Clinical and Translational Research (no. TL1TR000120). This study was presented as an oral platform presentation at the American College of Surgeons Clinical Congress (Washington, DC) in October 2013. The authors of this article have no conflicts to disclose. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health, National Center for Research Resources, or National Center for Advancing Translational Sciences. DOI: 10.1097/SHK.0000000000000241 Copyright Ó 2014 by the Shock Society
Study objective
Our objective in this study was to determine the effect of induced hypothermia during resuscitation from hemorrhagic shock on microvascular inflammation. Using intravital microscopy, we 518
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SHOCK DECEMBER 2014 have previously demonstrated increased leukocyte adherence in rat mesenteric venules following hemorrhagic shock/resuscitation, as well as an increase in mast cell degranulation (4). The present study was designed to evaluate whether mild hypothermia (32-CY34-C) induced during the period of shock and maintained during resuscitation would attenuate microvascular inflammation. This approach poses the least risk of coagulopathy and is easy to achieve in the trauma patient and maintain without aggressive cooling techniques (19). In addition, we examined the effect of rewarming following resuscitation to determine if the anti-inflammatory effect of hypothermia would persist after body temperature had been restored to normal, a question that has not previously been studied. We evaluated leukocyte adherence and mast cell degranulation, two important indices of microvascular inflammation and dysfunction (5, 20, 21). MATERIALS AND METHODS Regulatory considerations The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center. The University of Kansas Medical Center is accredited by the American Association for Accreditation of Laboratory Animal Care; all procedures were carried out in accordance with the National Institutes of Health guidelines.
Surgical procedures Sprague-Dawley rats weighing 250 to 350 g were obtained from Harlan Laboratories (Indianapolis, Ind). Animals were allowed full access to food and water prior to the experiment. Rats were anesthetized with sodium pentobarbital (40Y60 mg/kg i.m. injection, additional doses as needed). Jugular venous cannulation was performed for administration of fluids and blood with a polyethylene (PE) cannula (PE-50, 0.58-mm internal diameter). Carotid artery cannulation was likewise carried out for withdrawal of blood and monitoring of mean arterial pressure (MAP) with a continuous blood pressure analyzer (Micromed, Louisville, Ky). A tracheostomy was performed with PE-240 tubing to maintain airway patency. Midline laparotomy incision was conducted with radiocautery (Harvard Apparatus, Holliston, Mass).
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was measured using the ImageJ program (National Institutes of Health, Bethesda, Md), and the degree of degranulation was quantified as a mast cell degranulation index (MDI) equal to the ratio of the end of experiment to control period light intensities (5).
Experimental protocol The experimental groups (Fig. 1) consisted of normothermic normotensive animals (n = 6), hypothermic normotensive (n = 6), normothermic shock (n = 6), and hypothermic shock (n = 6). Temperature was recorded with a rectal thermometer and maintained at 36-C to 38-C with a homeothermic blanket and a heat lamp. Animals were allowed to stabilize after surgery for a 30-min control period prior to beginning the experiment. Hemorrhagic shock was induced by withdrawal of blood from the carotid artery into a syringe containing 100 U of heparin. A MAP of 40 to 45 mmHg was maintained for a period of 1 h. At the end of this period, the shed blood was returned to the animal via the jugular cannula, as well as two times the volume of blood in normal saline. For those undergoing hypothermia, animals were allowed to naturally begin cooling during hemorrhagic shock, with a target of 32-C to 34-C at the end of the shock period. Animals needing additional cooling at this point were subjected to air cooling via a fan. The hypothermia was maintained for a 2-h time period, after which the animals underwent rewarming to 36-C to 38-C for 2 h. Leukocyte adherence and physiologic variables were recorded every 15 min throughout the experiment. Mast cell degranulation was recorded at the end of the control period and at the end of the experiment.
Statistical analysis In order to detect at least a difference of four adherent leukocytes between groups, we calculated that six animals per group would be needed using an estimated SD of 2 with 80% power and a type I error of 0.05. Data were examined graphically using quantile plots and compared with historical controls in our laboratory to confirm that our assumption of normality was correct. Data were expressed as mean T SE for continuous variables. Mean arterial pressure and rectal temperature were analyzed with one-way analysis of variance with Bonferroni post hoc correction. Student t test was used to compare differences in leukocyte adherence between hypothermic shock and normothermic shock groups, normothermic normotensive and hypothermic normotensive groups, and each shock group individually with the normothermic normotensive group (acting as a longitudinal baseline comparison) at hourly time points during resuscitation. Shear rate was evaluated in the same manner.
Intravital microscopy The animal was placed in the right lateral decubitus position on a Plexiglas stage mounted to a Zeiss Axiovert inverted microscope. A loop of mesentery was draped over a glass cover slip in the center of the stage to view a mesenteric venule. Saline warmed to 37-C was superfused over the mesentery to prevent drying, and a square of Saran wrap was also placed over the exposed mesentery. Mesenteric venules were selected for study based on the following three criteria: (i) straight, unbranched vessels at least 100 2m in length; (ii) no adjacent vessels within 100 2m of the venule; and (iii) vessel diameter of 20 to 40 2m. Images of the mesentery (40 objective, 10 eyepiece) were recorded using Mini-DV videocassette recorder (Sony, Tokyo, Japan) using a video camera and time-date generator (Panasonic, Kadoma, Japan). Venular diameter was obtained using a video caliper, and an optical Doppler velocimeter was used to measure red blood cell (RBC) centerline velocity in mesenteric venules (Microcirculation Research Institute, Texas A&M University, College Station, Tex). An average RBC velocity was calculated by dividing centerline velocity by 1.6. Shear rate was then calculated as 8 multiplied by RBC velocity divided by venular diameter (22).
Leukocyte adherence and mast cell degranulation Leukocyte adherence and mast cell degranulation were measured during offline playback of recorded videotapes. Leukocyte adherence was measured over each minute by counting the number of leukocytes that remained stationary for longer than 30 s within a 100-2m length of venule (5). Tapes were analyzed two times by an experienced observer to ensure accurate counting of leukocytes; because analysis was carried out by the same staff performing the experiments, blinding of analysis was not performed. Mast cell degranulation was identified by superfusing the mesentery with ruthenium red (Sigma-Aldrich, St Louis, Mo), a chemical that stains degranulated mast cells. Between 13 and 15 mast cells were identified in the same mesenteric loop in relation to the venule identified for study and used to assess the extent of degranulation. Images of these mast cells were recorded during the control period, then once again at the conclusion of the experiment. These images were converted to digital grayscales and phase inverted. Relative light intensity of each mast cell
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FIG. 1. Experimental design and group configuration.
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Paired t test was used to compare shear rates from the control period to the final resuscitation time point within each group to determine change from the control period. Differences in MDI were compared using Student t test. Differences were considered significant when P e 0.05. Statistical analysis was carried out using SAS 9.3 (SAS Institute, Cary, NC).
RESULTS Physiological measures
Figure 2A displays rectal temperatures recorded throughout the experiment. Animals in the hypothermic groups gradually cooled during the 1-h period corresponding with hemorrhagic shock and were maintained at 32-C to 34-C for 2 h following resuscitation. One animal in each of the two hypothermic groups required additional passive cooling with a fan to reach goal at the end-of-the-hour period. Passive loss of heat was adequate to maintain the hypothermic state, and no further cooling measures were needed. The heat lamp and heating pad were used on all animals intermittently to maintain the goal temperature ranges. The rewarming took place over approximately 1 h in the hypothermic groups and was maintained at 36-C to 38-C for the
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remainder of the experiment. The hypothermic groups had significantly lower temperatures than did the normothermic groups from start of resuscitation to 30 min into the rewarming period (P G 0.05). Mean arterial pressure of the animals is displayed in Figure 2B. Shock was maintained for 60 min, and resuscitation provided initial pressures near baseline. This pressure gradually decreased as the experiment went on during the 4-h resuscitation period in all four groups. The shock groups had significantly lower MAPs than did the normotensive groups during the shock period (P G 0.05). In addition, the hypothermic normotensive group had a significantly higher MAP than did the other three groups from 30 min of the shock time period to 30 min into the rewarming period (P G 0.05). Calculated shear rates are shown in Figure 2C. Shear rates in the hypothermic shock group did not significantly change over the course of the experiment when compared with control time period measurements (P 9 0.05), whereas there was a significant decrease within the normothermic shock group over the course of the experiment from the control period measure to the end of 4 h of resuscitation (P G 0.05).
