ORIGINAL ARTICLE
Blood pressureYtargeted stepwise resuscitation for hemorrhagic shock in rats Jae Hyuk Lee, MD, PhD, Kyuseok Kim, MD, PhD, You Hwan Jo, MD, PhD, Min A Kim, MD, PhD, Kyoung-Bun Lee, MD, PhD, Joong Eui Rhee, MD, PhD, Ah-Reum Doo, PhD, Min Ji Lee, BSc, Chan Jong Park, MD, Joonghee Kim, MD, and Heajin Chung, MD, Seoul, Korea BACKGROUND: Generation of reactive oxygen species (ROS) is an important mechanism of ischemia-reperfusion injury. Abrupt reoxygenation compared with slow reoxygenation has been known to increase ROS generation. Thus, slow and stepwise reperfusion can reduce ROS generation and subsequent ischemia-reperfusion injury. This study investigated the effect of slow reperfusion by blood pressureYtargeted stepwise resuscitation (PSR) in hemorrhagic shock. METHODS: Pressure-controlled hemorrhagic shock was induced in male Sprague-Dawley rats for 1 hour. Rats were then allocated to one of three groups (no-resuscitation group, n = 14; PSR group, n = 15; rapid normalization of blood pressure (RR) group, n = 15). Survival time and hemodynamic changes were recorded and compared. Blood samples and liver tissue were harvested after 6 hours of resuscitation in surviving rats. RESULTS: All of the rats in the no-resuscitation group were expired before the end of the 6-hour observation period. Survival times were significantly longer in the PSR group than in the RR group (survival rates, 11 of 15 vs. 5 of 15, log rank p = 0.032). Plasma amino alanine transferase, histologic liver injury, and ROS generation in the liver tissue were significantly lower in the PSR group than in the RR group (all findings significant, p G 0.05). In addition, PSR significantly decreased plasma nitric oxide, liver interleukin 1A, and liver interleukin 6 compared with rapid resuscitation in addition to augmenting Akt survival pathways (all p G 0.05). CONCLUSION: Slow reperfusion by PSR decreased mortality, ROS generation, and liver injury in rats undergoing hemorrhagic shock. Stepwise resuscitation also decreased inflammatory cytokine production and augmented Akt survival pathways. (J Trauma Acute Care Surg. 2014;76: 771Y778. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: Ischemia-reperfusion injury; reactive oxygen species; hemorrhagic shock; rats.
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emorrhagic shock and resuscitation causes global ischemiareperfusion (I/R) injury and contributes to subsequent morbidity and mortality. Current guidelines for resuscitation of hemorrhagic shock include control of bleeding and achievement of adequate tissue perfusion with fluid and blood products.1Y3 However, no specific guidelines exist regarding reperfusion in hemorrhagic shock. Rapid normalization of blood pressure may be the general consensus when bleeding is controlled or when there is no ongoing blood loss. Cellular hypoxia and reoxygenation are a basic component of I/R injury. Cells or tissues injured by ischemia may recover following reperfusion. However, reperfusion itself may exacerbate injury by complex mechanisms including generation of reactive oxygen species (ROS),4 activation of destructive enzymatic reactions,5 activation of cell death pathways, and proinflammatory reactions.6,7 Of those, ROS generation is one of the most important mechanisms in I/R injury;4 thus, decreasing or scavenging ROS is an important method for reducing reperfusion injury.
ROS generation in endothelial cells has been reported to be much higher in abrupt reoxygenation than in slow reoxygenation.8 Moreover, in animal models of isolated coronary ischemia, gradual reperfusion produced less edema and functional loss compared with sudden reperfusion.9Y12 In lung transplantation models, high-flow reperfusion resulted in severe reperfusion injury.13Y15 Although the precise mechanisms have not been identified, decreased endothelial injury, transient acidosis, and inhibition of mitochondrial permeability transition pore were suggested as beneficial mechanisms of lowpressure reperfusion.9,16 Thus, reducing the reperfusion speed may attenuate injury in ischemic cells or tissues. Given this context, our study was designed to investigate the effect of reperfusion speed by blood pressureYtargeted stepwise reperfusion on mortality and liver injury in hemorrhagic shock. We hypothesized that pressure-targeted stepwise resuscitation would decrease mortality and liver injury in rats in hemorrhagic shock.
MATERIALS AND METHODS Submitted: August 25, 2013, Revised: October 28, 2013, Accepted: November 4, 2013. From the Department of Emergency Medicine (J.H.L., K.K., Y.H.J., J.E.R., A.R.D., M.J.L., C.J.P., J.K., H.C.), Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do; and Department of Pathology (M.A.K, K.B.L.), Seoul National University Hospital, Seoul, Korea. Address for reprints: Kyuseok Kim, MD, PhD, Department of Emergency Medicine, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-707, Korea; email: [email protected]. DOI: 10.1097/TA.0000000000000106
This study was approved by the Animal Care and Use Committee of our hospital and was conducted in accordance with National Institute of Health guidelines.
Animal Preparation Male Sprague-Dawley rats weighing 300 g to 350 g were used in the study. The rats were housed in a controlled
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environment with free access to food and water for 1 week before the experiment. Rats were anesthetized with an intramuscular injection of zoletil (3 mg/kg, Virbac, France) and xylazine (15 mg/kg, Bayer, Korea). Tracheostomy was performed using a 14-gauge catheter (BD Insyte, Autoguard, NJ), and rats were connected to a ventilator (tidal volume, 2 mL; respiratory rate, 55 breaths/min; FIO2, 0.21; Harvard rodent ventilator model 645, Harvard Apparatus, Holliston, MA). Anesthesia was maintained by inhalation of 1% isoflurane via mechanical ventilator. The minute ventilation was adjusted to ensure a PaCO2 between 35 mm Hg and 40 mm Hg according to the results of arterial blood gas analysis. Rectal temperature was monitored and controlled at 36.5-C to 37.0-C using a heating lamp. A sterile cut down was performed in the groin bilaterally, and 24-gauge intravascular catheters (BD Insyte) were inserted into both femoral arteries for blood pressure monitoring and for blood withdrawal or transfusion. Blood samples (0.3 mL at a time) were used for blood gas analysis and replaced with 0.9 mL of normal saline.
Experimental Procedure (Fig. 1) A pressure-controlled hemorrhagic shock rat model was prepared as described in a previous report.17 Briefly, hemorrhagic shock was induced by blood withdrawal. Mean arterial pressure (MAP) was rapidly decreased to 38 T 1 mm Hg and maintained for 60 minutes by blood withdrawal or reinfusion. Shed blood was collected in heparinized syringes (heparin, 7.5 IU/mL),18 kept warm, and stirred frequently. After 60 minutes of shock, rats were randomized into one of three groups (no-resuscitation group vs. rapid resuscitation [RR] group vs. pressure-targeted stepwise resuscitation [PSR] group). Baseline MAP values were approximately 70 mm Hg in our experimental setting. The no-resuscitation group was observed after the shock period without any fluid or blood transfusion. The RR group was resuscitated to reach a MAP of 70 mm Hg immediately by transfusion of shed blood, and MAP was maintained until the entire volume of shed blood was
transfused. The PSR group was resuscitated by increasing the MAP by 5 mm Hg for every 3 minutes up to 70 mm Hg and then maintained until the remaining shed blood was transfused. No additional fluid was administered to reach the target MAP value even if the target MAP was not achieved by transfusion of shed blood. Rats were observed until death or 6 hours after the shock period. MAP and heart rate (HR) were continuously monitored, and total transfusion times and survival time were recorded. Surviving rats were euthanized by exsanguination, and blood samples were obtained. The blood was centrifuged for 15 min at 3,000 rpm, and the plasma was stored at j70-C until analysis. The liver tissues were harvested and fixed with 4% formalin or stored in j70-C liquid nitrogen.
