THERAPEUTIC HYPOTHERMIA AND TEMPERATURE MANAGEMENT Volume 2, Number 4, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/ther.2012.0017
Drug-Induced Therapeutic Hypothermia After Asphyxial Cardiac Arrest in Swine Laurence M. Katz, M.D.,1 Gerald McGwin, Jr., Ph.D.,2 and Christopher J. Gordon, Ph.D.3
A feasibility study was performed to compare an investigational drug, HBN-1, to forced cooling to induce hypothermia after resuscitation in a translation model of asphyxial cardiac arrest in swine. Serum and cerebral spinal fluid neuron-specific enolase activity (sNSE and csfNSE) were measured after cardiac arrest as surrogate markers of brain injury. In a block design, swine resuscitated from 10 minutes of asphyxial cardiac arrest were infused intravenously with HBN-1 or iced saline vehicle (forced hypothermia [FH]) 5 to 45 minutes after return of spontaneous circulation (ROSC). External cooling in both groups was added 45 minutes after ROSC until hypothermia (T = 4C below baseline) was attained. Esophageal (core) temperature, shivering, cardiopulmonary parameters, and time to hypothermia after ROSC were monitored. sNSE and csfNSE were measured 180 minutes after ROSC. HBN-1 induced hypothermia significantly lowered temperature compared to FH 5–45 minutes after ROSC ( p < 0.0001). Time to hypothermia was reduced by HBN-1 (93 – 6 minutes) compared to FH (177 – 10 minutes) ( p < 0.0001). HBN-1 sNSE (0.7 – 1.9 ng/mL) and csfNSE (17.3 – 1.9 ng/mL) were lower compared to FH (6 – 1.6 ng/mL) and (49.7 – 32.0 ng/mL) ( p < 0.0001, p = 0.022, respectively). There was no shivering with HBN-1 cooling while all FH cooled swine shivered ( p < 0.0001). The time to reach target hypothermia after cardiac arrest was reduced by nearly 50% with HBN-1 compared to the FH method of inducing hypothermia. Moreover, surrogate biomarkers of brain injury were significantly reduced with HBN-1 as compared to FH. While HBN-1-induced hypothermia shows promise for being neuroprotective, survival studies are needed to confirm these preliminary findings.
Introduction
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ardiac arrest causes morbidity and death in hundreds of thousands of patients annually. Although mild hypothermia improves neurological outcome after cardiac arrest, it has been slow to be accepted in clinical practice, especially after asphyxial cardiac arrest because of concerns about the efficacy, safety, and complexity of the therapy. Hypothermia is clinically induced by forcing core temperature below its setpoint, thus overwhelming normal thermoregulatory mechanisms that resist cooling. These thermoeffector responses (shivering, vasoconstriction, and release of stress hormones) to forced cooling slow the onset and efficacy of hypothermia. On the other hand, regulated hypothermia induced by drugs may provide a simple alternative method for inducing hypothermia (Gordon, 2001). In theory, lowering the thermoregulatory setpoint would result in a rapid and controlled reduction in core temperature without activating thermoregulatory mechanisms that resist core cooling (Gordon, 1983).
1 2 3
HBN-1 is a combination of ethanol, vasopressin, and lidocaine. HBN-1 induced hypothermia in rodents has been shown to lower metabolic rate, blocks shivering, and minimizes peripheral vasoconstriction when compared to forced hypothermia, suggesting a regulated method of inducing hypothermia (Katz et al., 2012). Small mammals such as rats have a relatively large surface-to-body-mass ratio and small thermal inertia, allowing for a rapid reduction in body temperature when subjected to cold exposure. On the other hand, large mammals, including humans, have a low surfaceto-body-mass ratio with relatively high thermal inertia, making them comparatively more difficult to induce hypothermia compared to rodents (Anderson, 1999). Therefore, investigation of cooling methods in larger mammals such as swine with a surface-to-body-mass ratio closer to humans may provide improved translational value when developing methods for inducing therapeutic hypothermia in humans. The efficacy of neuroprotective therapies is most reliably evaluated by survival with a good neurological outcome.
Department Emergency Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina. Department of Epidemiology, University of Alabama at Birmingham School of Public Health, Birmingham, Alabama. Toxicity Assessment Division, Neurotoxicology Branch, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina.