FIG. 2. Physiological parameters: (A) rectal temperature, (B) MAP, (C) shear rate. NSignificant difference between hypothermic and normothermic groups (P G 0.05). *Significant difference between shock and normotensive groups (P G 0.05). #Significant difference between the hypothermic normotensive group and all other groups (P G 0.05).
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FIG. 3. Representative photomicrographs of leukocyte adherence in mesenteric venules: (A) normothermic normotensive group, (B) hypothermic normotensive group, (C) normothermic hypotensive group, (D) hypothermic hypotensive group. Blue arrows indicate adherent leukocytes. Black dots are a part of the optical Doppler velocimeter; white bars outline 100-2m length segment.
Leukocyte adherence
Figure 3 (AYD) shows representative photographs of rat mesenteric venules in the four groups at control, 2 h into resuscitation, and 4 h into resuscitation. The number of adherent leukocytes in a 100-2m length of venule is shown for all four experimental groups in Figure 4. Leukocyte adherence was significantly lower in the hypothermic shock group than in the normothermic shock group at 2 h after resuscitation (2.8 T 0.8 vs. 8.3 T 1.3 adherent leukocytes, P = 0.004), and the significant elevation continued through the end of the 4-h resuscitation time point (4.7 T 1.2 vs. 9.5 T 1.6 adherent leukocytes, P = 0.038). The increase in leukocyte adherence in the hemorrhagic shock groups as compared with the normothermic normotensive animals became significant at 1 h after beginning resuscitation in the normothermic shock group (P G 0.05), whereas the increase never reached significance in the hypothermic shock group (P 9 0.05). Normotensive animals did not have significant changes in leukocyte adherence throughout the experiment.
duced hypothermia during resuscitation reduces the extent of mast cell degranulation following hemorrhagic shock. The decreased leukocyte adherence between hypothermic and normothermic resuscitation was statistically significant after the hypothermic period and furthermore continued to be significantly lower after rewarming occurred. We also observed a significant decrease in shear rates following the resuscitation period as compared with the control period in the normothermic shock cohort, whereas there was no significant decrease during resuscitation of hypothermic animals; this could reflect greater endothelial function in animals undergoing hypothermic resuscitation because there was no significant difference in MAP between groups (23Y25).
Mast cell degranulation
Figure 5 (A and B) shows photographs of mast cells for the hypothermic shock group and the normothermic shock group during the control period and after 4 h of resuscitation. The MDI was significantly lower after the 4-h resuscitation period in the hypothermic shock group as compared with the normothermic shock group (1.02 T 0.04 vs. 1.22 T 0.07 MDI, P = 0.038). No significant degranulation occurred in the normotensive groups. DISCUSSION We have shown that induced hypothermia during resuscitation from hemorrhagic shock significantly attenuates leukocyte adherence and mast cell degranulation in the rat mesenteric microcirculation. This is the first report demonstrating that in-
FIG. 4. Leukocyte adherence in mesenteric venules. *Significant difference between normothermic shock group and normothermic normotensive group (P G 0.05). #Significant difference between normothermic shock group and hypothermic shock group (P G 0.05).
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FIG. 5. Mast cell degranulation: (A) hypothermic hypotensive group; (B) normothermic hypotensive group. Pink color represents uptake of ruthenium red dye due to mast cell degranulation. Blue arrows indicate mast cells being observed. Black dots are part of the optical Doppler velocimeter.
Leukocyte adherence has been shown previously to be a mediator of microvascular dysfunction during hemorrhagic shock (4). The increase in leukocyte adherence has been linked to several other indices of inflammation, such as mast cell degranulation, ROS formation, and increased vascular permeability (4Y6, 26). Mast cell degranulation has been shown to occur throughout the body during hemorrhagic shock (27Y30). We have shown in our laboratory that hypoxia, as well as hemorrhagic shock, causes mast cells to degranulate in response to oxidative stress as well as when inflammatory mediators extravasate through the damaged endothelium (5, 30). A previous study showed that when mast cells are inhibited chemically, ROS and leukocyte adherence still occur with injury, but to a much lesser degree (5). Therefore, the degranulation of mast cells increases ROS formation and leukocyte adherence in the microvasculature. This cycling cascade continues for several hours after injury and is likely responsible for much of the delayed morbidity and mortality in patients with hemorrhagic shock injury. Childs and colleagues (18) showed that pretreating animals with mild and moderate hypothermia prior to hemorrhagic shock attenuated ROS formation and vascular permeability during the resuscitation period. It is important to note that their study pretreated with hypothermia and maintained constant temperature throughout the experiment without examining the actual induction of hypothermia or rewarming. In contrast, we used a model that involved allowing animals to cool and maintaining the hypothermic range as might be done in actual resuscitation settings. We also found that decreased microvascular inflammation persisted after rewarming the animals, a finding not previously reported. This is a key finding for demonstrating the feasibility of the use of hypothermia in the clinical setting. In addition, it has been shown that moderate hypothermia decreases systemic oxidative stress during intestinal ischemia-reperfusion injury in the rat (31). Reduced ROS
formation likely plays a role in the protective effect of hypothermia during resuscitation, but the exact mechanism remains unclear. Future studies should examine which of these proposed mechanisms is responsible for the observed decrease in leukocyte adherence and mast cell degranulation associated with hypothermic resuscitation. Specific knockout animals, inflammatory mediator inhibitors, and well-designed and powered large survival studies will all be required to elucidate the ideal model of hypothermic resuscitation from hemorrhage. Inflammatory mediator modulation may be associated with the protective effect of hypothermia during resuscitation. A study in rats showed that IL-1" and IL-6 were decreased in hypothermic resuscitation, but the study was underpowered to show significant differences (32). Increased levels of IL-1" (9100ug) were associated with early mortality before 72 h in this animal study. The same study found that IL-10, an antiinflammatory cytokine, was elevated in hypothermia. An interesting finding was that TNF-! was significantly increased after hypothermic resuscitation. Although this response seems counterintuitive, delayed increased levels of TNF-! may help to decrease ROS levels via activation of superoxide dismutase and may decrease metabolic demand on myocardium and peripheral tissues (32, 33). These cytokines also play a role in modulating selectins, intracellular adhesion molecules, and mast cells. This could potentially contribute to our findings. Whereas we were unable to study mortality because of the invasive nature of our model, several studies indicate decreased early mortality with mild to moderate hypothermia during resuscitation from hemorrhage in rat models (10, 12, 16, 17). The few studies that did look at long-term outcomes showed either a decrease or no change in mortality (15, 17, 34). A recent porcine laboratory animal model has shown that environmental (natural cooling) hypothermia has similar mortality to normothermia (35). A separate porcine model demonstrated a reduction in mortality 48 h following hemorrhagic shock from