DCF-DA [2¶, 7¶-Dichlorofluorescein Diacetate] Assay The relative levels of ROS were measured by DCF-DA (Sigma, St. Louis, MO) assay. Frozen liver tissues were weighed, thawed, and homogenized in 500-KL ice-cold RIPA buffer (20-mM Tris-Hcl, pH 7.5, 150-mM NaCl, 1-mM EDTA, 0.5% Ninidet P-40, and 5 Kg/mL; Intron Biotechnology Inc.) and then centrifuged at 2,000 rpm at 4-C for 10 minutes. The supernatant was added to the reaction mixture containing DCFDA (10 KM/mL) and incubated for 60 minutes at 37-C. The fluorescence intensity was determined by a fluorescence plate reader, with an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Values were presented as fluorescence intensity per milligram of tissue or percentage of negative control.
Cytokines and Nitric Oxide Inflammatory cytokines including interleukin 1A (IL-1A) and IL-6 were measured in liver tissue using an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN). Plasma nitric oxide (NO) was measured using a commercial kit for nitrite/nitrate by using the Griess reaction (R&D Systems).
Figure 1. Study protocol of animal experiment. ABGA, arterial blood gas analysis; T, time. 772
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Western Blot Analysis Western blot analysis was performed in liver tissue samples. Primary antibodies against Bcl-2, Akt (total), phospho-Akt (Ser473), GSK-3A (total), phospho-GSK-3A (Ser9), Bad (total), phospho-Bad (Ser136), and cleaved caspase-3 (Asp175) were purchased from Cell Signaling Technology (MA). Primary antibodies against A-actin were purchased from Santa-Cruz Biotechnology. Proteins (approximately 100 Kg per lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were blocked in 0.05% PBSTween containing 5% milk (Bio-Rad Laboratories) and then incubated with the primary antibody at 4-C overnight. The primary antibody was detected by incubation with the horseradish peroxidaseYcoupled second antibody (1:3,000 in PBSTween with 5% milk) at room temperature for 2 hours. The chemiluminescence detection was performed by using Western Lighting Chemiluminescence Reagant Plus (Perkin Elmer, MA). Films were developed using a standard photographic
procedure, and quantitative analyses of bands were performed using Versa Doc Imaging System (Bio-Rad Laboratories, CA).
Blood Gas Analysis, Lactate, and Alanine Aminotransferase Arterial blood gas analysis and lactate measurements were performed at baseline, immediately after the shock period, at 2 hours of resuscitation, and at 6 hours of resuscitation. Plasma alanine aminotransferase (ALT) was measured with VetScan VS2 (Abaxis, Inc., CA).
Assessment of Histologic Liver Injury Formalin-fixed liver tissues were embedded in paraffin. The paraffin blocks were sectioned at 4-Km thickness and stained with hematoxylin and eosin. The stained samples were reviewed blindly by two pathologists using light microscope, and liver injury was scored using the previously reported morphologic criteria19: spotty necrosis, capsular inflammation, portal inflammation, ballooning degeneration, and steatosis. The liver Injury Severity Score (ISS) ranged from 0 (none) to 16 (severe).
TABLE 1. Bleeding Volume, Arterial Blood Gas Analysis, and Lactate Baseline Body weight, g No resuscitation RR PSR Bleeding volume, mL/100 g No resuscitation RR PSR pH No resuscitation RR PSR PCO2, mm Hg No resuscitation RR PSR PO2, mm Hg No resuscitation RR PSR HCO3j, mmol/L No resuscitation RR PSR Base excess, mmol/L No resuscitation RR PSR Lactate, mmol/L No resuscitation RR PSR
Shock
Transfusion 2 h
Transfusion 6 h
330.5 T 6.6 (14) 329.9 T 3.9 (15) 326.4 T 2.8 (15) 4.38 T 0.3 (14) 4.30 T 0.1 (15) 4.48 T 0.2 (15) 7.402 T 0.006 (14) 7.393 T 0.006 (15) 7.391 T 0.003 (15)
7.287 T 0.013 (14) 7.276 T 0.010 (15) 7.299 T 0.010 (15)
7.254 T 0.035 (4) 7.286 T 0.035 (8) 7.289 T 0.008 (12)
V 7.191 T 0.059 (5)* 7.299 T 0.012 (11)
35.1 T 0.9 (14) 35.0 T 1.7 (15) 35.1 T 0.8 (15)
31.6 T 1.7 (14) 31.4 T 1.8 (15) 27.5 T 1.3 (15)
28.7 T 3.3 (4) 30.6 T 1.5 (8) 33.7 T 2.3 (12)
V 37.8 T 3.3 (5) 32.6 T 2.0 (11)
73.2 T 2.9 (14) 79.0 T 2.7 (15) 71.6 T 5.1 (15)
68.2 T 2.4 (14) 68.4 T 2.4 (15) 70.1 T 2.4 (15)
72.3 T 4.3 (4) 58.7 T 5.1 (8) 50.7 T 2.0 (12)
V 61.8 T 3.8 (5) 68.3 T 7.0 (11)
21.5 T 0.9 (14) 21.5 T 0.9 (15) 21.5 T 0.4 (15)
15.1 T 0.5 (14) 14.6 T 0.7 (15) 13.7 T 0.5 (15)
12.7 T 1.3 (4) 14.8 T 1.2 (8) 16.3 T 1.1 (12)
V 14.7 T 1.7 (5) 16.2 T 1.1 (11)
j3.73 T 0.58 (14) j3.73 T 0.91 (15) j3.75 T 0.45 (15) 0.7 T 0.0 (14) 1.1 T 0.1 (15) 1.0 T 0.1 (15)
j11.84 T 0.44 (14) j12.47 T 0.62 (15) j12.86 T 0.49 (15) 4.5 T 0.3 (14) 5.3 T 0.4 (15) 4.6 T 0.3 (15)
j14.73 T 1.48 (4) j12.13 T 1.69 (8) j10.53 T 1.18 (12) 5.2 T 1.0 (4) 2.9 T 0.9 (8) 2.0 T 0.3 (12)
V j13.78 T 2.41 (5) j10.53 T 1.22 (11) V 4.3 T 2.4 (5) 1.4 T 0.2 (11)
*RR group versus PSR group at transfusion 6 hours, p G 0.05. Number in parenthesis indicates number of animals.
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STATISTICAL ANALYSIS Data were expressed as mean T SEM. Normality testing was performed using the Kolmogorov-Smirnov test. The Student’s t test for normally distributed data and the MannWhitney U-test for nonnormally distributed data were performed for comparison between the groups. For analysis of difference in histologic score, a modified Z score using median absolute deviation was used, and the histologic injury scores were compared with Mann-Whitney U-test.20 A repeatedmeasures analysis of variance was performed to compare hemodynamic changes between the groups. A Kaplan-Meier survival analysis was performed for the comparison of survival time. A p G 0.05 was considered significant.
RESULTS A total of 44 rats (no-resuscitation group [n = 14], RR group [n = 15], PSR group [n = 15]) were used for the study. Baseline characteristics including body weight, bleeding volume, arterial blood gas values, and lactate did not differ significantly between the groups (Table 1).