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DRUG-INDUCED THERAPEUTIC HYPOTHERMIA IN SWINE However, these studies in large mammals are not practical for screening of neuroprotective therapies because of costs and time. Therefore, nonsurvival studies are often used to screen for neuroprotective therapies. Serum neuron-specific enolase (sNSE) and cerebral spinal fluid neuron-specific enolase (csfNSE) are elevated after acute brain injury and may provide surrogate markers of acute brain injury early during reperfusion from acute brain injury (Auer et al., 2006; Berger et al., 2010). These quantitative markers may be helpful for screening of neuroprotective therapies in nonsurvival largeanimal studies of acute brain injury such as cardiac arrest. The purpose of this feasibility study was (1) to determine if HBN-1 can induce hypothermia after resuscitation from cardiac arrest in a translational, large-animal (swine) model of cardiac arrest, and (2) to compare the efficacy of HBN-1induced hypothermia to forced hypothermia. The study also screened the potential of HBN-1-induced hypothermia to afford neuroprotection during reperfusion from cardiac arrest using biomarkers of acute brain injury. Methods The study was approved by the Institutional Animal Care and Use Committee at the University of North Carolina School of Medicine (UNC). UNC animal facilities are accredited by the American Association for the Accreditation of Laboratory Animal Care. Treatment interventions of HBN-1 or forced hypothermia were assigned in a block design after surgical preparation. Farm-bred swine (N = 14) averaging *30 kg were fasted overnight and given water ad lib. Swine were anesthetized with intramuscular ketamine 500 mg, buprenorphine 0.1 mg/kg, and insufflation of 4% isoflurane anesthesia. The swine were then intubated, mechanically ventilated, and isoflurane was discontinued (duration of isoflurane was *5 minutes). A peripheral intravenous catheter was inserted, and Propofol was titrated (250–350 mcg/ kg/h) to maintain sedation before cardiac arrest. A SwanGanz catheter was placed for measurement of cardiac output and pulmonary capillary wedge pressure. An esophageal probe was inserted and designated as a measure of core body temperature. Right femoral artery and venous catheters were placed for cardiovascular monitoring and access for blood sampling. A second peripheral intravenous line was established for therapeutic interventions. Isoflurane was discontinued for at least 2 hours before cardiac arrest to assure adequate washout of isoflurane and stabilization of cardiovascular parameters and core temperature. Intravenous vecuronium (0.1 mg/kg) was given 10 minutes before discontinuation of ventilation. Discontinuation of ventilation induced apnea and asphyxia, which lead to cardiac arrest. Cardiac arrest was defined as no arterial pulse pressure and a mean arterial pressure (MAP) < 10 mmHg. Propofol was discontinued after cardiac arrest. Ten minutes after asphyxia, the animals were resuscitated with cardiopulmonary resuscitation (CPR) that included 100% oxygen, mechanical ventilation, chest compressions, intravenous epinephrine (0.04 mg/kg), and sodium bicarbonate (1 meq/kg) intravenously. Chest compressions at an estimated coronary perfusion pressure of 40 mmHg and rate of 100 compressions per minute were performed for 60-second intervals between defibrillation attempts and continued after defibrillation until return of spontaneous circulation (ROSC). ROSC was defined
177 as a sustained (> 5 minute) spontaneous heart beat with an MAP > 60 mmHg. Interventions HBN-1 is a combination of ethanol 63 g/L, vasopressin 2.7 U/L, and lidocaine 66 mg/L prepared in 0.9% physiological saline administered at room temperature. The forced hypothermia group received iced saline at 4C containing vasopressin 2.7 U/L and lidocaine 66 mg/L. HBN-1 or iced saline vehicle infusion began 5 minutes after ROSC and continued until 180 minutes after ROSC. Both groups received a volume of 30 mL/kg over 40 minutes (5–45 minutes after ROSC) through the peripheral intravenous line, and then the rate of the infusions was decreased to 5 mL/kg/h until the end of the experiment at 180 minutes after ROSC. Propofol was initiated at 150 mcg/kg/h at 10 minutes after ROSC. At 45 minutes after ROSC, both groups were externally cooled with 4C externally applied gel pads (Arctic Sun; Medivance, Louisville, CO) that covered 60% of total body surface area until the swine reached a core target temperature of 4C below baseline. When target temperature was reached, external cooling was discontinued (Arctic Sun; Medivance). Swine were maintained on mechanical ventilation and 100% oxygen throughout the 180 minutes of reperfusion after ROSC with evidence of spontaneous respirations *10 minutes after ROSC. Ventilator adjustments were made to maintain normocapnia. At the end of the protocol, swine were euthanized per the Division of Lab and Animal Medicine protocol. Blood gas values, blood glucose, and cardiovascular parameters were measured at baseline and 10, 30, 60, 90, 120, 150, and 180 minutes after ROSC. Core temperature was measure at 1-minute intervals until the end of the protocol. Serum NSE was obtained at baseline and 3 hours after ROSC. Cerebral spinal fluid NSE was obtained 3 hours after ROSC. Blood alcohol levels were obtained at 60 and 180 minutes after ROSC. Shivering was defined as any involuntary highfrequency contraction of skeletal muscles of the trunk and limbs and continually monitored during reperfusion. Due to pilot experiments where hypotension and recurrent ventricular fibrillation occurred after resuscitation in the forced hypothermia group that received iced saline, vasopressin and lidocaine were added to the saline to attenuate these changes observed during forced hypothermia in our model. The dose of ethanol, vasopressin, and lidocaine in HBN-1 was based on pilot experiments and earlier work in our laboratory (Katz et al., 2012). Statistical analysis Longitudinal measurements of core temperature, cardiovascular parameters, glucose, and arterial blood gas results were compared between groups with repeated-measures analysis of variance (ANOVA). ROSC time, time to target temperature, number of defibrillations, sNSE, and csfNSE were compared between groups with a one-way ANOVA. The number of defibrillations was compared between groups by a simple t-test. The incidence of shivering was compared between groups by a chi-square test. Spearman correlation coefficients were used to evaluate the association between glucose and NSE. p-values of p < 0.05 were considered statistically significant. All statistical analyses were performed with
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KATZ ET AL. Table 1. Baseline Cardiovascular, Temperature, and Metabolic Parameters
Physiological parameters Esophageal temperature Mean arterial pressure Heart rate Cardiac output PCWP pH pCO2 pO2 HCO3 Base deficit Glucose
Forced hypothermia (n = 7)
HBN-1 (n = 7)
p-Value
37.7C – 0.3C 100 – 13 mmHg 101 – 13 BPM 3.9 – 0.99 L/min 14 – 3 mmHg 7.43 – 0.03 mmHg 43 – 3 mmHg 97 – 9 mmHg 28 – 1 3.8 – 1.7 118 – 27
37.7C – 0.2C 99 – 9mmHg 105 – 12 BPM 4.3 – 0.97 L/min 12 – 3 mmHg 7.44 – 0.04 mmHg 43 – 5 mmHg 95 – 6 mmHg 28 – 2 3.7 – 1.9 128 – 23
0.91 0.89 0.59 0.50 0.42 0.31 0.86 0.60 0.43 0.98 0.42
a
p-Value
< 0.0001 0.40 0.86 0.51 0.22 0.71 0.38 0.55 0.60 0.82 0.003
a Physiological parameters 10–180 minutes after ROSC. Values are reported as means and standard deviations. p-value = physiological parameters at baseline. BPM, beats per minute; PCWP, pulmonary capillary wedge pressure; ROSC, return of spontaneous circulation.