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SHOCK DECEMBER 2014 66% in the normothermic resuscitation group to 7% in the 36-C resuscitation and 0% in the 33-C resuscitation groups (36). In this study, there was evidence of reduced oxygen consumption in the two hypothermic groups, but these two groups required more colloid resuscitation to maintain MAP in the postresuscitation period. These studies are routinely conducted with a small number of animals, and this could account for some of the variations in observed outcomes. It is also important to point out that the methods of hemorrhagic shock induction and hypothermia maintenance differ between studies, making comparing results difficult. We chose to use a pressure-controlled hemorrhagic shock model in our study because of earlier studies suggesting animals with hypothermia had increased MAPs during volumecontrolled shock, which is a confounding factor (11, 12, 17). Findings in humans regarding hypothermia in the trauma population have been largely negative (37). However, a recent case series reported successful outcomes following induced hypothermia after traumatic cardiac arrest (9). In addition, a neuroprotective effect of hypothermia in patients with traumatic brain injury has been demonstrated in several studies (38). These limited uses of induced hypothermia, along with an abundance of preclinical evidence, show that select trauma patients may benefit from appropriate applications of this therapy (39). In fact, one ongoing trial set to begin enrollment soon will be looking at profound induced hypothermia in patients with traumatic cardiac arrest secondary to hemorrhagic shock (Clinicaltrials.gov identifier: NCT01042015). It is imperative that we continue to seek a greater understanding of the mechanisms of induced hypothermia in order to optimize the therapy for the benefit of our patients. Our study has several strengths. We used a pressurecontrolled hemorrhagic shock model in our study in order to limit the increased MAP effect of hypothermia. We allowed the animal to cool during shock and maintained the hypothermia during the first 2 h of resuscitation, simulating real-world clinical conditions that patients may experience in the field or the hospital. We also examined 2 h of rewarming to see if the effect carried over after the therapeutic hypothermia period, a novel aspect specific to this study that is imperative to understand for translation to clinical practice. Our study was also powered adequately to identify the significant differences we observed, a weakness of the few other physiological studies available. There also exist several limitations to our model. Because our model involves nonsurvival surgical methods, we were unable to assess mortality. It remains unclear what the proper duration of hypothermia during the resuscitation period should be; we selected 2 h because of our current hemorrhagic shock models. In addition, our resuscitation technique may not replicate exactly what happens in a trauma-resuscitation scenario. However, this technique was standard in all animals undergoing resuscitation, minimizing opportunity for bias. Because we kept only the subjects hypothermic for the first 2 h of resuscitation, we are unable to predict what effects longer periods of hypothermia may have on microvascular inflammation. We also acknowledge that genetic differences in the response to hemorrhagic shock and resuscitation between laboratory models and humans exist; however, even with different genetic expression between species, similar physiological responses
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can be observed (40, 41). Further translational studies in large animal models, as well as humans, are needed to confirm the benefit of hypothermia during resuscitation from hemorrhage. We conclude that mild induced hypothermia (32-CY34-C) during resuscitation from hemorrhagic shock decreases leukocyte adherence and mast cell degranulation in the rat mesenteric circulation, and importantly this protective effect is carried over after rewarming of the animals. Hypothermia did not cause microvascular inflammation in normotensive animals. Further research is warranted to examine the optimal duration of hypothermia, the optimal degree of cooling, and the effect of rewarming on microvascular inflammation. ACKNOWLEDGMENTS The authors thank Naomi Holloway for invaluable technical support in conducting the experiments. In addition, they thank the Department of Surgery Research Fund, the Department of Molecular & Integrative Physiology, and the Department of Preventive Medicine and Public Health for financial support.
REFERENCES 1. US Department of Health and Human Services, CDC, National Center for Injury Prevention and Control: CDC: Web-Based Injury Statistics Query and Reporting System (WISQARS). Atlanta Georgia: Centers for Disease Control and Prevention, 2012. 2. Kauvar DS, Wade CE: The epidemiology and modern management of traumatic hemorrhage: US and international perspectives. Crit Care 9(Suppl 5): S1YS9, 2005. 3. Eltzschig HK, Eckle T: Ischemia and reperfusionVfrom mechanism to translation. Nat Med 17(11):1391Y1401, 2011. 4. Childs EW, Udobi KF, Wood JG, Hunter FA, Smalley DM, Cheung LY: In vivo visualization of reactive oxidants and leukocyte-endothelial adherence following hemorrhagic shock. Shock 18(5):423Y427, 2002. 5. Steiner DR, Gonzalez NC, Wood JG: Mast cells mediate the microvascular inflammatory response to systemic hypoxia. J Appl Physiol 94(1):325Y334, 2003. 6. Morita Y, Clemens MG, Miller LS, Rangan U, Kondo S, Miyasaka M, Yoshikawa T, Bulkley GB: Reactive oxidants mediate TNF-alphaYinduced leukocyte adhesion to rat mesenteric venular endothelium. Am J Physiol 269(6 Pt 2): H1833YH1842, 1995. 7. Kimura M, Mitani H, Bandoh T, Totsuka T, Hayashi S: Mast cell degranulation in rat mesenteric venule: effects of L-NAME, methylene blue and ketotifen. Pharmacol Res 39(5):397Y402, 1999. 8. Alam HB: Translational barriers and opportunities for emergency preservation and resuscitation in severe injuries. Br J Surg 99(Suppl 1):29Y39, 2012. 9. Tuma MA, Stansbury LG, Stein DM, McQuillan KA, Scalea TM: Induced hypothermia after cardiac arrest in trauma patients: a case series. J Trauma 71(6):1524Y1527, 2011. 10. Li T, Lin X, Zhu Y, Li L, Liu L: Short-term, mild hypothermia can increase the beneficial effect of permissive hypotension on uncontrolled hemorrhagic shock in rats. Anesthesiology 116(6):1288Y1298, 2012. 11. Nishi K, Takasu A, Shibata M, Uchino S, Yamamoto Y, Sakamoto T: Hypothermia reduces resuscitation fluid volumes required to maintain blood pressure in a rat hemorrhagic shock model. J Trauma Acute Care Surg 72(1):130Y135, 2012. 12. Prueckner S, Safar P, Kentner R, Stezoski J, Tisherman SA: Mild hypothermia increases survival from severe pressure-controlled hemorrhagic shock in rats. J Trauma 50(2):253Y262, 2001. 13. Sasaki H, Yukioka T, Ohta S, Fujikawa T, Noda M, Homma H, Mishima S: Is there a self-preserving hypothermic mechanism in shock? Shock 27(4):354Y357, 2007. 14. Subeq YM, Hsu BG, Lin NT, Yang FL, Chao YF, Peng TC, Kuo CH, Lee RP: Hypothermia caused by slow and limited-volume fluid resuscitation decreases organ damage by hemorrhagic shock. Cytokine 60(1):68Y75, 2012. 15. Takasu A, Yanagawa Y, Sakamoto T, Okada Y: Therapeutic hypothermia limited to the resuscitation period does not prolong survival after severe hemorrhagic shock in rats. Resuscitation 67(1):119Y126, 2005. 16. Wu X, Stezoski J, Safar P, Behringer W, Kentner R, Kochanek PM, Tisherman SA: Systemic hypothermia, but not regional gut hypothermia, improves survival from prolonged hemorrhagic shock in rats. J Trauma 53(4):654Y662, 2002. 17. Wu X, Stezoski J, Safar P, Nozari A, Tisherman SA: After spontaneous hypothermia during hemorrhagic shock, continuing mild hypothermia (34 degrees C) improves early but not late survival in rats. J Trauma 55(2):308Y316, 2003. 18. Childs EW, Udobi KF, Hunter FA: Hypothermia reduces microvascular permeability and reactive oxygen species expression after hemorrhagic shock. J Trauma 58(2):271Y277, 2005.