Effect of Pressure-Targeted Stepwise Resuscitation on the Survival Rates During the 6-hour observation period, all of the rats in the no-resuscitation group were expired (survival rate, 0.0%), 5 rats in the RR group survived (survival rate, 33.3%) and 11 rats in the PSR group survived (survival rate, 73.3%). Survival rates were significantly higher in the PSR group than in the RR group (log rank p = 0.032, Fig. 2A).
Effect of Pressure-Targeted Stepwise Resuscitation on Transfusion Time, Blood Gases, and Hemodynamic Change Total transfusion time was significantly longer in the PSR group than in the RR group (8.5 T 3.0 minutes in the RR group and 27.8 T 7.9 minutes in the PSR group, p = 0.024, Fig. 2B). Arterial blood gas values were not significantly different between the groups during the experimental period with the exception of blood pH at 6 hours of resuscitation, which was significantly lower in the RR group than in the PSR group (p = 0.020, Table 1). Figure 2 shows the observed hemodynamic changes. HR did not significantly differ between the groups (Fig. 2C). However, MAP was significantly higher in the PSR group than in the RR group (group, p G 0.05; time, p G 0.05; group time interaction, p G 0.05, Fig. 2D)
Effect of Pressure-Targeted Stepwise Resuscitation on Liver Injury, Generation of ROS, Inflammatory Cytokines, and NO Plasma ALT was significantly higher in the RR group than in the PSR group (p = 0.032, Fig. 3A), and liver injury score was significantly lower in the PSR group than in the RR group (p = 0.024, Fig. 3B). The ROS generation in liver tissue samples was significantly lower in the PSR group than in the RR group (p = 0.039, Fig. 3C). In addition, liver tissue IL-1A and IL-6 levels were significantly lower in the PSR group than in the RR group, while plasma NO was also significantly lower 774
in the PSR group than in the RR group (all p G 0.05, respectively, Fig. 3D).
Effect of Pressure-Targeted Stepwise Resuscitation on the Akt Survival Pathway in Liver Tissue Phosphorylation of Akt, GSK-3A, and Bad as well as expression of Bcl-2 were significantly increased in the PSR group as compared with the RR group (all p G 0.05, Fig. 4AYD). In addition, expression of cleaved caspase 3 was significantly lower in the PSR group (p G 0.05, Fig. 4E).
DISCUSSION This study demonstrated that blood pressureYtargeted stepwise resuscitation for the management of hemorrhagic shock in rats resulted in decreased ROS generation, mortality, and liver injury. Moreover, pressure-targeted stepwise resuscitation decreased inflammatory cytokine production and enhanced Akt survival pathways. Previous evidence demonstrated that a constant rate of crystalloid infusion for resuscitating fixed-volume hemorrhagic shock in dogs improved hemodynamic parameters when compared with impulse crystalloid infusion.21 Although the study did not investigate the precise mechanism, the blood volume restitution was significantly improved by a constant rate of infusion when compared with impulse infusion. In addition, Bark et al.22 also reported a greater plasma volume expansion with slow rates of colloid infusion than with faster infusion rates in septic rats. Thus, the improved hemodynamics and survival of the pressure-targeted stepwise resuscitation group in our study may be a result of improved blood volume restitution during resuscitation of hemorrhagic shock. Reintroduction of oxygen to ischemic cells inevitably results in reperfusion injury. Many possible mechanisms of reperfusion injury have been suggested, with ROS generation regarded as one of the most important mechanisms.4,23 Generation of ROS is known to begin with the onset of ischemia24 and gradually increases as the ischemic period is prolonged, reaching a peak for a short period after reperfusion.25,26 Oxygen delivered by reperfusion is an important source of ROS generation. Previous evidence demonstrates that the ischemic pattern of ROS generation is unchanged with hypoxic reperfusion and that ROS generation spikes with oxygenated reperfusion.27 Initially, endogenous antioxidant enzymes may balance the generation of ROS.28 However, increased ROS generation may overwhelm the capacity of antioxidant defense system and result in cellular injury.29 Considering that ROS generation is much higher following abrupt reoxygenation,8 it is possible that ROS generation during slow reoxygenation will be balanced by the endogenous antioxidant defense system, allowing post-reoxygenation survival of ischemic cells. Systemic oxygen delivery is dependent on cardiac output and the arterial blood oxygen content. Considering that blood pressure is dependent on cardiac output and systemic vascular resistance, a slow increase in systemic blood pressure by slow transfusion and subsequent increase in preload can lead to more gradual oxygen delivery as more compared with rapid normalization of blood pressure. In our study, the arterial * 2014 Lippincott Williams & Wilkins
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Figure 2. Survival and hemodynamic effect of blood pressureYtargeted stepwise resuscitation in hemorrhagic shock. A, Kaplan-Meier survival curve. B, Time required to transfuse whole shed blood (RR group, n = 15; PSR group, n = 15). C, HR change (no-resuscitation group, n = 14; RR group, n = 15; PSR group, n = 15). D, MAP change, PSR group showed significantly higher MAP than in RR group (group, p G 0.05; time, p G 0.05; group time interaction, p G 0.05) (no-resuscitation group, n = 14; RR group, n = 15; PSR group, n = 15).
Figure 3. Effect of blood pressureYtargeted stepwise resuscitation on liver injury and inflammation. A, Plasma ALT (RR group, n = 5; PSR group, n = 11). B, Representative photos and histologic liver injury score (original magnification 200, hematoxylin and eosin stain; 0 = none; 16 = severe; RR group, n = 4; PSR group, n = 11). C, Generation of ROS (RR group, n = 5; PSR group, n = 11). D, Plasma inflammatory cytokine and NO (RR group, n = 5; PSR group, n = 11). * 2014 Lippincott Williams & Wilkins
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Figure 4. Effect of blood pressureYtargeted stepwise resuscitation on Akt survival pathway in liver tissue. A, Phosphorylation of Akt. B, Phosphorylation of GSK-3A. C, Phosphorylation of Bad. D, Bcl-2 expression. E, Cleaved caspase 3 expression. RR group, n = 5; PSR group, n = 11.