the SPSS v17 statistical software package (IBM Solutions, Research Triangle Park, NC). Results Fourteen swine resuscitated from asphyxial cardiac arrest were assigned to the HBN-1 (n = 7) or forced hypothermia (n = 7) group. There were no statistically significant differences in baseline weight (HBN-1 29.3 – 8 kg; forced hypothermia 30.4 – 2.2 kg), cardiovascular parameters, arterial blood gases, blood glucose, and core temperature between groups (Table 1). There was no statistically significant difference in time to cardiac arrest from onset of asphyxia, number of defibrillations, or time to ROSC between groups (Table 2). The initial rhythm before resuscitation was pulseless electrical activity (PEA) in 5 of 7 animals in the HBN-1 group and 6 of 7 in the forced hypothermia group. The remaining animals spontaneously went into ventricular fibrillation from PEA during asphyxia before resuscitation. Ventricular fibrillation occurred in all animals during CPR. Response to HBN-1 There were marked differences in the hypothermic response to HBN-1 compared to forced cooling (Fig. 1). Core temperature in the HBN-1 group was significantly lower than the forced hypothermia group during the initial infusion from 5–45 minutes after ROSC ( p < 0.0001) when there was no ex-
Table 2. Time to Cardiac Arrest from Onset of Asphyxia Resuscitation parameters
Forced hypothermia
HBN-1
Time to cardiac 7.2 – 0.5 minutes 7.2 – 0.8 minutes arrest Number of 2 (median) 2 (median) defibrillations Time to ROSC 4.5 – 4.4 minutes 4.6 – 2.6 minutes
pValue 0.97 0.59 0.97
Number of defibrillations before sinus rhythm. Values reported as means and standard deviations unless otherwise noted.
ternal cooling. The time from initiation of cooling (5 minutes after resuscitation) until target temperature was reached (4C below baseline) was significantly shorter in the HBN-1 (93 – 6 minutes) compared to the forced hypothermia group (177 – 10 minutes) ( p < 0.0001). There was no significant difference during reperfusion (Table 1) in MAP (Fig. 2), cardiac output (Fig. 3), pulmonary capillary wedge pressure (Fig. 4), or arterial blood gases between groups. There were significant differences in blood glucose and biomarkers of brain injury between the HBN-1 and forced hypothermia groups (Table 3). Blood glucose in the HBN-1 group was significantly lower compared to the forced hypothermia group ( p = 0.003). Serum NSE was zero in all groups before cardiac arrest. The serum NSE level was lower in the HBN-1-induced hypothermia group (0.7 – 1.9 ng/mL) compared to the forced hypothermia group (6.0 – 1.6 ng/mL) at 180 minutes after ROSC ( p < 0.0001). Cerebral spinal fluid NSE was also lower in the HBN-1-induced hypothermia group (17.3 – 1.9 ng/mL) compared to the forced hypothermia group (49.7 – 32 ng/mL) at 180 minutes after resuscitation ( p = 0.02). There were no significant correlations between glucose and NSE for either the forced (sNSE r = 0.27, p = 0.56, csfNSE r = - 0.62, p = 0.14) or HBN-1 (sNSE r = - 0.41, p = 0.36, csfNSE r = 0.04, p = 0.94) groups. Blood alcohol levels averaged 322 – 48 mg/dL std dev (60 minutes) and 356 – 37 mg/dL (180 minutes) after ROSC in the HBN-1-induced hypothermia group. No animals in the HBN-1 group (0 of 7) shivered after resuscitation, whereas all animals (7 of 7) in the forced hypothermia group showed profound shivering after resuscitation ( p < 0.001). The onset of shivering averaged 22 – 3 minutes after ROSC and continued until sacrifice at 180 minutes after resuscitation. Discussion This feasibility study, using a translational large-animal model of asphyxial cardiac arrest, demonstrated that peripheral intravenous infusion of HBN-1 at room temperature in nonparalyzed animals was superior to iced saline vehicle infusion in lowering core temperature 5–45 minutes after resuscitation from cardiac arrest. The hypothermic response of HBN-1 compared to forced hypothermia also reduced the time to reach the target hypothermic temperature. Blood
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FIG. 1. Mean core (esophageal) temperature in swine treated with HBN-1 or forced hypothermia after resuscitation from asphyxial cardiac arrest. HBN-1 was infused at room temperature, and iced saline vehicle in the forced hypothermia group was infused at 4C starting 5 minutes after resuscitation. Both groups were externally cooled with gel pads at 4C applied 45 minutes after resuscitation. Error bars represent standard deviations.
glucose was significantly lower in the HBN-1 group. Moreover, surrogate biomarkers of brain injury observed during early reperfusion were significantly reduced in swine made hypothermic with HBN-1. The 5–45-minute time period was chosen for the initial rapid intravenous therapy, because it corresponds to a reasonable time and volume of fluid administered by Emergency Medical Services (EMS) before arriving in the Emergency Department (ED) (Bernard et al., 2003). HBN-1 may provide EMS with the potential to initiate induction of hypothermia before reaching the hospital, since it can be administered intravenously without any special equipment.