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19. Alam HB, Pusateri AE, Kindzelski A, Egan D, Hoots K, Andrews MT, Rhee P, Tisherman S, Mann K, Vostal J, et al.: Hypothermia and hemostasis in severe trauma: a new crossroads workshop report. J Trauma Acute Care Surg 73(4): 809Y817, 2012. 20. Zilberberg J, Harris NR: Role of shear and leukocyte adherence on venular permeability in the rat mesentery. Microvasc Res 62(3):215Y225, 2001. 21. Morita Y, Clemens MG, Bulkley GB: Delayed Bspontaneous[ adhesion of leukocytes to rat mesenteric venules is dependent upon reactive oxidants. Shock 4(1):41Y44, 1995. 22. House SD, Lipowsky HH: Leukocyte-endothelium adhesion: microhemodynamics in mesentery of the cat. Microvasc Res 34(3):363Y379, 1987. 23. Wang P, Hauptman JG, Chaudry IH: Hemorrhage produces depression in microvascular blood flow which persists despite fluid resuscitation. Circ Shock 32(4):307Y318, 1990. 24. Schaser KD, Stover JF, Melcher I, Lauffer A, Haas NP, Bail HJ, Stockle U, Puhl G, Mittlmeier TW: Local cooling restores microcirculatory hemodynamics after closed soft-tissue trauma in rats. J Trauma 61(3):642Y649, 2006. 25. Barroso-Aranda J, Schmid-Schonbein GW, Zweifach BW, Engler RL: Granulocytes and no-reflow phenomenon in irreversible hemorrhagic shock. Circ Res 63(2):437Y447, 1988. 26. Childs EW, Udobi KF, Hunter FA, Dhevan V: Evidence of transcellular albumin transport after hemorrhagic shock. Shock 23(6):565Y570, 2005. 27. Kasacka I, Humenczyk-Zybala AM, Niczyporuk M, Mycko G: Morphometric evaluation of murine pulmonary mast cells in experimental hemorrhagic shock. Eur J Histochem 48(2):167Y172, 2004. 28. Santone DJ, Shahani R, Rubin BB, Romaschin AD, Lindsay TF: Mast cell stabilization improves cardiac contractile function following hemorrhagic shock and resuscitation. Am J Physiol Heart Circ Physiol 294(6):H2456YH2464, 2008. 29. Tikiz H, Tuncel N, Gurer F, Baycu C: Mast cell degranulation in hemorrhagic shock in rats and the effects of vasoactive intestinal peptide, aprotinin and H1 and H2-receptor blockers on degranulation. Pharmacology 43(1):47Y52, 1991. 30. Dix R, Orth T, Allen J, Wood JG, Gonzalez NC: Activation of mast cells by systemic hypoxia, but not by local hypoxia, mediates increased leukocyte-endothelial adherence in cremaster venules. J Appl Physiol 95(6):2495Y2502, 2003.
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31. Stefanutti G, Pierro A, Vinardi S, Spitz L, Eaton S: Moderate hypothermia protects against systemic oxidative stress in a rat model of intestinal ischemia and reperfusion injury. Shock 24(2):159Y164, 2005. 32. Kentner R, Rollwagen FM, Prueckner S, Behringer W, Wu X, Stezoski J, Safar P, Tisherman SA: Effects of mild hypothermia on survival and serum cytokines in uncontrolled hemorrhagic shock in rats. Shock 17(6):521Y526, 2002. 33. Sullivan PG, Bruce-Keller AJ, Rabchevsky AG, Christakos S, Clair DK, Mattson MP, Scheff SW: Exacerbation of damage and altered NF-kappaB activation in mice lacking tumor necrosis factor receptors after traumatic brain injury. J Neurosci 19(15):6248Y6256, 1999. 34. Takasu A, Stezoski SW, Stezoski J, Safar P, Tisherman SA: Mild or moderate hypothermia, but not increased oxygen breathing, increases long-term survival after uncontrolled hemorrhagic shock in rats. Crit Care Med 28(7):2465Y2474, 2000. 35. Iyegha UP, Greenberg JJ, Mulier KE, Chipman J, George M, Beilman GJ: Environmental Hypothermia in Porcine Polytrauma and Hemorrhagic Shock is Safe. Shock 38(4):387Y394, 2012. 36. George ME, Mulier KE, Beilman GJ: Hypothermia is associated with improved outcomes in a porcine model of hemorrhagic shock. J Trauma 68(3): 662Y668, 2010. 37. Neligan PJ, Baranov D: Trauma and aggressive homeostasis management. Anesthesiol Clin 31(1):21Y39, 2013. 38. Crossley S, Reid J, McLatchie R, Hayton J, Clark C, Macdougall M, Andrews PJ: A systematic review of therapeutic hypothermia for adult patients following traumatic brain injury. Crit Care 18(2):R75, 2014. 39. Tisherman SA: Salvage techniques in traumatic cardiac arrest: thoracotomy, extracorporeal life support, and therapeutic hypothermia. Curr Opin Crit Care 19(6):594Y598, 2013. 40. Seok J, Warren HS, Cuenca AG, Mindrinos MN, Baker HV, Xu W, Richards DR, McDonald-Smith GP, Gao H, Hennessy L, et al.: Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci U S A 110(9):3507Y3512, 2013. 41. Abonia JP, Friend DS, Austen WG Jr, Moore FD Jr, Carroll MC, Chan R, Afnan J, Humbles A, Gerard C, Knight P, et al.: Mast cell protease 5 mediates ischemia-reperfusion injury of mouse skeletal muscle. J Immunol 174(11): 7285Y7291, 2005.
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INDUCED HYPOTHERMIA DURING RESUSCITATION FROM HEMORRHAGIC SHOCK ATTENUATES MICROVASCULAR INFLAMMATION IN THE RAT MESENTERIC MICROCIRCULATION Garrett N. Coyan,*† Michael Moncure,* James H. Thomas,* and John G. Wood*‡ Departments of *Surgery, † Preventive Medicine and Public Health, and ‡ Molecular & Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas Received 4 May 2014; first review completed 21 May 2014; accepted in final form 16 Jul 2014 ABSTRACT— Microvascular inflammation occurs during resuscitation following hemorrhagic shock, causing multiple organ dysfunction and mortality. Preclinical evidence suggests that hypothermia may have some benefit in selected patients by decreasing this inflammation, but this effect has not been extensively studied. Intravital microscopy was used to visualize mesenteric venules of anesthetized rats in real time to evaluate leukocyte adherence and mast cell degranulation. Animals were randomly allocated to normotensive or hypotensive groups and further subdivided into hypothermic and normothermic resuscitation (n = 6 per group). Animals in the shock groups underwent mean arterial blood pressure reduction to 40 to 45 mmHg for 1 h via blood withdrawal. During the first 2 h following resuscitation by infusion of shed blood plus double that volume of normal saline, rectal temperature of the hypothermic groups was maintained at 32-C to 34-C, whereas the normothermic groups were maintained between 36-C to 38-C. The hypothermic group was then rewarmed for the final 2 h of resuscitation. Leukocyte adherence was significantly lower after 2 h of hypothermic resuscitation compared with normothermic resuscitation: (2.8 T 0.8 vs. 8.3 T 1.3 adherent leukocytes, P = 0.004). Following rewarming, leukocyte adherence remained significantly different between hypothermic and normothermic shock groups: (4.7 T 1.2 vs. 9.5 T 1.6 adherent leukocytes, P = 0.038). Mast cell degranulation index (MDI) was significantly decreased in the hypothermic (1.02 T 0.04 MDI) versus normothermic (1.22 T 0.07 MDI) shock groups (P = 0.038) after the experiment. Induced hypothermia during resuscitation following hemorrhagic shock attenuates microvascular inflammation in rat mesentery. Furthermore, this decrease in inflammation is carried over after rewarming takes place. KEYWORDS—Hemorrhagic shock, resuscitation, hypothermia, leukocyte adherence, mast cell degranulation, mesenteric microcirculation
INTRODUCTION
related systemic hypoxia (3, 5). The generation of reactive oxygen species (ROS) initiates cytokine formation through mediators such as tumor necrosis factor ! (TNF-!) and interleukin 1" (IL-1"), followed by endothelial activation (6). This increases leukocyte adherence and emigration. In addition, mast cells located in the perivascular tissue are activated to release granules via immunoglobulin E (7). These granules contain histamine, additional inflammatory cytokines, and even some anti-inflammatory markers that modulate immune function. This complex pathway leads to localized tissue death due to edema and apoptosis, ending with multiple organ dysfunction and death of the patient. Hypothermia has seen a recent increase in usage as a therapeutic modality (8). A recent case series has shown that inducedhypothermia protocols may be used after traumatic cardiac arrest with positive outcomes (9). Several laboratory studies have sought to clarify the role of hypothermia during resuscitation from hemorrhagic shock and trauma (10Y17). In one study directly examining microvascular inflammation Childs et al. induced mild-moderate hypothermia in rats prior to hemorrhagic shock and resuscitation, and noted a decrease in ROS formation and vascular permeability (18). In this study, however, the effects of hypothermia on shock-induced mast cell degranulation or of rewarming to normal body temperature were not examined.