oxygen content may have been temporarily much lower in the PSR group than in the RR group because the time required to transfuse the shed blood was significantly longer in the PSR group than in the RR group. Overall, stepwise resuscitation may deliver oxygen more slowly and gradually than rapid resuscitation, leading to reduced ROS production and subsequent reduced tissue injury and mortality. Production of ROS was estimated by DCF-DA assay in this study. Although the difference in DCF-DA fluorescence intensity between groups was minor, it was significantly decreased in the stepwise resuscitation group compared with the rapid resuscitation group. Cellular ischemic stress results in ROS production, which may be inevitable in hemorrhage patients. However, reintroduction of oxygen by abrupt reperfusion increases ROS production, causing additional reperfusion injury. Thus, decreasing ROS generation by stepwise resuscitation may be a somewhat effective strategy in resuscitating hemorrhage patients. In this study, slow reperfusion was performed according to predetermined target of blood pressure and duration. There may be several considerations to determine reperfusion methods. First, prolonged reperfusion duration can increase the relative ischemic period, which may counteract the protective effects of slow reoxygenation in animals. Second, individual animals may respond differently to the transfusion rate and amount. These differences could originate from differences in cardiac function and systemic vascular resistance. Thus, a constant rate of transfusion would result in differences in tissue perfusion in experimental animals. To overcome this limitation, we used a blood pressureYtargeted strategy. In addition, considering that the objective of our study was to achieve a relatively 776
slow oxygen delivery to cells or tissues in a reproducible manner, our method might be a more appropriate approach. In this study, slow reperfusion with a predetermined pressure target decreased histologic liver injury and augmented Akt survival pathways. Previous reports demonstrate that staged reperfusion for coronary ischemia in a canine model reduced myocardial stunning, suggesting that this phenomenon may be caused by transient acidosis caused by slow reperfusion in ischemic tissues.9 Transient cellular acidosis has been suggested as one of the protective mechanisms of ischemic conditioning.30,31 Although we did not examine the effect of slow reperfusion on cellular or tissue acidosis in our study, we measured the protein expression associated with Akt survival pathways and found that stepwise resuscitation augmented Akt survival pathways. Ischemic postconditioning has been reported to be associated with augmentation of PI3K/Akt survival pathways in various disease models, including stroke, coronary ischemia, and hepatic I/R injury.32Y34 Although the precise mechanism of ischemic postconditioning is unclear, we previously postulated that a gradual and stepwise increase in blood pressure for the management of hemorrhagic shock could decrease tissue injury and mortality by mimicking ischemic postconditioning.35 Ischemic postconditioning can be performed by initiating a short period of interruption of perfusion before full restoration of perfusion. It is possible that ischemic postconditioning may produce a transitional period of oxygen supply between ischemia and full perfusion restoration. Thus, a gradual and stepwise increase in blood pressure for the management of hemorrhagic shock may at least partially mimic ischemic postconditioning with regard to oxygen delivery to * 2014 Lippincott Williams & Wilkins
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ischemic tissues. To define the association between stepwise resuscitation and the mechanism of ischemic postconditioning in hemorrhagic shock, further studies are required, including the change in cellular acidosis and the opening of mitochondrial permeability transition pore associated with slow reperfusion. In this study, adequate blood pressure was maintained for a significantly longer period in the PSR group than in the RR group. While the precise mechanism of this phenomenon was not investigated in this study, a possible cause is NO production. Plasma NO levels were significantly lower in the PSR group than in the RR group. It has been reported that I/R results in profound production of NO, which can decrease systemic vasomotor tone.36 In addition, decreased NO production during delayed reoxygenation of ischemic myocardium has been reported.37 Thus, decreased NO production might be related to vasomotor tone and could contribute to the long transfusion time in the PSR group in our model. Although oxygen is absolutely necessary for all vertebrate animals, excessive oxygen can cause harm by increasing ROS. In this study, supplemental oxygen was not administered throughout the experiment, as we postulated that supplemental oxygen might mitigate the effects of a gradual increase in oxygen delivery in the PSR group. However, hemorrhagic shock and resuscitation causes acute lung injury, which can disturb gas exchange and cause mortality. Thus, supplemental oxygen should be administered and, if systemic oxygenation is impaired by acute lung injury, titrated accordingly. In this study, we measured proinflammatory cytokines and found that slow reperfusion decreased levels of IL-1A and IL-6. IL-1A production is known to be mediated by the NLRP3 inflammasome, which is activated by ROS.38 IL-6 production is known to be associated with released damage-associated molecular pattern molecules released from injured cells via toll-like receptor signaling.7 Although the precise mechanism associated with inflammatory cytokine production was not investigated in our study, reperfusion speed might be associated with proinflammatory cytokine production. Our study has several limitations. First, the exact and precise mechanism of slow reperfusion leading to decreased cellular and tissue injury was not identified with the exception of ROS generation and plasma NO. In addition, different rates of reperfusion speed on I/R injury were not investigated, and further studies are warranted. Second, supplemental oxygen was not administered owing to the reasoning discussed previously. Supplemental oxygen is known to be helpful in the management of systemic hypoxemia caused by disturbances in gas exchange. Third, the pressure-controlled hemorrhagic shock model used in this study may not be clinically relevant. However, the model of hemorrhagic shock used was chosen because a similar amount of ischemic insults was mandatory to compare the effects of gradual reperfusion based on predetermined pressure targets with rapid normalization of blood pressure. Further studies in clinically relevant large animal model are warranted. In conclusion, slow reperfusion by blood pressureY targeted stepwise reperfusion decreased mortality and liver injury in the management of hemorrhagic shock. This stepwise
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method also decreased ROS production and inflammatory cytokine expression as well as augmented Akt survival pathways. AUTHORSHIP J.H.L., K.K., Y.H.J., M.A.K., K.-B.L., M.J.L., A.-R.D., and J.E.R. contributed in the conception and design. J.H.L., C.J.P., J.K., H.C., M.J.L., M.A.K., K.B.L., and A.-R.D. conducted the acquisition of data. J.H.L., M.A.K., C.J.P., J.K., H.C., K.-B.L., Y.H.J., and K.K. conducted the analysis and interpretation. J.H.L., K.K., Y.H.J., J.E.R., M.A.K., and K.-B.L. drafted the manuscript for important intellectual content.
DISCLOSURE This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A2042541).
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17. Lee JH, Jo YH, Kim K, Lee JH, Rim KP, Kwon WY, Suh GJ, Rhee JE. Effect of N-acetylcysteine (NAC) on acute lung injury and acute kidney injury in hemorrhagic shock. Resuscitation. 2013;84(1):121Y127. 18. Kim K, Kim W, Rhee JE, Jo YH, Lee JH, Kim KS, Kwon WY, Suh GJ, Lee CC, Singer AJ. Induced hypothermia attenuates the acute lung injury in hemorrhagic shock. J Trauma. 2010;68(2):373Y381. 19. Muftuoglu MA, Aktekin A, Ozdemir NC, Saglam A. Liver injury in sepsis and abdominal compartment syndrome in rats. Surg Today. 2006;36(6): 519Y524. 20. Iglewicz B, Hoaglin DC. How to Detect and Handle Outliers. Milwaukee, WI: ASQC Quality Press; 1993:ix, 87. 21. Lilly MP, Gala GJ, Carlson DE, Sutherland BE, Gann DS. Saline resuscitation after fixed-volume hemorrhage. Role of resuscitation volume and rate of infusion. Ann Surg. 1992;216(2):161Y171. 22. Bark BP, Persson J, Grande PO. Importance of the infusion rate for the plasma expanding effect of 5% albumin, 6% HES 130/0.4, 4% gelatin, and 0.9% NaCl in the septic rat. Crit Care Med. 2013;41(3):857Y866. 23. Raedschelders K, Ansley DM, Chen DD. The cellular and molecular origin of reactive oxygen species generation during myocardial ischemia and reperfusion. Pharmacol Ther. 2012;133(2):230Y255. 24. Peters O, Back T, Lindauer U, Busch C, Megow D, Dreier J, Dirnagl U. Increased formation of reactive oxygen species after permanent and reversible middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab. 1998;18(2):196Y205. 25. Nelson CW, Wei EP, Povlishock JT, Kontos HA, Moskowitz MA. Oxygen radicals in cerebral ischemia. Am J Physiol. 1992;263(5 Pt 2): H1356YH1362. 26. Garlick PB, Davies MJ, Hearse DJ, Slater TF. Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy. Circ Res. 1987;61(5):757Y760. 27. Zweier JL, Flaherty JT, Weisfeldt ML. Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci U S A. 1987;84(5):1404Y1407.
