Hemorrhagic shock (HS) resulting from trauma remains a leading cause of preventable death in civilian and military populations (1). Acute and massive blood loss results in reduced cardiac output and cardiovascular function leading to inadequate tissue perfusion and impaired oxygen delivery to essentially all organ systems (2, 3). Pathophysiological responses to HS include the formation of reactive oxygen and nitrogen species that promote irreversible cellular damage and cell death (for review, see Chan ). Taylor and colleagues (5) found an increase in glycolytic intermediates, lactate, and hydrogen ions in skeletal muscle of pigs subjected to HS, suggesting a switch from aerobic to anaerobic glycolysis and the failure of mitochondrial respiration. Clinically, there is general consensus for the use of resuscitation fluids for treating HS, but their composition, timing, volume administered, and mechanism of action remain controversial and uncertain (1).
Evolution has generated a myriad of biochemical pathways that have allowed living organisms to adapt to a variety of environmental extremes. Natural hibernation is an example of an adaptation that has promoted survival of wild animals that also has the potential to improve patient care in the clinical setting. Hibernation in mammals is composed of long periods of inactivity (days - weeks) and depressed body temperature ([T b] <10°C) called torpor, interrupted by brief periods of activity and normothermia called interbout arousals ([IBAs] reviewed in Andrews ). Torpor is characterized by a decrease in cardiac output and cerebral blood flow that is similar in magnitude to HS, and IBAs resemble reperfusion when blood flow to ischemic tissues resumes.
In this article, we describe experiments that investigate the use of hibernation strategies as a possible therapeutic approach for protection against conditions associated with HS. These strategies, including a passive decrease in T b, an increase in circulating ketone bodies (alternative energy substrates), and an elevation of antioxidant activity, are evaluated in a nonhibernating mammal: the Sprague-Dawley rat subjected to 60% blood loss and monitored for improvement of survival and recovery. This model was used to develop a low-volume therapy that can extend the so-called golden hour immediately after traumatic injury-a time when the probability of preventing death from blood loss is highest. The therapeutic is a simple solution containing two substances elevated in the serum of hibernating mammals, the d-stereoisomer of the ketone β-hydroxybutyrate (d-BHB) and, an antioxidant, melatonin. We found that this mixture is effective for prolonging recoverable survival by administering a volume as small as 4% of the total blood removed. The portability of this therapy was developed for future evaluation in a large-animal model and intended for use by first responders in a nonhospital setting.
MATERIALS AND METHODS
Eighty-five male Sprague-Dawley rats were purchased from Harlan Teklad (Madison, Wis) and maintained at the University of Minnesota Medical School Duluth on a 12:12 light-dark cycle with standard rat chow (Harlan Teklad) and tap water ad libitum for a minimum acclimation period of 7 days before the experiment. After acclimation, animals weighing 320 ± 23 g were used in experiments approved by the University of Minnesota Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication no. 80-23).
Animal preparation and instrumentation
Animals were briefly anesthetized with 2% isoflurane in breathing grade air followed by i.m. injection with a 100/20 mg/kg mixture of ketamine/xylazine. The left femoral artery was aseptically cannulated with polyethylene tubing (PE 50 connected to PE 10) containing heparinized saline (10 units/mL heparin and 0.9% saline) and connected to a blood pressure transducer (PowerLab, ADInstruments, Colorado Springs, Colo) for measuring mean arterial blood pressure (MAP) and heart rate. Body temperature was monitored using a rectal probe (RET-2, Physitemp Instruments, Inc, Clifton, NJ) connected to PowerLab. The right femoral artery was aseptically cannulated to facilitate blood withdrawal and sampling. The right femoral vein was aseptically cannulated to facilitate solution infusion when applicable.