Currently, induction of hypothermia is often delayed until hospital arrival, since most external or endovascular cooling devices are large, heavy, and are not readily available until arrival in the ED. In addition, chemical paralysis, an intervention not available to many EMS services, is required for use with these cooling devices, since they trigger shivering and other thermoregulatory mechanisms that slow or prevent cooling without paralysis. Paralysis has the disadvantage of limiting the ability to follow progress of neurological changes during reperfusion. The HBN-1 group continued to cool rapidly and showed no evidence of shivering with the addition of external cooling. In
FIG. 2. Mean arterial pressure in swine treated with HBN-1 or forced hypothermia after resuscitation from asphyxial cardiac arrest. ROSC, return of spontaneous circulation.
FIG. 3. Mean cardiac output in swine treated with HBN-1 or forced hypothermia after resuscitation from asphyxial cardiac arrest.
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KATZ ET AL.
FIG. 4. Mean pulmonary capillary wedge pressure in swine treated with HBN-1 or forced hypothermia after resuscitation from asphyxial cardiac arrest. PCWP, pulmonary capillary wedge pressure. contrast, the forced hypothermia group continued to shiver with the addition of external cooling. The rapid cooling and lack of shivering observed with HBN-1 administration in the current study support our earlier work in rodents that HBN-1 may induce regulated hypothermia (Katz et al., 2012). These results are important, because they suggest that HBN-1 induces similar thermoregulatory responses in small and large mammals. Future studies in humans are needed to establish the translational value of these results. Long-term survival and improved cognitive function are the best measures of neuroprotective therapies. However, these outcome measures are complex and expensive to perform in large animals. Therefore, sNSE and csfNSE were used in this nonsurvival study to screen for surrogate markers of brain injury (Auer et al., 2006; Berger et al., 2010). NSE is a soluble protein enolase enzyme (2-phopho-D-glyceride hydrolase) of the glycolytic pathway found in high concentrations in neurons and released into the cerebral spinal fluid and Table 3. Biomarkers of Brain Injury Forced hypothermia
HBN-1 hypothermia
p-Value
0 ng/mL 0 ng/mL Baseline serum NSE Serum NSE 6.0 – 1.6 ng/mL 0.7 – 1.9 ng/mL < 0.0001 Cerebral spinal 49.7 – 32 ng/mL 17.3 – 1.9 ng/mL 0.02 fluid NSE Glucose 213 – 32 123 – 7 mg/dL 0.003 Shivering 7/7 0/7 < 0.0001 Baseline serum neuron-specific enolase obtained at normothermia before cardiac arrest. Serum NSE obtained 180 minutes after ROSC. Cerebral spinal fluid NSE obtained 180 minutes after ROSC. Serum glucose 180 minutes after ROSC. Incidence of shivering during the 180-minute period after ROSC. Values reported as means and standard deviations. NSE, neuron-specific enolase.
blood during acute brain injury (Persson et al., 1987). NSE measured early after resuscitation from cardiac arrest in humans is considered to be a biomarker of hypoxic brain injury and a two- to nine fold higher NSE level was predictive of poor outcome (death or persistent coma) compared to the neurologically intact group in humans (Daubin et al., 2011). The relative lower NSE levels with HBN-1 versus the forced hypothermia group in our study were comparable to the differences predictive of good neurological outcome in the human study. Despite the encouraging results of lower sNSE and csfNSE levels with HBN-1-induced hypothermia, the biological significance of lower NSE levels requires long-term survival and neurocognitive function studies to adequately evaluate the neuroprotective potential of HBN-1 after acute brain injury. Elevated glucose is associated with worsened neurological outcome after acute brain injury (Warner et al., 1992). Glucose levels were unexpectedly lower in the HBN-1 group compared to the forced hypothermia group during reperfusion from cardiac arrest. The reason for this finding is unclear, but HBN-1 may reduce the stress response and insulin resistance induced by forced hypothermia (Macho et al., 2003; Polderman, 2009). There is an association between elevated glucose and serum NSE levels in stroke patients (Pandey et al., 2011). However, there were no significant correlations between the glucose and NSE levels in our study. There are no practical pharmacological methods to induce therapeutic hypothermia after resuscitation from cardiac arrest. General anesthetics such as isoflurane and barbiturates can lower body temperature, but do not induce regulated hypothermia and are not practical for Prehospital or Emergency Department administration, because it must be carefully titrated by an experienced anesthesiologists and can cause respiratory arrest and hypotension (Sessler et al., 1991). Hydrogen sulfide can lower body temperature in rodents, but failed to lower body temperature in anesthetized and paralyzed pigs, presumably due to failure to reduce metabolism (Li et al., 2008; Drabek et al., 2011). The combination of meperidine, buspirone, granisetron, and magnesium has been used in attempts to induce hypothermia in humans (Testori et al., 2011). Despite the addition of external cooling during the combination drug therapy, therapeutic levels of hypothermia could not be consistently obtained. A number of drug strategies have been developed to attenuate or eliminate shivering during forced cooling, but these drugs have the potential for respiratory depression and hypotension (Sessler, 2009). However, despite eliminating shivering during cooling, none of these pharmacological strategies reach therapeutic hypothermia temperatures without paralysis or the addition of forced cooling methods. HBN-1-induced hypothermia holds the advantage of being given as a single agent through one peripheral intravenous line, requires no paralysis, and does not cause respiratory depression or hypotension. In addition, HBN-1 promotes heat loss and reduces heat production (metabolism) consistent with regulated hypothermia demonstrated in earlier work (Katz et al., 2012). The individual drugs in HBN-1 are FDA approved and used in clinical practice. Ethanol has a well-defined toxicity, safety margin, and pharmacology in humans and other mammals. Ethanol induces a regulated hypothermia by lowering the thermoregulatory setpoint (Gordon and Stead, 1986). Recent human studies have shown that ethanol elicits a