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28. Zhang Y, Du Y, Le W, Wang K, Kieffer N, Zhang J. Redox control of the survival of healthy and diseased cells. Antioxid Redox Signal. 2011; 15(11):2867Y2908. 29. Gutteridge JM. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem. 1995;41(12 Pt 2):1819Y1828. 30. Inserte J, Barba I, Hernando V, Garcia-Dorado D. Delayed recovery of intracellular acidosis during reperfusion prevents calpain activation and determines protection in postconditioned myocardium. Cardiovasc Res. 2009;81(1):116Y122. 31. Cohen MV, Yang XM, Downey JM. The pH hypothesis of postconditioning: staccato reperfusion reintroduces oxygen and perpetuates myocardial acidosis. Circulation. 2007;115(14):1895Y1903. 32. Xie R, Wang P, Ji X, Zhao H. Ischemic post-conditioning facilitates brain recovery after stroke by promoting Akt/mTOR activity in nude rats. J Neurochem. 2013;127:723Y732. 33. Wu QL, Shen T, Shao LL, Ma H, Wang JK. Ischemic postconditioning mediates cardioprotection via PI3K/GSK-3A/A-catenin signaling pathway in ischemic rat myocardium. Shock. 2012;38(2):165Y169. 34. Guo JY, Yang T, Sun XG, Zhou NY, Li FS, Long D, Lin T, Li PY, Feng L. Ischemic postconditioning attenuates liver warm ischemia-reperfusion injury through Akt-eNOS-NO-HIF pathway. J Biomed Sci. 2011;18:79. 35. Lee JH, Kim K, Jo YH, Kang KW, Rhee JE, Park CJ, Kim J, Chung H. Gradual and stepwise increase of blood pressure in hemorrhagic shock: mimicking ischemic post-conditioning. Med Hypotheses. 2013;81(4): 701Y703. 36. Thiemermann C, Szabo C, Mitchell JA, Vane JR. Vascular hyporeactivity to vasoconstrictor agents and hemodynamic decompensation in hemorrhagic shock is mediated by nitric oxide. Proc Natl Acad Sci U S A. 1993;90(1):267Y271. 37. Morita K, Ihnken K, Buckberg GD, Sherman MP, Young HH, Ignarro LJ. Role of controlled cardiac reoxygenation in reducing nitric oxide production and cardiac oxidant damage in cyanotic infantile hearts. J Clin Invest. 1994;93(6):2658Y2666. 38. Chen GY, Nunez G. Sterile inflammation: sensing and reacting to damage. Nat Rev Immunol. 2010;10(12):826Y837.
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Blood pressureYtargeted stepwise resuscitation for hemorrhagic shock in rats Jae Hyuk Lee, MD, PhD, Kyuseok Kim, MD, PhD, You Hwan Jo, MD, PhD, Min A Kim, MD, PhD, Kyoung-Bun Lee, MD, PhD, Joong Eui Rhee, MD, PhD, Ah-Reum Doo, PhD, Min Ji Lee, BSc, Chan Jong Park, MD, Joonghee Kim, MD, and Heajin Chung, MD, Seoul, Korea BACKGROUND: Generation of reactive oxygen species (ROS) is an important mechanism of ischemia-reperfusion injury. Abrupt reoxygenation compared with slow reoxygenation has been known to increase ROS generation. Thus, slow and stepwise reperfusion can reduce ROS generation and subsequent ischemia-reperfusion injury. This study investigated the effect of slow reperfusion by blood pressureYtargeted stepwise resuscitation (PSR) in hemorrhagic shock. METHODS: Pressure-controlled hemorrhagic shock was induced in male Sprague-Dawley rats for 1 hour. Rats were then allocated to one of three groups (no-resuscitation group, n = 14; PSR group, n = 15; rapid normalization of blood pressure (RR) group, n = 15). Survival time and hemodynamic changes were recorded and compared. Blood samples and liver tissue were harvested after 6 hours of resuscitation in surviving rats. RESULTS: All of the rats in the no-resuscitation group were expired before the end of the 6-hour observation period. Survival times were significantly longer in the PSR group than in the RR group (survival rates, 11 of 15 vs. 5 of 15, log rank p = 0.032). Plasma amino alanine transferase, histologic liver injury, and ROS generation in the liver tissue were significantly lower in the PSR group than in the RR group (all findings significant, p G 0.05). In addition, PSR significantly decreased plasma nitric oxide, liver interleukin 1A, and liver interleukin 6 compared with rapid resuscitation in addition to augmenting Akt survival pathways (all p G 0.05). CONCLUSION: Slow reperfusion by PSR decreased mortality, ROS generation, and liver injury in rats undergoing hemorrhagic shock. Stepwise resuscitation also decreased inflammatory cytokine production and augmented Akt survival pathways. (J Trauma Acute Care Surg. 2014;76: 771Y778. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: Ischemia-reperfusion injury; reactive oxygen species; hemorrhagic shock; rats.
H
emorrhagic shock and resuscitation causes global ischemiareperfusion (I/R) injury and contributes to subsequent morbidity and mortality. Current guidelines for resuscitation of hemorrhagic shock include control of bleeding and achievement of adequate tissue perfusion with fluid and blood products.1Y3 However, no specific guidelines exist regarding reperfusion in hemorrhagic shock. Rapid normalization of blood pressure may be the general consensus when bleeding is controlled or when there is no ongoing blood loss. Cellular hypoxia and reoxygenation are a basic component of I/R injury. Cells or tissues injured by ischemia may recover following reperfusion. However, reperfusion itself may exacerbate injury by complex mechanisms including generation of reactive oxygen species (ROS),4 activation of destructive enzymatic reactions,5 activation of cell death pathways, and proinflammatory reactions.6,7 Of those, ROS generation is one of the most important mechanisms in I/R injury;4 thus, decreasing or scavenging ROS is an important method for reducing reperfusion injury.
ROS generation in endothelial cells has been reported to be much higher in abrupt reoxygenation than in slow reoxygenation.8 Moreover, in animal models of isolated coronary ischemia, gradual reperfusion produced less edema and functional loss compared with sudden reperfusion.9Y12 In lung transplantation models, high-flow reperfusion resulted in severe reperfusion injury.13Y15 Although the precise mechanisms have not been identified, decreased endothelial injury, transient acidosis, and inhibition of mitochondrial permeability transition pore were suggested as beneficial mechanisms of lowpressure reperfusion.9,16 Thus, reducing the reperfusion speed may attenuate injury in ischemic cells or tissues. Given this context, our study was designed to investigate the effect of reperfusion speed by blood pressureYtargeted stepwise reperfusion on mortality and liver injury in hemorrhagic shock. We hypothesized that pressure-targeted stepwise resuscitation would decrease mortality and liver injury in rats in hemorrhagic shock.
MATERIALS AND METHODS Submitted: August 25, 2013, Revised: October 28, 2013, Accepted: November 4, 2013. From the Department of Emergency Medicine (J.H.L., K.K., Y.H.J., J.E.R., A.R.D., M.J.L., C.J.P., J.K., H.C.), Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do; and Department of Pathology (M.A.K, K.B.L.), Seoul National University Hospital, Seoul, Korea. Address for reprints: Kyuseok Kim, MD, PhD, Department of Emergency Medicine, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-707, Korea; email: [email protected]. DOI: 10.1097/TA.0000000000000106
This study was approved by the Animal Care and Use Committee of our hospital and was conducted in accordance with National Institute of Health guidelines.