Trauma continues to be a major cause of morbidity and mortality in the general population (1). Along with traumatic brain injury, hemorrhagic shock shares a large responsibility for these poor outcomes (2). Even with optimal resuscitation, delayed morbidity and mortality occur in this population secondary to microvascular inflammation. Although major efforts have been made to overcome this inflammatory response to injury and resuscitation, little improvement in standardized care has been realized (3). Hemorrhagic shock has been shown to induce microvascular inflammatory responses that are not attenuated with volume resuscitation (4). Various mechanisms of this injury have been hypothesized, most notably ischemia-reperfusion injury with Address reprint requests to John G. Wood PhD, 3901 Rainbow Blvd, Mailstop 3043, Kansas City, KS 66160. E-mail: [email protected]. Co-correspondence: Garrett N. Coyan, MS, 3901 Rainbow Blvd, Mailstop 3043, Kansas City, KS 66160. E-mail: [email protected]. Funding support provided by the Department of Surgery Research Fund and the Department of Molecular & Integrative Physiology, University of Kansas Medical Center. G.C. is supported in part by a CTSA grant from National Center for Research Resources and National Center for Advancing Translational Sciences awarded to the University of Kansas Medical Center for Frontiers: The Heartland Institute for Clinical and Translational Research (no. TL1TR000120). This study was presented as an oral platform presentation at the American College of Surgeons Clinical Congress (Washington, DC) in October 2013. The authors of this article have no conflicts to disclose. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health, National Center for Research Resources, or National Center for Advancing Translational Sciences. DOI: 10.1097/SHK.0000000000000241 Copyright Ó 2014 by the Shock Society
Study objective
Our objective in this study was to determine the effect of induced hypothermia during resuscitation from hemorrhagic shock on microvascular inflammation. Using intravital microscopy, we 518
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SHOCK DECEMBER 2014 have previously demonstrated increased leukocyte adherence in rat mesenteric venules following hemorrhagic shock/resuscitation, as well as an increase in mast cell degranulation (4). The present study was designed to evaluate whether mild hypothermia (32-CY34-C) induced during the period of shock and maintained during resuscitation would attenuate microvascular inflammation. This approach poses the least risk of coagulopathy and is easy to achieve in the trauma patient and maintain without aggressive cooling techniques (19). In addition, we examined the effect of rewarming following resuscitation to determine if the anti-inflammatory effect of hypothermia would persist after body temperature had been restored to normal, a question that has not previously been studied. We evaluated leukocyte adherence and mast cell degranulation, two important indices of microvascular inflammation and dysfunction (5, 20, 21). MATERIALS AND METHODS Regulatory considerations The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center. The University of Kansas Medical Center is accredited by the American Association for Accreditation of Laboratory Animal Care; all procedures were carried out in accordance with the National Institutes of Health guidelines.
Surgical procedures Sprague-Dawley rats weighing 250 to 350 g were obtained from Harlan Laboratories (Indianapolis, Ind). Animals were allowed full access to food and water prior to the experiment. Rats were anesthetized with sodium pentobarbital (40Y60 mg/kg i.m. injection, additional doses as needed). Jugular venous cannulation was performed for administration of fluids and blood with a polyethylene (PE) cannula (PE-50, 0.58-mm internal diameter). Carotid artery cannulation was likewise carried out for withdrawal of blood and monitoring of mean arterial pressure (MAP) with a continuous blood pressure analyzer (Micromed, Louisville, Ky). A tracheostomy was performed with PE-240 tubing to maintain airway patency. Midline laparotomy incision was conducted with radiocautery (Harvard Apparatus, Holliston, Mass).
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was measured using the ImageJ program (National Institutes of Health, Bethesda, Md), and the degree of degranulation was quantified as a mast cell degranulation index (MDI) equal to the ratio of the end of experiment to control period light intensities (5).
Experimental protocol The experimental groups (Fig. 1) consisted of normothermic normotensive animals (n = 6), hypothermic normotensive (n = 6), normothermic shock (n = 6), and hypothermic shock (n = 6). Temperature was recorded with a rectal thermometer and maintained at 36-C to 38-C with a homeothermic blanket and a heat lamp. Animals were allowed to stabilize after surgery for a 30-min control period prior to beginning the experiment. Hemorrhagic shock was induced by withdrawal of blood from the carotid artery into a syringe containing 100 U of heparin. A MAP of 40 to 45 mmHg was maintained for a period of 1 h. At the end of this period, the shed blood was returned to the animal via the jugular cannula, as well as two times the volume of blood in normal saline. For those undergoing hypothermia, animals were allowed to naturally begin cooling during hemorrhagic shock, with a target of 32-C to 34-C at the end of the shock period. Animals needing additional cooling at this point were subjected to air cooling via a fan. The hypothermia was maintained for a 2-h time period, after which the animals underwent rewarming to 36-C to 38-C for 2 h. Leukocyte adherence and physiologic variables were recorded every 15 min throughout the experiment. Mast cell degranulation was recorded at the end of the control period and at the end of the experiment.
Statistical analysis In order to detect at least a difference of four adherent leukocytes between groups, we calculated that six animals per group would be needed using an estimated SD of 2 with 80% power and a type I error of 0.05. Data were examined graphically using quantile plots and compared with historical controls in our laboratory to confirm that our assumption of normality was correct. Data were expressed as mean T SE for continuous variables. Mean arterial pressure and rectal temperature were analyzed with one-way analysis of variance with Bonferroni post hoc correction. Student t test was used to compare differences in leukocyte adherence between hypothermic shock and normothermic shock groups, normothermic normotensive and hypothermic normotensive groups, and each shock group individually with the normothermic normotensive group (acting as a longitudinal baseline comparison) at hourly time points during resuscitation. Shear rate was evaluated in the same manner.
Intravital microscopy The animal was placed in the right lateral decubitus position on a Plexiglas stage mounted to a Zeiss Axiovert inverted microscope. A loop of mesentery was draped over a glass cover slip in the center of the stage to view a mesenteric venule. Saline warmed to 37-C was superfused over the mesentery to prevent drying, and a square of Saran wrap was also placed over the exposed mesentery. Mesenteric venules were selected for study based on the following three criteria: (i) straight, unbranched vessels at least 100 2m in length; (ii) no adjacent vessels within 100 2m of the venule; and (iii) vessel diameter of 20 to 40 2m. Images of the mesentery (40 objective, 10 eyepiece) were recorded using Mini-DV videocassette recorder (Sony, Tokyo, Japan) using a video camera and time-date generator (Panasonic, Kadoma, Japan). Venular diameter was obtained using a video caliper, and an optical Doppler velocimeter was used to measure red blood cell (RBC) centerline velocity in mesenteric venules (Microcirculation Research Institute, Texas A&M University, College Station, Tex). An average RBC velocity was calculated by dividing centerline velocity by 1.6. Shear rate was then calculated as 8 multiplied by RBC velocity divided by venular diameter (22).