DRUG-INDUCED THERAPEUTIC HYPOTHERMIA IN SWINE heat-dissipating response, characterized by sweating, peripheral vasodilation, and preference for cool ambient temperatures (Yoda et al., 2005). Ethanol alone is unable to sustain a long-term hypothermia, because hypothermia tolerance rapidly develops (Froehlich et al., 2001). The addition of vasopressin and lidocaine to ethanol used to formulate HBN-1 provides prolonged hypothermia by minimizing ethanol hypothermia tolerance, but the mechanism for this attenuated tolerance is unknown (Katz et al., 2012). The vasopressin component of HBN-1 also increases blood pressure during shock and may improve cerebral blood flow, but is not known to lower body temperature (Banet and Wieland, 1985; Wenzel et al., 1999). Lidocaine raises ventricular fibrillation threshold, but also does not lower body temperature (Harris et al., 1989). The combination of ethanol, vasopressin, and lidocaine in HBN-1 holds the potential for adverse affects, including sedation, respiratory depression, hypertension, hypotension, and lowering of the seizure threshold. However, none of these adverse affects were noted during this study. Future studies are required to determine the safety profile of HBN-1 in humans, especially when considered for use in ambulances and other clinical settings. Limitations This study has several limitations. The study utilized an assigned block design rather than a randomized protocol. This was due to a need to conduct pilot experiments to adjust the HBN-1 dosing while performing concurrent forced hypothermia experiments. However, the duration of cardiac arrest and resuscitation was similar between groups before initiation of therapeutic interventions. In addition, the observer who recorded cardiovascular, shivering, and temperature data was blinded to intervention and did not participate in surgical preparation. There was no normothermia group or HBN-1 group maintained normothermic in this study because of cost limitations; therefore, normothermic cardiopulmonary, glucose, sNSE, and csfNSE values during normothermic reperfusion in this cardiac arrest model in our laboratory are unknown. Since forced hypothermia can lower NSE levels after cardiac arrest (Tiainen et al., 2003; Sun et al., 2012), there is a value in demonstrating that HBN-1 did not raise NSE or glucose levels compared to forced hypothermia, but interpretation of the glucose and NSE values are limited without a normothermic control group. However, the focus of this feasibility study was to compare different methods of inducing hypothermia in large mammals after resuscitation from cardiac arrest in swine. Spontaneous hypothermia can occur in normothermic rats after cardiac arrest, but is unlikely to occur early after cardiac arrest in swine because of the relatively large thermal inertia compared to rodents. The study design was unable to determine whether HBN-1 alone can lower core temperature to target hypothermia, and future studies are needed to determine the efficiency of HBN1 alone to induce therapeutic hypothermia. A cooling device (external cooling pad) was added to both groups 40 minutes after initiation of infusion therapy to simulate the current clinical practice of inducing hypothermia in many EDs with cooling devices. Future studies with HBN-1 are planned to evaluate the ability to induce target hypothermia rapidly without cooling devices.
181 The cerebral insult for this model was mild with only 10 minutes of asphyxia (*3 minutes of no flow) and an average of 4 minutes of low flow (CPR). Previous work with asphyxial cardiac arrest has demonstrated that asphyxial cardiac arrest causes significant permanent brain injury, which may be relatively more severe compared to ventricular fibrillation cardiac arrest of the same duration (Vaagenes et al., 1997). Since the primary outcome of this study was comparing different methods of inducing hypothermia after cardiac arrest, a more severe insult may have reduced the chances of resuscitation and cardiovascular stability during reperfusion. These potential confounding variables could limit the ability to compare the different methods of inducing hypothermia. Animals were not paralyzed after cardiac arrest, although they all received anesthesia as is appropriate for the humane treatment of experimental animals. Anesthesia can affect thermoregulation, so extrapolation of these results to humans without anesthesia should be approached with caution (Farber et al., 1994). Lastly, the short duration of reperfusion in this study was inadequate to allow maturation of brain injury; thus, the study cannot adequately assess the neuroprotective effects of HBN-1 (Pulsinelli et al., 1982; Kirino et al., 1984; Katz et al., 1995). HBN-1-induced hypothermia was associated with a shortened time to hypothermia and decreased sNSE and csfNSE levels during early reperfusion when compared with forced hypothermia. These results are encouraging, but a normothermic control group and longer reperfusion times are needed to determine the values of NSE for predicting brain damage during early reperfusion in this model before drawing any conclusions regarding the potential neuroprotective properties of HBN-1. In addition, long-term survival studies will be needed to determine whether HBN-1 or the shortened time to hypothermia induced by HBN-1 provides neuroprotection. Conclusions Administration of intravenous HBN-1 in a translational swine model of cardiac arrest lowered core temperature and shortened the time to reach target hypothermia compared to the forced hypothermia method. HBN-1 prevented shivering and reduced blood glucose during induction of hypothermia. Surrogate biomarkers of brain injury sNSE and csfNSE were attenuated with HBN-1-induced hypothermia as compared to forced hypothermia. While HBN-1 induced hypothermia showed promise for being neuroprotective, survival studies are needed to confirm these preliminary findings. Acknowledgments The study was funded by a grant from the Laerdal Foundation for Acute Medicine. We thank Bonita L. Marks, PhD., for her review of the manuscript. We would also like to thank Jonathan E. Frank, Shane McCurdy, and Chad Spruill for their technical support. Disclosure Statement Dr. Katz submitted a patent entitled Methods and Compositions for the Induction of Hypothermia with the University of North Carolina at Chapel Hill. Dr. Katz owns stock in the University of North Carolina spin-off company Hibernaid. No other author has a conflict of interest.