Animal Preparation Male Sprague-Dawley rats weighing 300 g to 350 g were used in the study. The rats were housed in a controlled
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environment with free access to food and water for 1 week before the experiment. Rats were anesthetized with an intramuscular injection of zoletil (3 mg/kg, Virbac, France) and xylazine (15 mg/kg, Bayer, Korea). Tracheostomy was performed using a 14-gauge catheter (BD Insyte, Autoguard, NJ), and rats were connected to a ventilator (tidal volume, 2 mL; respiratory rate, 55 breaths/min; FIO2, 0.21; Harvard rodent ventilator model 645, Harvard Apparatus, Holliston, MA). Anesthesia was maintained by inhalation of 1% isoflurane via mechanical ventilator. The minute ventilation was adjusted to ensure a PaCO2 between 35 mm Hg and 40 mm Hg according to the results of arterial blood gas analysis. Rectal temperature was monitored and controlled at 36.5-C to 37.0-C using a heating lamp. A sterile cut down was performed in the groin bilaterally, and 24-gauge intravascular catheters (BD Insyte) were inserted into both femoral arteries for blood pressure monitoring and for blood withdrawal or transfusion. Blood samples (0.3 mL at a time) were used for blood gas analysis and replaced with 0.9 mL of normal saline.
Experimental Procedure (Fig. 1) A pressure-controlled hemorrhagic shock rat model was prepared as described in a previous report.17 Briefly, hemorrhagic shock was induced by blood withdrawal. Mean arterial pressure (MAP) was rapidly decreased to 38 T 1 mm Hg and maintained for 60 minutes by blood withdrawal or reinfusion. Shed blood was collected in heparinized syringes (heparin, 7.5 IU/mL),18 kept warm, and stirred frequently. After 60 minutes of shock, rats were randomized into one of three groups (no-resuscitation group vs. rapid resuscitation [RR] group vs. pressure-targeted stepwise resuscitation [PSR] group). Baseline MAP values were approximately 70 mm Hg in our experimental setting. The no-resuscitation group was observed after the shock period without any fluid or blood transfusion. The RR group was resuscitated to reach a MAP of 70 mm Hg immediately by transfusion of shed blood, and MAP was maintained until the entire volume of shed blood was
transfused. The PSR group was resuscitated by increasing the MAP by 5 mm Hg for every 3 minutes up to 70 mm Hg and then maintained until the remaining shed blood was transfused. No additional fluid was administered to reach the target MAP value even if the target MAP was not achieved by transfusion of shed blood. Rats were observed until death or 6 hours after the shock period. MAP and heart rate (HR) were continuously monitored, and total transfusion times and survival time were recorded. Surviving rats were euthanized by exsanguination, and blood samples were obtained. The blood was centrifuged for 15 min at 3,000 rpm, and the plasma was stored at j70-C until analysis. The liver tissues were harvested and fixed with 4% formalin or stored in j70-C liquid nitrogen.
DCF-DA [2¶, 7¶-Dichlorofluorescein Diacetate] Assay The relative levels of ROS were measured by DCF-DA (Sigma, St. Louis, MO) assay. Frozen liver tissues were weighed, thawed, and homogenized in 500-KL ice-cold RIPA buffer (20-mM Tris-Hcl, pH 7.5, 150-mM NaCl, 1-mM EDTA, 0.5% Ninidet P-40, and 5 Kg/mL; Intron Biotechnology Inc.) and then centrifuged at 2,000 rpm at 4-C for 10 minutes. The supernatant was added to the reaction mixture containing DCFDA (10 KM/mL) and incubated for 60 minutes at 37-C. The fluorescence intensity was determined by a fluorescence plate reader, with an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Values were presented as fluorescence intensity per milligram of tissue or percentage of negative control.
Cytokines and Nitric Oxide Inflammatory cytokines including interleukin 1A (IL-1A) and IL-6 were measured in liver tissue using an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN). Plasma nitric oxide (NO) was measured using a commercial kit for nitrite/nitrate by using the Griess reaction (R&D Systems).
Figure 1. Study protocol of animal experiment. ABGA, arterial blood gas analysis; T, time. 772
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Western Blot Analysis Western blot analysis was performed in liver tissue samples. Primary antibodies against Bcl-2, Akt (total), phospho-Akt (Ser473), GSK-3A (total), phospho-GSK-3A (Ser9), Bad (total), phospho-Bad (Ser136), and cleaved caspase-3 (Asp175) were purchased from Cell Signaling Technology (MA). Primary antibodies against A-actin were purchased from Santa-Cruz Biotechnology. Proteins (approximately 100 Kg per lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were blocked in 0.05% PBSTween containing 5% milk (Bio-Rad Laboratories) and then incubated with the primary antibody at 4-C overnight. The primary antibody was detected by incubation with the horseradish peroxidaseYcoupled second antibody (1:3,000 in PBSTween with 5% milk) at room temperature for 2 hours. The chemiluminescence detection was performed by using Western Lighting Chemiluminescence Reagant Plus (Perkin Elmer, MA). Films were developed using a standard photographic
procedure, and quantitative analyses of bands were performed using Versa Doc Imaging System (Bio-Rad Laboratories, CA).
Blood Gas Analysis, Lactate, and Alanine Aminotransferase Arterial blood gas analysis and lactate measurements were performed at baseline, immediately after the shock period, at 2 hours of resuscitation, and at 6 hours of resuscitation. Plasma alanine aminotransferase (ALT) was measured with VetScan VS2 (Abaxis, Inc., CA).
Assessment of Histologic Liver Injury Formalin-fixed liver tissues were embedded in paraffin. The paraffin blocks were sectioned at 4-Km thickness and stained with hematoxylin and eosin. The stained samples were reviewed blindly by two pathologists using light microscope, and liver injury was scored using the previously reported morphologic criteria19: spotty necrosis, capsular inflammation, portal inflammation, ballooning degeneration, and steatosis. The liver Injury Severity Score (ISS) ranged from 0 (none) to 16 (severe).