Acute experiments-no infusion
Blood volume was calculated using the formula that total blood volume is equal to 6% of body weight (7). Hemorrhagic shock was induced via controlled hypotension with a heparinized syringe from the right femoral artery. Forty percent of the total calculated blood volume was removed within a period of 10 min resulting in a MAP of about 35 mmHg. The animal's blood was further removed during an additional 10 min until the total amount of shed blood was equal to 60% of total blood volume. After blood loss, one group of animals (n = 5) was connected to a rectal thermometer that thermostatically controlled a heat lamp to maintain a T b at 37°C, whereas another set of animals (n = 6) lacked thermostatic control and were kept at room temperature (22°C) after blood loss. The end point of each animal experiment was death as determined by loss of corneal reflex, cessation of breathing, and absence of detectable pulse.
Acute experiments-solution infusion
Hemorrhagic shock was induced as previously described. For six animals, a volume of either 4 M d-BHB or 4 M NaCl equal to 1 mL/kg of animal body weight was administered via the right femoral vein for a period of 10 min after achieving 40% blood loss. The solution concentration of 4 M was determined by calculating a desired blood concentration of 4 mM d-BHB resulting from infusion of the 1 mL/kg bolus. A concentration range of 2 to 4 mM d-BHB is important because this concentration is found in serum of hibernating ground squirrels during torpor (8). In a separate experiment, the initial bolus infusion was immediately followed by a 100-μL/h infusion of 4 M d-BHB or 4 M NaCl that continued until the end of the experiment (n = 6). In both experiments, the animal's blood volume was then further lowered until 60% of total blood was removed. The initial bolus was administered after 40% blood loss for two reasons: (a) animals quickly died after 60% blood loss and administration of any therapy would have been futile in some animals, and (b) an effort was made to mimic real-life trauma scenarios where patient bleeding continues even after intervention by emergency personnel. The end point of this study was death as described in the previous section. Blood samples (150 μL) were taken at the following time points: before hypotension, at 40% blood loss, after the bolus solution infusion, at 60% blood loss, and every 30 min post-60% blood loss until the animal either died or no more blood could be sampled from the right femoral artery. Representative time points during the surgical procedure are shown in Figure 1A.
Return of shed blood experiments
Hemorrhagic shock was induced as previously described for the acute experiments. For 10 animals, a solution of 4 M d-BHB, 4 M NaCl, 4 M d-BHB plus 43 mM melatonin, or 4 M NaCl plus 43 mM melatonin was tested. A 5× melatonin stock solution at a concentration of 215 mM in 100% DMSO was used to make the final formulation. Therefore, 20% DMSO was present in the final solutions containing melatonin. The concentration of melatonin was calculated to be 10 mg/kg after the initial bolus of fluid. This concentration was chosen based on previous ischemia studies (9-11), as well as levels of melatonin found during IBAs in hibernating animals (12). Each solution was administered at a volume equal to 1 mL/kg animal body weight after 40% blood loss was achieved and was immediately followed by a continuous infusion of 100 μL/h via the right femoral vein until blood return. The animal's blood was then further removed until the total amount of shed blood was equal to 60% of blood volume. Blood samples (150 μL) were taken at the following time points during the experiment: before hypotension, after the animal reached 40% blood loss, after the bolus solution infusion, after 60% blood loss, and immediately after shed blood return when the venous cannula was still in place. Respective time points during the surgical procedure are shown in Figure 1B. The shed blood was kept in a rocking water bath at a temperature equivalent to the animal's T b (∼26°C-28°C after HS). The shed blood was returned 60 min after HS at a constant rate of 0.5 mL/min using an infusion pump (Syringe Pump Model 341; Sage Instruments, Freedom, Calif). After blood return, all animals had the cannulas removed, wounds sutured, and were monitored for 4 h postsurgery during the recovery period. Sham-operated animals were subject to the same anesthesia and cannulation protocols previously described in Animal Preparation and Instrumentation, but blood was not removed. The end point of this study was death during the 10-day observation period or by euthanasia on the 10th day postsurgery by decapitation under deep isoflurane anesthesia.
Blood samples collected from the acute experiment of animals were kept on ice until spun in a microcentrifuge (Hermle MR-2; National Labnet Co, Edison, NJ) at 3,500 rpm for 5 min, and serum was frozen at −80°C in 20-μL aliquots until analysis. Blood samples from animals in the blood return experiments were prepared similarly and analyzed for d-BHB, glucose, and lactate concentrations. The d-BHB was measured using the Stanbio LiquiColor testing kit (no. 2440; Boerne, Tex). Glucose measurements were obtained using the Stanbio LiquiColor testing kit (no. 1070). Lactate concentrations were obtained using the Biomedical Research Service Center lactate assay kit (no. A-108L; University of Buffalo, Buffalo, NY).