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KATZ ET AL. Macho L, Zorad S, Radikova Z, Patterson-Buckedahl P, Kvetnansky R. Ethanol consumption affects stress response and insulin binding in tissues of rats. Endocr Regul 2003;37: 195–202. Pandey A, Saxena K, Verma M, Bharosay A. Correlative study between neuron-specific enolase and blood sugar level in ischemic stroke patients. J Neurosci Rural Pract 2011;2:50–54. Persson L, Hardemark HG, Gustafsson J, Rundstrom G, MendelHartvig I, Esscher T, et al. S-100 protein and neuron-specific enolase in cerebrospinal fluid and serum: markers of cell damage in human central nervous system. Stroke 1987;18:911–918. Polderman KH. Mechanisms of action, physiological effects, and complications of hypothermia. Crit Care Med 2009;37: S186–S202. Pulsinelli WA, Levy DE, Duffy TE. Regional cerebral blood flow and glucose metabolism following transient forebrain ischemia. Ann Neurol 1982;11:499–502. Sessler DI. Defeating normal thermoregulatory defenses: induction of therapeutic hypothermia. Stroke 2009;40:e614–e621. Sessler DI, Rubinstein EH, Moayeri A. Physiologic responses to mild perianesthetic hypothermia in humans. Anesthesiology 1991;75:594–610. Sun J, Li J, Cheng G, Sha B, Zhou W. Effects of hypothermia on NSE and S-100 protein levels in CSF in neonates following hypoxic/ischaemic brain damage. Acta Paediatr 2012;101: e316–e320. Testori C, Sterz F, Behringer W, Spiel A, Firbas C, Jilma B. Surface cooling for induction of mild hypothermia in conscious healthy volunteers—a feasibility trial. Crit Care 2011; 15:1–7. Tiainen M, Roine RO, Pettila V, Takkunen O. Serum neuronspecific enolase and S-100B protein in cardiac arrest patients treated with hypothermia. Stroke 2003;34:2881–2886. Vaagenes P, Safar P, Moossy J, Rao G, Diven W, Ravi C, et al. Asphyxiation versus ventricular fibrillation cardiac arrest in dogs. Differences in cerebral resuscitation effects—a preliminary study. Resuscitation 1997;35:41–52. Warner DS, Gionet TX, Todd MM, McAllister AM. Insulininduced normoglycemia improves ischemic outcome in hyperglycemic rats. Stroke 1992;23:1775–1780, discussion 1781. Wenzel V, Lindner KH, Prengel AW, Maier C, Voelckel W, Lurie KG, et al. Vasopressin improves vital organ blood flow after prolonged cardiac arrest with postcountershock pulseless electrical activity in pigs. Crit Care Med 1999;27:486–492. Yoda T, Crawshaw LI, Nakamura M, Saito K, Konishi A, Nagashima K, et al. Effects of alcohol on thermoregulation during mild heat exposure in humans. Alcohol 2005;36:195–200.
Address correspondence to: Laurence M. Katz, M.D. Department Emergency Medicine University of North Carolina School of Medicine 170 Manning Drive CB 7594 Chapel Hill, NC 27599 E-mail: [email protected]
Drug-Induced Therapeutic Hypothermia After Asphyxial Cardiac Arrest in Swine Laurence M. Katz, M.D.,1 Gerald McGwin, Jr., Ph.D.,2 and Christopher J. Gordon, Ph.D.3
A feasibility study was performed to compare an investigational drug, HBN-1, to forced cooling to induce hypothermia after resuscitation in a translation model of asphyxial cardiac arrest in swine. Serum and cerebral spinal fluid neuron-specific enolase activity (sNSE and csfNSE) were measured after cardiac arrest as surrogate markers of brain injury. In a block design, swine resuscitated from 10 minutes of asphyxial cardiac arrest were infused intravenously with HBN-1 or iced saline vehicle (forced hypothermia [FH]) 5 to 45 minutes after return of spontaneous circulation (ROSC). External cooling in both groups was added 45 minutes after ROSC until hypothermia (T = 4C below baseline) was attained. Esophageal (core) temperature, shivering, cardiopulmonary parameters, and time to hypothermia after ROSC were monitored. sNSE and csfNSE were measured 180 minutes after ROSC. HBN-1 induced hypothermia significantly lowered temperature compared to FH 5–45 minutes after ROSC ( p < 0.0001). Time to hypothermia was reduced by HBN-1 (93 – 6 minutes) compared to FH (177 – 10 minutes) ( p < 0.0001). HBN-1 sNSE (0.7 – 1.9 ng/mL) and csfNSE (17.3 – 1.9 ng/mL) were lower compared to FH (6 – 1.6 ng/mL) and (49.7 – 32.0 ng/mL) ( p < 0.0001, p = 0.022, respectively). There was no shivering with HBN-1 cooling while all FH cooled swine shivered ( p < 0.0001). The time to reach target hypothermia after cardiac arrest was reduced by nearly 50% with HBN-1 compared to the FH method of inducing hypothermia. Moreover, surrogate biomarkers of brain injury were significantly reduced with HBN-1 as compared to FH. While HBN-1-induced hypothermia shows promise for being neuroprotective, survival studies are needed to confirm these preliminary findings.
Introduction
C
ardiac arrest causes morbidity and death in hundreds of thousands of patients annually. Although mild hypothermia improves neurological outcome after cardiac arrest, it has been slow to be accepted in clinical practice, especially after asphyxial cardiac arrest because of concerns about the efficacy, safety, and complexity of the therapy. Hypothermia is clinically induced by forcing core temperature below its setpoint, thus overwhelming normal thermoregulatory mechanisms that resist cooling. These thermoeffector responses (shivering, vasoconstriction, and release of stress hormones) to forced cooling slow the onset and efficacy of hypothermia. On the other hand, regulated hypothermia induced by drugs may provide a simple alternative method for inducing hypothermia (Gordon, 2001). In theory, lowering the thermoregulatory setpoint would result in a rapid and controlled reduction in core temperature without activating thermoregulatory mechanisms that resist core cooling (Gordon, 1983).