TABLE 1. Bleeding Volume, Arterial Blood Gas Analysis, and Lactate Baseline Body weight, g No resuscitation RR PSR Bleeding volume, mL/100 g No resuscitation RR PSR pH No resuscitation RR PSR PCO2, mm Hg No resuscitation RR PSR PO2, mm Hg No resuscitation RR PSR HCO3j, mmol/L No resuscitation RR PSR Base excess, mmol/L No resuscitation RR PSR Lactate, mmol/L No resuscitation RR PSR
Shock
Transfusion 2 h
Transfusion 6 h
330.5 T 6.6 (14) 329.9 T 3.9 (15) 326.4 T 2.8 (15) 4.38 T 0.3 (14) 4.30 T 0.1 (15) 4.48 T 0.2 (15) 7.402 T 0.006 (14) 7.393 T 0.006 (15) 7.391 T 0.003 (15)
7.287 T 0.013 (14) 7.276 T 0.010 (15) 7.299 T 0.010 (15)
7.254 T 0.035 (4) 7.286 T 0.035 (8) 7.289 T 0.008 (12)
V 7.191 T 0.059 (5)* 7.299 T 0.012 (11)
35.1 T 0.9 (14) 35.0 T 1.7 (15) 35.1 T 0.8 (15)
31.6 T 1.7 (14) 31.4 T 1.8 (15) 27.5 T 1.3 (15)
28.7 T 3.3 (4) 30.6 T 1.5 (8) 33.7 T 2.3 (12)
V 37.8 T 3.3 (5) 32.6 T 2.0 (11)
73.2 T 2.9 (14) 79.0 T 2.7 (15) 71.6 T 5.1 (15)
68.2 T 2.4 (14) 68.4 T 2.4 (15) 70.1 T 2.4 (15)
72.3 T 4.3 (4) 58.7 T 5.1 (8) 50.7 T 2.0 (12)
V 61.8 T 3.8 (5) 68.3 T 7.0 (11)
21.5 T 0.9 (14) 21.5 T 0.9 (15) 21.5 T 0.4 (15)
15.1 T 0.5 (14) 14.6 T 0.7 (15) 13.7 T 0.5 (15)
12.7 T 1.3 (4) 14.8 T 1.2 (8) 16.3 T 1.1 (12)
V 14.7 T 1.7 (5) 16.2 T 1.1 (11)
j3.73 T 0.58 (14) j3.73 T 0.91 (15) j3.75 T 0.45 (15) 0.7 T 0.0 (14) 1.1 T 0.1 (15) 1.0 T 0.1 (15)
j11.84 T 0.44 (14) j12.47 T 0.62 (15) j12.86 T 0.49 (15) 4.5 T 0.3 (14) 5.3 T 0.4 (15) 4.6 T 0.3 (15)
j14.73 T 1.48 (4) j12.13 T 1.69 (8) j10.53 T 1.18 (12) 5.2 T 1.0 (4) 2.9 T 0.9 (8) 2.0 T 0.3 (12)
V j13.78 T 2.41 (5) j10.53 T 1.22 (11) V 4.3 T 2.4 (5) 1.4 T 0.2 (11)
*RR group versus PSR group at transfusion 6 hours, p G 0.05. Number in parenthesis indicates number of animals.
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STATISTICAL ANALYSIS Data were expressed as mean T SEM. Normality testing was performed using the Kolmogorov-Smirnov test. The Student’s t test for normally distributed data and the MannWhitney U-test for nonnormally distributed data were performed for comparison between the groups. For analysis of difference in histologic score, a modified Z score using median absolute deviation was used, and the histologic injury scores were compared with Mann-Whitney U-test.20 A repeatedmeasures analysis of variance was performed to compare hemodynamic changes between the groups. A Kaplan-Meier survival analysis was performed for the comparison of survival time. A p G 0.05 was considered significant.
RESULTS A total of 44 rats (no-resuscitation group [n = 14], RR group [n = 15], PSR group [n = 15]) were used for the study. Baseline characteristics including body weight, bleeding volume, arterial blood gas values, and lactate did not differ significantly between the groups (Table 1).
Effect of Pressure-Targeted Stepwise Resuscitation on the Survival Rates During the 6-hour observation period, all of the rats in the no-resuscitation group were expired (survival rate, 0.0%), 5 rats in the RR group survived (survival rate, 33.3%) and 11 rats in the PSR group survived (survival rate, 73.3%). Survival rates were significantly higher in the PSR group than in the RR group (log rank p = 0.032, Fig. 2A).
Effect of Pressure-Targeted Stepwise Resuscitation on Transfusion Time, Blood Gases, and Hemodynamic Change Total transfusion time was significantly longer in the PSR group than in the RR group (8.5 T 3.0 minutes in the RR group and 27.8 T 7.9 minutes in the PSR group, p = 0.024, Fig. 2B). Arterial blood gas values were not significantly different between the groups during the experimental period with the exception of blood pH at 6 hours of resuscitation, which was significantly lower in the RR group than in the PSR group (p = 0.020, Table 1). Figure 2 shows the observed hemodynamic changes. HR did not significantly differ between the groups (Fig. 2C). However, MAP was significantly higher in the PSR group than in the RR group (group, p G 0.05; time, p G 0.05; group time interaction, p G 0.05, Fig. 2D)
Effect of Pressure-Targeted Stepwise Resuscitation on Liver Injury, Generation of ROS, Inflammatory Cytokines, and NO Plasma ALT was significantly higher in the RR group than in the PSR group (p = 0.032, Fig. 3A), and liver injury score was significantly lower in the PSR group than in the RR group (p = 0.024, Fig. 3B). The ROS generation in liver tissue samples was significantly lower in the PSR group than in the RR group (p = 0.039, Fig. 3C). In addition, liver tissue IL-1A and IL-6 levels were significantly lower in the PSR group than in the RR group, while plasma NO was also significantly lower 774
in the PSR group than in the RR group (all p G 0.05, respectively, Fig. 3D).
Effect of Pressure-Targeted Stepwise Resuscitation on the Akt Survival Pathway in Liver Tissue Phosphorylation of Akt, GSK-3A, and Bad as well as expression of Bcl-2 were significantly increased in the PSR group as compared with the RR group (all p G 0.05, Fig. 4AYD). In addition, expression of cleaved caspase 3 was significantly lower in the PSR group (p G 0.05, Fig. 4E).
DISCUSSION This study demonstrated that blood pressureYtargeted stepwise resuscitation for the management of hemorrhagic shock in rats resulted in decreased ROS generation, mortality, and liver injury. Moreover, pressure-targeted stepwise resuscitation decreased inflammatory cytokine production and enhanced Akt survival pathways. Previous evidence demonstrated that a constant rate of crystalloid infusion for resuscitating fixed-volume hemorrhagic shock in dogs improved hemodynamic parameters when compared with impulse crystalloid infusion.21 Although the study did not investigate the precise mechanism, the blood volume restitution was significantly improved by a constant rate of infusion when compared with impulse infusion. In addition, Bark et al.22 also reported a greater plasma volume expansion with slow rates of colloid infusion than with faster infusion rates in septic rats. Thus, the improved hemodynamics and survival of the pressure-targeted stepwise resuscitation group in our study may be a result of improved blood volume restitution during resuscitation of hemorrhagic shock. Reintroduction of oxygen to ischemic cells inevitably results in reperfusion injury. Many possible mechanisms of reperfusion injury have been suggested, with ROS generation regarded as one of the most important mechanisms.4,23 Generation of ROS is known to begin with the onset of ischemia24 and gradually increases as the ischemic period is prolonged, reaching a peak for a short period after reperfusion.25,26 Oxygen delivered by reperfusion is an important source of ROS generation. Previous evidence demonstrates that the ischemic pattern of ROS generation is unchanged with hypoxic reperfusion and that ROS generation spikes with oxygenated reperfusion.27 Initially, endogenous antioxidant enzymes may balance the generation of ROS.28 However, increased ROS generation may overwhelm the capacity of antioxidant defense system and result in cellular injury.29 Considering that ROS generation is much higher following abrupt reoxygenation,8 it is possible that ROS generation during slow reoxygenation will be balanced by the endogenous antioxidant defense system, allowing post-reoxygenation survival of ischemic cells. Systemic oxygen delivery is dependent on cardiac output and the arterial blood oxygen content. Considering that blood pressure is dependent on cardiac output and systemic vascular resistance, a slow increase in systemic blood pressure by slow transfusion and subsequent increase in preload can lead to more gradual oxygen delivery as more compared with rapid normalization of blood pressure. In our study, the arterial * 2014 Lippincott Williams & Wilkins
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Figure 2. Survival and hemodynamic effect of blood pressureYtargeted stepwise resuscitation in hemorrhagic shock. A, Kaplan-Meier survival curve. B, Time required to transfuse whole shed blood (RR group, n = 15; PSR group, n = 15). C, HR change (no-resuscitation group, n = 14; RR group, n = 15; PSR group, n = 15). D, MAP change, PSR group showed significantly higher MAP than in RR group (group, p G 0.05; time, p G 0.05; group time interaction, p G 0.05) (no-resuscitation group, n = 14; RR group, n = 15; PSR group, n = 15).