Statistical analysis was performed by either using JMP or SAS (SAS Institute, Cary, NC). Differences in body weights, shed blood d-BHB levels, and shed blood glucose levels between animal groups were calculated using analysis of variance (ANOVA) and a Tukey post hoc to find differences between groups (JMP). Acute survival differences for the nonsolution infusion animals were computed by using single-factor ANOVA (JMP). The acute survival differences for the solution-infused animals were computed using ANOVA with unequal variances between groups using Satterwhaite approximation for degrees of freedom (SAS). Survival curves for the blood return experiments were analyzed using the log-rank and Wilcoxon tests individually, and a step-down Bonferroni to correct for multiple tests (JMP). Mean arterial pressure, heart rate, T b, serum d-BHB, glucose, and lactate concentrations were all analyzed using repeated-measures ANOVA with contrasts between treatment groups (SAS). Significant differences in values before hypotension and after blood return for MAP, heart rate, and T b were done using Wilcoxon signed rank matched pairs test (JMP). Sample sizes for groups were calculated using power calculations, and all tests were performed using a 95% confidence interval.
To investigate the hibernation strategy of reduced T b, we compared the survival of hemorrhaged rats maintained at T b = 37°C with rats receiving no intervention. We found that rats thermostatically maintained at 37°C after 60% blood loss showed a mean survival of only 7 min (Fig. 2, left bar). In nonthermostatically controlled rats at a room temperature of 22°C, the T b gradually decreased after hemorrhage to T b = 24°C-28°C. These animals survived an average of 35.4 min without active cooling (Fig. 2, right bar)-a 5-fold increase when compared with the animals maintained at 37°C (P < 0.001). Therefore, survival time was significantly extended after 60% blood loss by not artificially maintaining a warm (37°C) T b.
Based on the observation that a passive decrease in T b resembling natural hibernation prolongs survival during hemorrhagic shock (Fig. 2), subsequent experiments were performed without maintaining a fixed T b. The timeline of the solution infusion experiments described in this section is shown in Figure 1A. Figure 3 and the corresponding Kaplan-Meier plot (Fig. 4) show that the infusion of a single bolus (1 mL/kg) of 4 M d-BHB into rats subjected to 60% blood loss was more effective at extending survival than infusion of equal osmolar 4 M NaCl. Infusion of 4 M d-BHB significantly extended the survival time when compared with no infusion (P < 0.01). Therefore, after removing approximately 11 mL of blood, survival was significantly improved by a fluid replacement of only 300 μL of 4 M d-BHB. Most impressively, the addition of a slow continuous infusion of 4 M d-BHB at 100 μL/h after the 1 mL/kg bolus increased the survival time to a mean of 183 min (Fig. 3, bar on far right), which is significantly longer than the same treatment using 4 M NaCl (P < 0.01). The total fluid replacement with the bolus plus slow infusion during the 3-h period was approximately 600 μL or 5.5% of the total 11 mL of blood volume removed. Interestingly, two of the animals treated with d-BHB lived up to 4 h post-60% blood loss (Fig. 4).
Blood return after 60% blood loss
After establishing the survival benefit of reduced T b and elevated serum d-BHB levels, we wanted to determine whether these hibernation strategies would improve recovery from HS after returning the shed blood. An added complication of blood return after 60% blood loss is the potential for reperfusion injury that results from the sudden increase in oxygen delivery to tissues (13). Hibernators avoid damage from reperfusion injury during arousal from torpor by a variety of potential mechanisms, including an elevation in circulating antioxidants (14). An increase in serum melatonin levels during arousal (reviewed in Tan et al. ) suggested that this particular molecule could be an effective additive to improve survival after blood return.