Leukocyte adherence and mast cell degranulation Leukocyte adherence and mast cell degranulation were measured during offline playback of recorded videotapes. Leukocyte adherence was measured over each minute by counting the number of leukocytes that remained stationary for longer than 30 s within a 100-2m length of venule (5). Tapes were analyzed two times by an experienced observer to ensure accurate counting of leukocytes; because analysis was carried out by the same staff performing the experiments, blinding of analysis was not performed. Mast cell degranulation was identified by superfusing the mesentery with ruthenium red (Sigma-Aldrich, St Louis, Mo), a chemical that stains degranulated mast cells. Between 13 and 15 mast cells were identified in the same mesenteric loop in relation to the venule identified for study and used to assess the extent of degranulation. Images of these mast cells were recorded during the control period, then once again at the conclusion of the experiment. These images were converted to digital grayscales and phase inverted. Relative light intensity of each mast cell
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FIG. 1. Experimental design and group configuration.
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Paired t test was used to compare shear rates from the control period to the final resuscitation time point within each group to determine change from the control period. Differences in MDI were compared using Student t test. Differences were considered significant when P e 0.05. Statistical analysis was carried out using SAS 9.3 (SAS Institute, Cary, NC).
RESULTS Physiological measures
Figure 2A displays rectal temperatures recorded throughout the experiment. Animals in the hypothermic groups gradually cooled during the 1-h period corresponding with hemorrhagic shock and were maintained at 32-C to 34-C for 2 h following resuscitation. One animal in each of the two hypothermic groups required additional passive cooling with a fan to reach goal at the end-of-the-hour period. Passive loss of heat was adequate to maintain the hypothermic state, and no further cooling measures were needed. The heat lamp and heating pad were used on all animals intermittently to maintain the goal temperature ranges. The rewarming took place over approximately 1 h in the hypothermic groups and was maintained at 36-C to 38-C for the
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remainder of the experiment. The hypothermic groups had significantly lower temperatures than did the normothermic groups from start of resuscitation to 30 min into the rewarming period (P G 0.05). Mean arterial pressure of the animals is displayed in Figure 2B. Shock was maintained for 60 min, and resuscitation provided initial pressures near baseline. This pressure gradually decreased as the experiment went on during the 4-h resuscitation period in all four groups. The shock groups had significantly lower MAPs than did the normotensive groups during the shock period (P G 0.05). In addition, the hypothermic normotensive group had a significantly higher MAP than did the other three groups from 30 min of the shock time period to 30 min into the rewarming period (P G 0.05). Calculated shear rates are shown in Figure 2C. Shear rates in the hypothermic shock group did not significantly change over the course of the experiment when compared with control time period measurements (P 9 0.05), whereas there was a significant decrease within the normothermic shock group over the course of the experiment from the control period measure to the end of 4 h of resuscitation (P G 0.05).
FIG. 2. Physiological parameters: (A) rectal temperature, (B) MAP, (C) shear rate. NSignificant difference between hypothermic and normothermic groups (P G 0.05). *Significant difference between shock and normotensive groups (P G 0.05). #Significant difference between the hypothermic normotensive group and all other groups (P G 0.05).
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FIG. 3. Representative photomicrographs of leukocyte adherence in mesenteric venules: (A) normothermic normotensive group, (B) hypothermic normotensive group, (C) normothermic hypotensive group, (D) hypothermic hypotensive group. Blue arrows indicate adherent leukocytes. Black dots are a part of the optical Doppler velocimeter; white bars outline 100-2m length segment.
Leukocyte adherence
Figure 3 (AYD) shows representative photographs of rat mesenteric venules in the four groups at control, 2 h into resuscitation, and 4 h into resuscitation. The number of adherent leukocytes in a 100-2m length of venule is shown for all four experimental groups in Figure 4. Leukocyte adherence was significantly lower in the hypothermic shock group than in the normothermic shock group at 2 h after resuscitation (2.8 T 0.8 vs. 8.3 T 1.3 adherent leukocytes, P = 0.004), and the significant elevation continued through the end of the 4-h resuscitation time point (4.7 T 1.2 vs. 9.5 T 1.6 adherent leukocytes, P = 0.038). The increase in leukocyte adherence in the hemorrhagic shock groups as compared with the normothermic normotensive animals became significant at 1 h after beginning resuscitation in the normothermic shock group (P G 0.05), whereas the increase never reached significance in the hypothermic shock group (P 9 0.05). Normotensive animals did not have significant changes in leukocyte adherence throughout the experiment.
duced hypothermia during resuscitation reduces the extent of mast cell degranulation following hemorrhagic shock. The decreased leukocyte adherence between hypothermic and normothermic resuscitation was statistically significant after the hypothermic period and furthermore continued to be significantly lower after rewarming occurred. We also observed a significant decrease in shear rates following the resuscitation period as compared with the control period in the normothermic shock cohort, whereas there was no significant decrease during resuscitation of hypothermic animals; this could reflect greater endothelial function in animals undergoing hypothermic resuscitation because there was no significant difference in MAP between groups (23Y25).
Mast cell degranulation
Figure 5 (A and B) shows photographs of mast cells for the hypothermic shock group and the normothermic shock group during the control period and after 4 h of resuscitation. The MDI was significantly lower after the 4-h resuscitation period in the hypothermic shock group as compared with the normothermic shock group (1.02 T 0.04 vs. 1.22 T 0.07 MDI, P = 0.038). No significant degranulation occurred in the normotensive groups. DISCUSSION We have shown that induced hypothermia during resuscitation from hemorrhagic shock significantly attenuates leukocyte adherence and mast cell degranulation in the rat mesenteric microcirculation. This is the first report demonstrating that in-
FIG. 4. Leukocyte adherence in mesenteric venules. *Significant difference between normothermic shock group and normothermic normotensive group (P G 0.05). #Significant difference between normothermic shock group and hypothermic shock group (P G 0.05).
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FIG. 5. Mast cell degranulation: (A) hypothermic hypotensive group; (B) normothermic hypotensive group. Pink color represents uptake of ruthenium red dye due to mast cell degranulation. Blue arrows indicate mast cells being observed. Black dots are part of the optical Doppler velocimeter.