Figure 3. Effect of blood pressureYtargeted stepwise resuscitation on liver injury and inflammation. A, Plasma ALT (RR group, n = 5; PSR group, n = 11). B, Representative photos and histologic liver injury score (original magnification 200, hematoxylin and eosin stain; 0 = none; 16 = severe; RR group, n = 4; PSR group, n = 11). C, Generation of ROS (RR group, n = 5; PSR group, n = 11). D, Plasma inflammatory cytokine and NO (RR group, n = 5; PSR group, n = 11). * 2014 Lippincott Williams & Wilkins
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Figure 4. Effect of blood pressureYtargeted stepwise resuscitation on Akt survival pathway in liver tissue. A, Phosphorylation of Akt. B, Phosphorylation of GSK-3A. C, Phosphorylation of Bad. D, Bcl-2 expression. E, Cleaved caspase 3 expression. RR group, n = 5; PSR group, n = 11.
oxygen content may have been temporarily much lower in the PSR group than in the RR group because the time required to transfuse the shed blood was significantly longer in the PSR group than in the RR group. Overall, stepwise resuscitation may deliver oxygen more slowly and gradually than rapid resuscitation, leading to reduced ROS production and subsequent reduced tissue injury and mortality. Production of ROS was estimated by DCF-DA assay in this study. Although the difference in DCF-DA fluorescence intensity between groups was minor, it was significantly decreased in the stepwise resuscitation group compared with the rapid resuscitation group. Cellular ischemic stress results in ROS production, which may be inevitable in hemorrhage patients. However, reintroduction of oxygen by abrupt reperfusion increases ROS production, causing additional reperfusion injury. Thus, decreasing ROS generation by stepwise resuscitation may be a somewhat effective strategy in resuscitating hemorrhage patients. In this study, slow reperfusion was performed according to predetermined target of blood pressure and duration. There may be several considerations to determine reperfusion methods. First, prolonged reperfusion duration can increase the relative ischemic period, which may counteract the protective effects of slow reoxygenation in animals. Second, individual animals may respond differently to the transfusion rate and amount. These differences could originate from differences in cardiac function and systemic vascular resistance. Thus, a constant rate of transfusion would result in differences in tissue perfusion in experimental animals. To overcome this limitation, we used a blood pressureYtargeted strategy. In addition, considering that the objective of our study was to achieve a relatively 776
slow oxygen delivery to cells or tissues in a reproducible manner, our method might be a more appropriate approach. In this study, slow reperfusion with a predetermined pressure target decreased histologic liver injury and augmented Akt survival pathways. Previous reports demonstrate that staged reperfusion for coronary ischemia in a canine model reduced myocardial stunning, suggesting that this phenomenon may be caused by transient acidosis caused by slow reperfusion in ischemic tissues.9 Transient cellular acidosis has been suggested as one of the protective mechanisms of ischemic conditioning.30,31 Although we did not examine the effect of slow reperfusion on cellular or tissue acidosis in our study, we measured the protein expression associated with Akt survival pathways and found that stepwise resuscitation augmented Akt survival pathways. Ischemic postconditioning has been reported to be associated with augmentation of PI3K/Akt survival pathways in various disease models, including stroke, coronary ischemia, and hepatic I/R injury.32Y34 Although the precise mechanism of ischemic postconditioning is unclear, we previously postulated that a gradual and stepwise increase in blood pressure for the management of hemorrhagic shock could decrease tissue injury and mortality by mimicking ischemic postconditioning.35 Ischemic postconditioning can be performed by initiating a short period of interruption of perfusion before full restoration of perfusion. It is possible that ischemic postconditioning may produce a transitional period of oxygen supply between ischemia and full perfusion restoration. Thus, a gradual and stepwise increase in blood pressure for the management of hemorrhagic shock may at least partially mimic ischemic postconditioning with regard to oxygen delivery to * 2014 Lippincott Williams & Wilkins
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ischemic tissues. To define the association between stepwise resuscitation and the mechanism of ischemic postconditioning in hemorrhagic shock, further studies are required, including the change in cellular acidosis and the opening of mitochondrial permeability transition pore associated with slow reperfusion. In this study, adequate blood pressure was maintained for a significantly longer period in the PSR group than in the RR group. While the precise mechanism of this phenomenon was not investigated in this study, a possible cause is NO production. Plasma NO levels were significantly lower in the PSR group than in the RR group. It has been reported that I/R results in profound production of NO, which can decrease systemic vasomotor tone.36 In addition, decreased NO production during delayed reoxygenation of ischemic myocardium has been reported.37 Thus, decreased NO production might be related to vasomotor tone and could contribute to the long transfusion time in the PSR group in our model. Although oxygen is absolutely necessary for all vertebrate animals, excessive oxygen can cause harm by increasing ROS. In this study, supplemental oxygen was not administered throughout the experiment, as we postulated that supplemental oxygen might mitigate the effects of a gradual increase in oxygen delivery in the PSR group. However, hemorrhagic shock and resuscitation causes acute lung injury, which can disturb gas exchange and cause mortality. Thus, supplemental oxygen should be administered and, if systemic oxygenation is impaired by acute lung injury, titrated accordingly. In this study, we measured proinflammatory cytokines and found that slow reperfusion decreased levels of IL-1A and IL-6. IL-1A production is known to be mediated by the NLRP3 inflammasome, which is activated by ROS.38 IL-6 production is known to be associated with released damage-associated molecular pattern molecules released from injured cells via toll-like receptor signaling.7 Although the precise mechanism associated with inflammatory cytokine production was not investigated in our study, reperfusion speed might be associated with proinflammatory cytokine production. Our study has several limitations. First, the exact and precise mechanism of slow reperfusion leading to decreased cellular and tissue injury was not identified with the exception of ROS generation and plasma NO. In addition, different rates of reperfusion speed on I/R injury were not investigated, and further studies are warranted. Second, supplemental oxygen was not administered owing to the reasoning discussed previously. Supplemental oxygen is known to be helpful in the management of systemic hypoxemia caused by disturbances in gas exchange. Third, the pressure-controlled hemorrhagic shock model used in this study may not be clinically relevant. However, the model of hemorrhagic shock used was chosen because a similar amount of ischemic insults was mandatory to compare the effects of gradual reperfusion based on predetermined pressure targets with rapid normalization of blood pressure. Further studies in clinically relevant large animal model are warranted. In conclusion, slow reperfusion by blood pressureY targeted stepwise reperfusion decreased mortality and liver injury in the management of hemorrhagic shock. This stepwise
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method also decreased ROS production and inflammatory cytokine expression as well as augmented Akt survival pathways. AUTHORSHIP J.H.L., K.K., Y.H.J., M.A.K., K.-B.L., M.J.L., A.-R.D., and J.E.R. contributed in the conception and design. J.H.L., C.J.P., J.K., H.C., M.J.L., M.A.K., K.B.L., and A.-R.D. conducted the acquisition of data. J.H.L., M.A.K., C.J.P., J.K., H.C., K.-B.L., Y.H.J., and K.K. conducted the analysis and interpretation. J.H.L., K.K., Y.H.J., J.E.R., M.A.K., and K.-B.L. drafted the manuscript for important intellectual content.
DISCLOSURE This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A2042541).
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