The timeline of the blood return experiments described in this section is shown in Figure 1B. A period of 1 h of 60% blood loss was chosen because most rats that were continuously infused with either d-BHB or NaCl survived 60% blood loss for at least 1 h (Fig. 4). Four solutions were tested: 4 M d-BHB, 4 M d-BHB plus 43 mM melatonin, 4M NaCl, and 4 M NaCl plus 43 mM melatonin. Stock solutions of 215 mM melatonin were made in 100% DMSO, therefore all test solutions with melatonin contained 20% DMSO. During the 1 h of HS, the 1 mL/kg bolus followed by slow infusion (100 μL/h) of the test solutions resulted in a total fluid replacement of only 400 μL or approximately 4% of the blood volume removed.
During the period of 60% blood loss through blood return, 4 M d-BHB plus 43 mM melatonin solution showed statistically similar levels of elevated serum d-BHB as that seen in rats infused with 4 M d-BHB alone (Fig. 5). The total blood removed from the 4 M d-BHB infused groups (approximately 11 mL) also had statistically higher levels of d-BHB than blood removed from both the NaCl-infused control animals (Table 1) and higher than all animals before 40% blood loss (Fig. 5). This is because solution infusion began at 40% blood loss and continued through collection of the remaining 60% blood volume (Fig. 1B). The sham animals were not included in this comparison of shed blood because they were not subjected to blood removal.
After showing that we could maintain elevated d-BHB during 1 h of 60% blood loss (Fig. 5), we then monitored survival for up to 10 days after blood return without i.v. fluid resuscitation or other interventions. Eighty percent (8/10) of the animals treated with 4 M d-BHB plus 43 mM melatonin survived 10 days compared with 20% survival with 4 M d-BHB alone (Fig. 6). In fact, the 4 M d-BHB plus 43 mM melatonin-treated animals are statistically different (P < 0.05) than the other three groups: 4 M d-BHB, 4 M NaCl, and 4 M NaCl plus 43 mM melatonin groups. As early as the first 24 h after blood return, these same three treatment groups showed a sharp decline in survival. Effects attributed to melatonin were seen 60 h after blood return as the 4 M d-BHB plus 43 mM melatonin and 4 M NaCl plus 43 mM melatonin groups showed 90% and 60% survival, respectively. This is in contrast to the 60-h survival of the two groups not receiving melatonin: 20% with 4 M d-BHB and 10% with 4 M NaCl.
Serum glucose concentrations in animals subjected to HS rose from approximately 10 mM before hypotension to greater than 15 mM in all groups after HS (data not shown). Differences in glucose concentrations between hemorrhaged animals were not significantly different, but levels in all hemorrhaged groups were statistically higher when compared with the sham-operated animals (P < 0.05). Although serum lactate is typically elevated during HS, we found serum lactate concentrations were approximately 1.5 mM for all animal groups before hypotension, remained constant throughout the experiment, and were not significantly different from each other or the sham-operated animals (data not shown). In addition, a limited number of serum enzyme assays used as markers for global tissue damage (alanine transferase, aspartate aminotransferase, and lactate dehydrogenase) showed no differences among sham and hemorrhaged groups (data not shown). As with the d-BHB assays (Fig. 5), these other serum measurements were made at six different points taken before hypotension, during 40% and 60% blood loss, and after return of shed blood.
At the start of each experiment (before hypotension in Table 2), MAP of all animals was within normal rat physiological levels (15). During HS, the average MAP decreased to between 35 mmHg and 40 mmHg in all animal groups. After blood return, MAP in all groups of animals returned to prehypotension levels with no statistical difference in MAP from before blood removal and after blood return. Mean arterial blood pressure for sham-operated animals (no blood removed) was statistically different when compared with the groups of hemorrhaged animals (Table 2, P < 0.001, repeated ANOVA), but no other statistical significance was found among groups.
Heart rates were within normal rat physiological levels (15) for all groups at the start of the experiment (Table 3). The heart rate decreased by more than 100 beats/min at 1 h after 60% blood loss in all treatment groups. Upon blood return, the heart rate of the hemorrhaged animals increased slightly. Heart rates after blood return were significantly lower than the rates before hypotension. The heart rate for sham-operated animals was statistically different when compared with that of hemorrhaged animals (Table 3, P < 0.001, repeated ANOVA).