Leukocyte adherence has been shown previously to be a mediator of microvascular dysfunction during hemorrhagic shock (4). The increase in leukocyte adherence has been linked to several other indices of inflammation, such as mast cell degranulation, ROS formation, and increased vascular permeability (4Y6, 26). Mast cell degranulation has been shown to occur throughout the body during hemorrhagic shock (27Y30). We have shown in our laboratory that hypoxia, as well as hemorrhagic shock, causes mast cells to degranulate in response to oxidative stress as well as when inflammatory mediators extravasate through the damaged endothelium (5, 30). A previous study showed that when mast cells are inhibited chemically, ROS and leukocyte adherence still occur with injury, but to a much lesser degree (5). Therefore, the degranulation of mast cells increases ROS formation and leukocyte adherence in the microvasculature. This cycling cascade continues for several hours after injury and is likely responsible for much of the delayed morbidity and mortality in patients with hemorrhagic shock injury. Childs and colleagues (18) showed that pretreating animals with mild and moderate hypothermia prior to hemorrhagic shock attenuated ROS formation and vascular permeability during the resuscitation period. It is important to note that their study pretreated with hypothermia and maintained constant temperature throughout the experiment without examining the actual induction of hypothermia or rewarming. In contrast, we used a model that involved allowing animals to cool and maintaining the hypothermic range as might be done in actual resuscitation settings. We also found that decreased microvascular inflammation persisted after rewarming the animals, a finding not previously reported. This is a key finding for demonstrating the feasibility of the use of hypothermia in the clinical setting. In addition, it has been shown that moderate hypothermia decreases systemic oxidative stress during intestinal ischemia-reperfusion injury in the rat (31). Reduced ROS
formation likely plays a role in the protective effect of hypothermia during resuscitation, but the exact mechanism remains unclear. Future studies should examine which of these proposed mechanisms is responsible for the observed decrease in leukocyte adherence and mast cell degranulation associated with hypothermic resuscitation. Specific knockout animals, inflammatory mediator inhibitors, and well-designed and powered large survival studies will all be required to elucidate the ideal model of hypothermic resuscitation from hemorrhage. Inflammatory mediator modulation may be associated with the protective effect of hypothermia during resuscitation. A study in rats showed that IL-1" and IL-6 were decreased in hypothermic resuscitation, but the study was underpowered to show significant differences (32). Increased levels of IL-1" (9100ug) were associated with early mortality before 72 h in this animal study. The same study found that IL-10, an antiinflammatory cytokine, was elevated in hypothermia. An interesting finding was that TNF-! was significantly increased after hypothermic resuscitation. Although this response seems counterintuitive, delayed increased levels of TNF-! may help to decrease ROS levels via activation of superoxide dismutase and may decrease metabolic demand on myocardium and peripheral tissues (32, 33). These cytokines also play a role in modulating selectins, intracellular adhesion molecules, and mast cells. This could potentially contribute to our findings. Whereas we were unable to study mortality because of the invasive nature of our model, several studies indicate decreased early mortality with mild to moderate hypothermia during resuscitation from hemorrhage in rat models (10, 12, 16, 17). The few studies that did look at long-term outcomes showed either a decrease or no change in mortality (15, 17, 34). A recent porcine laboratory animal model has shown that environmental (natural cooling) hypothermia has similar mortality to normothermia (35). A separate porcine model demonstrated a reduction in mortality 48 h following hemorrhagic shock from
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SHOCK DECEMBER 2014 66% in the normothermic resuscitation group to 7% in the 36-C resuscitation and 0% in the 33-C resuscitation groups (36). In this study, there was evidence of reduced oxygen consumption in the two hypothermic groups, but these two groups required more colloid resuscitation to maintain MAP in the postresuscitation period. These studies are routinely conducted with a small number of animals, and this could account for some of the variations in observed outcomes. It is also important to point out that the methods of hemorrhagic shock induction and hypothermia maintenance differ between studies, making comparing results difficult. We chose to use a pressure-controlled hemorrhagic shock model in our study because of earlier studies suggesting animals with hypothermia had increased MAPs during volumecontrolled shock, which is a confounding factor (11, 12, 17). Findings in humans regarding hypothermia in the trauma population have been largely negative (37). However, a recent case series reported successful outcomes following induced hypothermia after traumatic cardiac arrest (9). In addition, a neuroprotective effect of hypothermia in patients with traumatic brain injury has been demonstrated in several studies (38). These limited uses of induced hypothermia, along with an abundance of preclinical evidence, show that select trauma patients may benefit from appropriate applications of this therapy (39). In fact, one ongoing trial set to begin enrollment soon will be looking at profound induced hypothermia in patients with traumatic cardiac arrest secondary to hemorrhagic shock (Clinicaltrials.gov identifier: NCT01042015). It is imperative that we continue to seek a greater understanding of the mechanisms of induced hypothermia in order to optimize the therapy for the benefit of our patients. Our study has several strengths. We used a pressurecontrolled hemorrhagic shock model in our study in order to limit the increased MAP effect of hypothermia. We allowed the animal to cool during shock and maintained the hypothermia during the first 2 h of resuscitation, simulating real-world clinical conditions that patients may experience in the field or the hospital. We also examined 2 h of rewarming to see if the effect carried over after the therapeutic hypothermia period, a novel aspect specific to this study that is imperative to understand for translation to clinical practice. Our study was also powered adequately to identify the significant differences we observed, a weakness of the few other physiological studies available. There also exist several limitations to our model. Because our model involves nonsurvival surgical methods, we were unable to assess mortality. It remains unclear what the proper duration of hypothermia during the resuscitation period should be; we selected 2 h because of our current hemorrhagic shock models. In addition, our resuscitation technique may not replicate exactly what happens in a trauma-resuscitation scenario. However, this technique was standard in all animals undergoing resuscitation, minimizing opportunity for bias. Because we kept only the subjects hypothermic for the first 2 h of resuscitation, we are unable to predict what effects longer periods of hypothermia may have on microvascular inflammation. We also acknowledge that genetic differences in the response to hemorrhagic shock and resuscitation between laboratory models and humans exist; however, even with different genetic expression between species, similar physiological responses
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can be observed (40, 41). Further translational studies in large animal models, as well as humans, are needed to confirm the benefit of hypothermia during resuscitation from hemorrhage. We conclude that mild induced hypothermia (32-CY34-C) during resuscitation from hemorrhagic shock decreases leukocyte adherence and mast cell degranulation in the rat mesenteric circulation, and importantly this protective effect is carried over after rewarming of the animals. Hypothermia did not cause microvascular inflammation in normotensive animals. Further research is warranted to examine the optimal duration of hypothermia, the optimal degree of cooling, and the effect of rewarming on microvascular inflammation. ACKNOWLEDGMENTS The authors thank Naomi Holloway for invaluable technical support in conducting the experiments. In addition, they thank the Department of Surgery Research Fund, the Department of Molecular & Integrative Physiology, and the Department of Preventive Medicine and Public Health for financial support.