1 2 3
HBN-1 is a combination of ethanol, vasopressin, and lidocaine. HBN-1 induced hypothermia in rodents has been shown to lower metabolic rate, blocks shivering, and minimizes peripheral vasoconstriction when compared to forced hypothermia, suggesting a regulated method of inducing hypothermia (Katz et al., 2012). Small mammals such as rats have a relatively large surface-to-body-mass ratio and small thermal inertia, allowing for a rapid reduction in body temperature when subjected to cold exposure. On the other hand, large mammals, including humans, have a low surfaceto-body-mass ratio with relatively high thermal inertia, making them comparatively more difficult to induce hypothermia compared to rodents (Anderson, 1999). Therefore, investigation of cooling methods in larger mammals such as swine with a surface-to-body-mass ratio closer to humans may provide improved translational value when developing methods for inducing therapeutic hypothermia in humans. The efficacy of neuroprotective therapies is most reliably evaluated by survival with a good neurological outcome.
Department Emergency Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina. Department of Epidemiology, University of Alabama at Birmingham School of Public Health, Birmingham, Alabama. Toxicity Assessment Division, Neurotoxicology Branch, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina.
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DRUG-INDUCED THERAPEUTIC HYPOTHERMIA IN SWINE However, these studies in large mammals are not practical for screening of neuroprotective therapies because of costs and time. Therefore, nonsurvival studies are often used to screen for neuroprotective therapies. Serum neuron-specific enolase (sNSE) and cerebral spinal fluid neuron-specific enolase (csfNSE) are elevated after acute brain injury and may provide surrogate markers of acute brain injury early during reperfusion from acute brain injury (Auer et al., 2006; Berger et al., 2010). These quantitative markers may be helpful for screening of neuroprotective therapies in nonsurvival largeanimal studies of acute brain injury such as cardiac arrest. The purpose of this feasibility study was (1) to determine if HBN-1 can induce hypothermia after resuscitation from cardiac arrest in a translational, large-animal (swine) model of cardiac arrest, and (2) to compare the efficacy of HBN-1induced hypothermia to forced hypothermia. The study also screened the potential of HBN-1-induced hypothermia to afford neuroprotection during reperfusion from cardiac arrest using biomarkers of acute brain injury. Methods The study was approved by the Institutional Animal Care and Use Committee at the University of North Carolina School of Medicine (UNC). UNC animal facilities are accredited by the American Association for the Accreditation of Laboratory Animal Care. Treatment interventions of HBN-1 or forced hypothermia were assigned in a block design after surgical preparation. Farm-bred swine (N = 14) averaging *30 kg were fasted overnight and given water ad lib. Swine were anesthetized with intramuscular ketamine 500 mg, buprenorphine 0.1 mg/kg, and insufflation of 4% isoflurane anesthesia. The swine were then intubated, mechanically ventilated, and isoflurane was discontinued (duration of isoflurane was *5 minutes). A peripheral intravenous catheter was inserted, and Propofol was titrated (250–350 mcg/ kg/h) to maintain sedation before cardiac arrest. A SwanGanz catheter was placed for measurement of cardiac output and pulmonary capillary wedge pressure. An esophageal probe was inserted and designated as a measure of core body temperature. Right femoral artery and venous catheters were placed for cardiovascular monitoring and access for blood sampling. A second peripheral intravenous line was established for therapeutic interventions. Isoflurane was discontinued for at least 2 hours before cardiac arrest to assure adequate washout of isoflurane and stabilization of cardiovascular parameters and core temperature. Intravenous vecuronium (0.1 mg/kg) was given 10 minutes before discontinuation of ventilation. Discontinuation of ventilation induced apnea and asphyxia, which lead to cardiac arrest. Cardiac arrest was defined as no arterial pulse pressure and a mean arterial pressure (MAP) < 10 mmHg. Propofol was discontinued after cardiac arrest. Ten minutes after asphyxia, the animals were resuscitated with cardiopulmonary resuscitation (CPR) that included 100% oxygen, mechanical ventilation, chest compressions, intravenous epinephrine (0.04 mg/kg), and sodium bicarbonate (1 meq/kg) intravenously. Chest compressions at an estimated coronary perfusion pressure of 40 mmHg and rate of 100 compressions per minute were performed for 60-second intervals between defibrillation attempts and continued after defibrillation until return of spontaneous circulation (ROSC). ROSC was defined