Looking closely at animal T b before hypotension (Table 4), we found them slightly lower than the accepted physiological levels of 36°C to 37.5°C normally seen in rats (16). We attribute these differences to the effects of anesthesia on T b. The T b of all hemorrhaged groups decreased approximately 6°C after 1 h of 60% blood loss. Statistical significance (P < 0.001, repeated ANOVA) was only seen when comparing the T b of the sham animals with those of all the treatment groups (Table 4).
In this article we describe a new small-volume therapy for severe HS (60% blood loss) based on the biochemistry of hibernating mammals. Infusion of 4 M d-BHB at a volume equal to 5.5% of the total blood removed was effective in prolonging survival up to 3 h after blood loss. In blood return experiments, infusion of 4 M d-BHB plus 43 mM melatonin at a volume equal to 4% of the total blood removed prolonged survival up to 10 days after 1 h of 60% blood loss. We conclude that the addition of melatonin has a positive outcome on the long-term survival of rats after 60% blood loss for 1 h, and that the d-BHB plus melatonin-treated group showed the best survival. This low-volume therapy is designed for purposes of portability so that it can be administered by first-responders at the scene of a trauma to extend the survival time of the critically wounded in a nonhospital setting.
As a nonhibernating species, we found the Sprague-Dawley rat tolerated and thus benefited from the hibernation strategies used in this study. The passive cooling that occurred upon blood loss was clearly beneficial compared with actively maintaining T b at 37°C. The protective effect of hypothermia resulting from blood loss may have evolved as a means to protect injured mammals by reducing metabolism under conditions of reduced oxygen delivery to tissues. This could explain the reason for the absence of an increase in serum lactate when animals were subjected to 60% blood loss. The authors acknowledge that the decrease in T b seen in these experiments may not be clinically achieved in humans, but the authors also stress the potential life-saving benefits of decreasing T b during HS. The effects of mild to moderate hypothermia are not novel and have been previously reported in animal experiments (17, 18), and the potential benefits to humans (19) are attributed to reduced metabolism and amelioration of events secondary to ischemia and reperfusion, although detrimental effects such as cardiac complications and coagulopathies are also recognized (20).
There is biological precedence for the use of ketones such as d-BHB as a primary fuel by mammals under stress. When hibernators enter torpor, they switch from a carbohydrate-based metabolism to a lipid-driven metabolism, and many studies support the fact that ketones become a primary energy source (21, 22). Furthermore, during arousal from torpor, d-BHB is preferred over glucose as a fuel in hibernating 13-lined ground squirrels (Spermophilus tridecemlineatus) (8). Ketone bodies are naturally formed in the liver from fatty acid catabolism after peripheral lipolysis (23) and consist of d-BHB, acetoacetate, and acetone (24). Rats infused with d-BHB exhibited an increase in serum d-BHB concentration from approximately 0.3 mM to more than 5 mM (Fig. 5), an increase similar to that seen during entrance into torpor by a hibernating ground squirrel (8). Animals given 4 M NaCl retained a low d-BHB concentration of approximately 0.2 mM, similar to basal serum levels previously reported for rats (25).
We found when HS rats were maintained at elevated d-BHB levels (3-6 mM) by continuous infusion after the bolus injection, survival was not only significant when compared with the no-infusion group, but also when compared with 4 M NaCl-administered controls (Fig. 3). Administration of d-BHB also improved survival in animals in which shed blood was returned after 1 h of 60% blood loss when compared with NaCl controls (Fig. 6). The importance of the controls, infused with a hypertonic NaCl solution, is that the benefit cannot be solely attributed to a vascular volume expansion resulting from the hypertonic solution drawing fluid from the extravascular space because the osmolarities of the test solutions were identical.