REFERENCES 1. US Department of Health and Human Services, CDC, National Center for Injury Prevention and Control: CDC: Web-Based Injury Statistics Query and Reporting System (WISQARS). Atlanta Georgia: Centers for Disease Control and Prevention, 2012. 2. Kauvar DS, Wade CE: The epidemiology and modern management of traumatic hemorrhage: US and international perspectives. Crit Care 9(Suppl 5): S1YS9, 2005. 3. Eltzschig HK, Eckle T: Ischemia and reperfusionVfrom mechanism to translation. Nat Med 17(11):1391Y1401, 2011. 4. Childs EW, Udobi KF, Wood JG, Hunter FA, Smalley DM, Cheung LY: In vivo visualization of reactive oxidants and leukocyte-endothelial adherence following hemorrhagic shock. Shock 18(5):423Y427, 2002. 5. Steiner DR, Gonzalez NC, Wood JG: Mast cells mediate the microvascular inflammatory response to systemic hypoxia. J Appl Physiol 94(1):325Y334, 2003. 6. Morita Y, Clemens MG, Miller LS, Rangan U, Kondo S, Miyasaka M, Yoshikawa T, Bulkley GB: Reactive oxidants mediate TNF-alphaYinduced leukocyte adhesion to rat mesenteric venular endothelium. Am J Physiol 269(6 Pt 2): H1833YH1842, 1995. 7. Kimura M, Mitani H, Bandoh T, Totsuka T, Hayashi S: Mast cell degranulation in rat mesenteric venule: effects of L-NAME, methylene blue and ketotifen. Pharmacol Res 39(5):397Y402, 1999. 8. Alam HB: Translational barriers and opportunities for emergency preservation and resuscitation in severe injuries. Br J Surg 99(Suppl 1):29Y39, 2012. 9. Tuma MA, Stansbury LG, Stein DM, McQuillan KA, Scalea TM: Induced hypothermia after cardiac arrest in trauma patients: a case series. J Trauma 71(6):1524Y1527, 2011. 10. Li T, Lin X, Zhu Y, Li L, Liu L: Short-term, mild hypothermia can increase the beneficial effect of permissive hypotension on uncontrolled hemorrhagic shock in rats. Anesthesiology 116(6):1288Y1298, 2012. 11. Nishi K, Takasu A, Shibata M, Uchino S, Yamamoto Y, Sakamoto T: Hypothermia reduces resuscitation fluid volumes required to maintain blood pressure in a rat hemorrhagic shock model. J Trauma Acute Care Surg 72(1):130Y135, 2012. 12. Prueckner S, Safar P, Kentner R, Stezoski J, Tisherman SA: Mild hypothermia increases survival from severe pressure-controlled hemorrhagic shock in rats. J Trauma 50(2):253Y262, 2001. 13. Sasaki H, Yukioka T, Ohta S, Fujikawa T, Noda M, Homma H, Mishima S: Is there a self-preserving hypothermic mechanism in shock? Shock 27(4):354Y357, 2007. 14. Subeq YM, Hsu BG, Lin NT, Yang FL, Chao YF, Peng TC, Kuo CH, Lee RP: Hypothermia caused by slow and limited-volume fluid resuscitation decreases organ damage by hemorrhagic shock. Cytokine 60(1):68Y75, 2012. 15. Takasu A, Yanagawa Y, Sakamoto T, Okada Y: Therapeutic hypothermia limited to the resuscitation period does not prolong survival after severe hemorrhagic shock in rats. Resuscitation 67(1):119Y126, 2005. 16. Wu X, Stezoski J, Safar P, Behringer W, Kentner R, Kochanek PM, Tisherman SA: Systemic hypothermia, but not regional gut hypothermia, improves survival from prolonged hemorrhagic shock in rats. J Trauma 53(4):654Y662, 2002. 17. Wu X, Stezoski J, Safar P, Nozari A, Tisherman SA: After spontaneous hypothermia during hemorrhagic shock, continuing mild hypothermia (34 degrees C) improves early but not late survival in rats. J Trauma 55(2):308Y316, 2003. 18. Childs EW, Udobi KF, Hunter FA: Hypothermia reduces microvascular permeability and reactive oxygen species expression after hemorrhagic shock. J Trauma 58(2):271Y277, 2005.
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19. Alam HB, Pusateri AE, Kindzelski A, Egan D, Hoots K, Andrews MT, Rhee P, Tisherman S, Mann K, Vostal J, et al.: Hypothermia and hemostasis in severe trauma: a new crossroads workshop report. J Trauma Acute Care Surg 73(4): 809Y817, 2012. 20. Zilberberg J, Harris NR: Role of shear and leukocyte adherence on venular permeability in the rat mesentery. Microvasc Res 62(3):215Y225, 2001. 21. Morita Y, Clemens MG, Bulkley GB: Delayed Bspontaneous[ adhesion of leukocytes to rat mesenteric venules is dependent upon reactive oxidants. Shock 4(1):41Y44, 1995. 22. House SD, Lipowsky HH: Leukocyte-endothelium adhesion: microhemodynamics in mesentery of the cat. Microvasc Res 34(3):363Y379, 1987. 23. Wang P, Hauptman JG, Chaudry IH: Hemorrhage produces depression in microvascular blood flow which persists despite fluid resuscitation. Circ Shock 32(4):307Y318, 1990. 24. Schaser KD, Stover JF, Melcher I, Lauffer A, Haas NP, Bail HJ, Stockle U, Puhl G, Mittlmeier TW: Local cooling restores microcirculatory hemodynamics after closed soft-tissue trauma in rats. J Trauma 61(3):642Y649, 2006. 25. Barroso-Aranda J, Schmid-Schonbein GW, Zweifach BW, Engler RL: Granulocytes and no-reflow phenomenon in irreversible hemorrhagic shock. Circ Res 63(2):437Y447, 1988. 26. Childs EW, Udobi KF, Hunter FA, Dhevan V: Evidence of transcellular albumin transport after hemorrhagic shock. Shock 23(6):565Y570, 2005. 27. Kasacka I, Humenczyk-Zybala AM, Niczyporuk M, Mycko G: Morphometric evaluation of murine pulmonary mast cells in experimental hemorrhagic shock. Eur J Histochem 48(2):167Y172, 2004. 28. Santone DJ, Shahani R, Rubin BB, Romaschin AD, Lindsay TF: Mast cell stabilization improves cardiac contractile function following hemorrhagic shock and resuscitation. Am J Physiol Heart Circ Physiol 294(6):H2456YH2464, 2008. 29. Tikiz H, Tuncel N, Gurer F, Baycu C: Mast cell degranulation in hemorrhagic shock in rats and the effects of vasoactive intestinal peptide, aprotinin and H1 and H2-receptor blockers on degranulation. Pharmacology 43(1):47Y52, 1991. 30. Dix R, Orth T, Allen J, Wood JG, Gonzalez NC: Activation of mast cells by systemic hypoxia, but not by local hypoxia, mediates increased leukocyte-endothelial adherence in cremaster venules. J Appl Physiol 95(6):2495Y2502, 2003.
COYAN ET
AL.
31. Stefanutti G, Pierro A, Vinardi S, Spitz L, Eaton S: Moderate hypothermia protects against systemic oxidative stress in a rat model of intestinal ischemia and reperfusion injury. Shock 24(2):159Y164, 2005. 32. Kentner R, Rollwagen FM, Prueckner S, Behringer W, Wu X, Stezoski J, Safar P, Tisherman SA: Effects of mild hypothermia on survival and serum cytokines in uncontrolled hemorrhagic shock in rats. Shock 17(6):521Y526, 2002. 33. Sullivan PG, Bruce-Keller AJ, Rabchevsky AG, Christakos S, Clair DK, Mattson MP, Scheff SW: Exacerbation of damage and altered NF-kappaB activation in mice lacking tumor necrosis factor receptors after traumatic brain injury. J Neurosci 19(15):6248Y6256, 1999. 34. Takasu A, Stezoski SW, Stezoski J, Safar P, Tisherman SA: Mild or moderate hypothermia, but not increased oxygen breathing, increases long-term survival after uncontrolled hemorrhagic shock in rats. Crit Care Med 28(7):2465Y2474, 2000. 35. Iyegha UP, Greenberg JJ, Mulier KE, Chipman J, George M, Beilman GJ: Environmental Hypothermia in Porcine Polytrauma and Hemorrhagic Shock is Safe. Shock 38(4):387Y394, 2012. 36. George ME, Mulier KE, Beilman GJ: Hypothermia is associated with improved outcomes in a porcine model of hemorrhagic shock. J Trauma 68(3): 662Y668, 2010. 37. Neligan PJ, Baranov D: Trauma and aggressive homeostasis management. Anesthesiol Clin 31(1):21Y39, 2013. 38. Crossley S, Reid J, McLatchie R, Hayton J, Clark C, Macdougall M, Andrews PJ: A systematic review of therapeutic hypothermia for adult patients following traumatic brain injury. Crit Care 18(2):R75, 2014. 39. Tisherman SA: Salvage techniques in traumatic cardiac arrest: thoracotomy, extracorporeal life support, and therapeutic hypothermia. Curr Opin Crit Care 19(6):594Y598, 2013. 40. Seok J, Warren HS, Cuenca AG, Mindrinos MN, Baker HV, Xu W, Richards DR, McDonald-Smith GP, Gao H, Hennessy L, et al.: Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci U S A 110(9):3507Y3512, 2013. 41. Abonia JP, Friend DS, Austen WG Jr, Moore FD Jr, Carroll MC, Chan R, Afnan J, Humbles A, Gerard C, Knight P, et al.: Mast cell protease 5 mediates ischemia-reperfusion injury of mouse skeletal muscle. J Immunol 174(11): 7285Y7291, 2005.
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