The benefit from d-BHB is likely a result of the favorable metabolism of this substrate relative to glucose, the prime metabolic energy source for most tissues in the absence of ketones. Sato and colleagues (26) found that the addition of 4 mM d-BHB and 1 mM acetoacetate to a perfused working rat heart helped to increase efficiency by 28% more than glucose alone. These data suggest that ketone bodies are an effective and efficient fuel source for the working heart and possibly other organs as well. Metabolism of d-BHB is energetically favorable because all 4 carbons of this ketone enter the mitochondrial tricarboxylic acid cycle as 2 molecules of acetyl-CoA, resulting in greater energy yield (adenosine triphosphate) with less oxygen consumption (27). Ketones have also been used in resuscitation mostly as a modification of traditional Ringer i.v. solutions (28-31). Furthermore, when ketones such as d-BHB become a major metabolic substrate, formation of excess lactate and H+ (lactic acidosis) commonly seen with HS is inhibited (32). Other studies of ketone metabolism in HS resuscitation fluids corroborate these observations (30, 33).
A significant parallel to our HS therapy exists in natural hibernators with respect to circulating melatonin levels, energy metabolism, and oxygen demand (12). A reduction in energy metabolism and oxygen consumption occurs without pathological consequences in mammals that undergo natural hibernation. The unchanging lactate levels seen in our rat HS study may be caused by the mild hypothermia in all groups and resembles the static lactate levels seen in the brain of active and torpid ground squirrels throughout the hibernation season (34). Periods of near-freezing torpor lasting several days in hibernating mammals such as ground squirrels are regularly interrupted by relatively brief periods of normothermic, physiological, and metabolic activity called interbout arousals (IBAs). During IBAs, blood flow, cardiac function, oxygen demand, and T b are rapidly increased during a period of minutes to hours without reperfusion injury (reviewed in Carey et al. ). Elevations of melatonin seem to be important both immediately before and during arousal from torpor and have been documented in both the golden-mantled ground squirrel (Spermophilus lateralis) (35) and Siberian hamster (Phodopus sungorus) (36).
Increased survival after blood return may result from melatonin's ability to act as an antioxidant and therefore reduce reperfusion injury. Within the cardiovascular system, the actions of melatonin are not fully understood, but melatonin is believed to directly neutralize free radicals, stimulate antioxidant enzymes, and stabilize cellular membranes (37). Some actions of melatonin are receptor mediated, which may be an additional mechanism at play in this study. Evidence for melatonin as a neuroprotectant against hypoxia/ischemia has been recently reviewed (38, 39), and its multiple mechanisms of action probably apply systemically in the case of HS. Because of melatonin's hydrophobic character, a small volume of approximately 80 μL DMSO was used during blood return experiments as a vehicle for melatonin. Much larger volumes of 100% DMSO have been used as a potential neuroprotective agent in ischemia studies (40-42). In our study, 80 μL of 100% DMSO in a 300-g rat results in a whole-body concentration of 0.03%. We found that this level of DMSO alone did not provide a beneficial effect when 60% blood loss was followed by blood return in a limited number of animals (data not shown).
The findings reported here may provide guidance for future clinical studies. There is great room for additional research in larger animals to examine the dose dependency and route of administration of the therapies outlined in this study. Although controlled blood loss animal models do not reflect tissue damage normally incurred during traumatic injuries (43), this type of model is more reproducible over uncontrolled blood loss (44) and seemed more appropriate to achieve the goals of this study. The Committee on Fluid Resuscitation for Combat Casualties has previously recommended that modifications to existing Ringer's lactate solutions include a reduction in l-lactate load, addition of ketones as an energy source, and the addition of free radical scavengers (45). The use of small-volume solutions as shown in the present study appear promising to future hemorrhage therapies in cases of both civilian and military trauma.
The authors thank Mr Ian Melander and Ms Mary Sneve for their technical assistance during these experiments. Special consideration goes out to Dr Ronald Regal, Mathematics and Statistics Department, University of Minnesota Duluth, for assistance with statistics determinations using SAS and repeated measures analysis. The authors also thank Mr Brian Kirkpatrick and Dr Richard Melvin of the Andrews laboratory, and Dr Gregory Beilman of the Department of Surgery at the University of Minnesota, for their input and advice on this manuscript.
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Hibernation; melatonin; hemorrhage; ketone; hypothermia; fluid resuscitation