177 as a sustained (> 5 minute) spontaneous heart beat with an MAP > 60 mmHg. Interventions HBN-1 is a combination of ethanol 63 g/L, vasopressin 2.7 U/L, and lidocaine 66 mg/L prepared in 0.9% physiological saline administered at room temperature. The forced hypothermia group received iced saline at 4C containing vasopressin 2.7 U/L and lidocaine 66 mg/L. HBN-1 or iced saline vehicle infusion began 5 minutes after ROSC and continued until 180 minutes after ROSC. Both groups received a volume of 30 mL/kg over 40 minutes (5–45 minutes after ROSC) through the peripheral intravenous line, and then the rate of the infusions was decreased to 5 mL/kg/h until the end of the experiment at 180 minutes after ROSC. Propofol was initiated at 150 mcg/kg/h at 10 minutes after ROSC. At 45 minutes after ROSC, both groups were externally cooled with 4C externally applied gel pads (Arctic Sun; Medivance, Louisville, CO) that covered 60% of total body surface area until the swine reached a core target temperature of 4C below baseline. When target temperature was reached, external cooling was discontinued (Arctic Sun; Medivance). Swine were maintained on mechanical ventilation and 100% oxygen throughout the 180 minutes of reperfusion after ROSC with evidence of spontaneous respirations *10 minutes after ROSC. Ventilator adjustments were made to maintain normocapnia. At the end of the protocol, swine were euthanized per the Division of Lab and Animal Medicine protocol. Blood gas values, blood glucose, and cardiovascular parameters were measured at baseline and 10, 30, 60, 90, 120, 150, and 180 minutes after ROSC. Core temperature was measure at 1-minute intervals until the end of the protocol. Serum NSE was obtained at baseline and 3 hours after ROSC. Cerebral spinal fluid NSE was obtained 3 hours after ROSC. Blood alcohol levels were obtained at 60 and 180 minutes after ROSC. Shivering was defined as any involuntary highfrequency contraction of skeletal muscles of the trunk and limbs and continually monitored during reperfusion. Due to pilot experiments where hypotension and recurrent ventricular fibrillation occurred after resuscitation in the forced hypothermia group that received iced saline, vasopressin and lidocaine were added to the saline to attenuate these changes observed during forced hypothermia in our model. The dose of ethanol, vasopressin, and lidocaine in HBN-1 was based on pilot experiments and earlier work in our laboratory (Katz et al., 2012). Statistical analysis Longitudinal measurements of core temperature, cardiovascular parameters, glucose, and arterial blood gas results were compared between groups with repeated-measures analysis of variance (ANOVA). ROSC time, time to target temperature, number of defibrillations, sNSE, and csfNSE were compared between groups with a one-way ANOVA. The number of defibrillations was compared between groups by a simple t-test. The incidence of shivering was compared between groups by a chi-square test. Spearman correlation coefficients were used to evaluate the association between glucose and NSE. p-values of p < 0.05 were considered statistically significant. All statistical analyses were performed with
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KATZ ET AL. Table 1. Baseline Cardiovascular, Temperature, and Metabolic Parameters
Physiological parameters Esophageal temperature Mean arterial pressure Heart rate Cardiac output PCWP pH pCO2 pO2 HCO3 Base deficit Glucose
Forced hypothermia (n = 7)
HBN-1 (n = 7)
p-Value
37.7C – 0.3C 100 – 13 mmHg 101 – 13 BPM 3.9 – 0.99 L/min 14 – 3 mmHg 7.43 – 0.03 mmHg 43 – 3 mmHg 97 – 9 mmHg 28 – 1 3.8 – 1.7 118 – 27
37.7C – 0.2C 99 – 9mmHg 105 – 12 BPM 4.3 – 0.97 L/min 12 – 3 mmHg 7.44 – 0.04 mmHg 43 – 5 mmHg 95 – 6 mmHg 28 – 2 3.7 – 1.9 128 – 23
0.91 0.89 0.59 0.50 0.42 0.31 0.86 0.60 0.43 0.98 0.42
a
p-Value
< 0.0001 0.40 0.86 0.51 0.22 0.71 0.38 0.55 0.60 0.82 0.003
a Physiological parameters 10–180 minutes after ROSC. Values are reported as means and standard deviations. p-value = physiological parameters at baseline. BPM, beats per minute; PCWP, pulmonary capillary wedge pressure; ROSC, return of spontaneous circulation.
the SPSS v17 statistical software package (IBM Solutions, Research Triangle Park, NC). Results Fourteen swine resuscitated from asphyxial cardiac arrest were assigned to the HBN-1 (n = 7) or forced hypothermia (n = 7) group. There were no statistically significant differences in baseline weight (HBN-1 29.3 – 8 kg; forced hypothermia 30.4 – 2.2 kg), cardiovascular parameters, arterial blood gases, blood glucose, and core temperature between groups (Table 1). There was no statistically significant difference in time to cardiac arrest from onset of asphyxia, number of defibrillations, or time to ROSC between groups (Table 2). The initial rhythm before resuscitation was pulseless electrical activity (PEA) in 5 of 7 animals in the HBN-1 group and 6 of 7 in the forced hypothermia group. The remaining animals spontaneously went into ventricular fibrillation from PEA during asphyxia before resuscitation. Ventricular fibrillation occurred in all animals during CPR. Response to HBN-1 There were marked differences in the hypothermic response to HBN-1 compared to forced cooling (Fig. 1). Core temperature in the HBN-1 group was significantly lower than the forced hypothermia group during the initial infusion from 5–45 minutes after ROSC ( p < 0.0001) when there was no ex-
Table 2. Time to Cardiac Arrest from Onset of Asphyxia Resuscitation parameters
Forced hypothermia
HBN-1
Time to cardiac 7.2 – 0.5 minutes 7.2 – 0.8 minutes arrest Number of 2 (median) 2 (median) defibrillations Time to ROSC 4.5 – 4.4 minutes 4.6 – 2.6 minutes
pValue 0.97 0.59 0.97
Number of defibrillations before sinus rhythm. Values reported as means and standard deviations unless otherwise noted.
ternal cooling. The time from initiation of cooling (5 minutes after resuscitation) until target temperature was reached (4C below baseline) was significantly shorter in the HBN-1 (93 – 6 minutes) compared to the forced hypothermia group (177 – 10 minutes) ( p < 0.0001). There was no significant difference during reperfusion (Table 1) in MAP (Fig. 2), cardiac output (Fig. 3), pulmonary capillary wedge pressure (Fig. 4), or arterial blood gases between groups. There were significant differences in blood glucose and biomarkers of brain injury between the HBN-1 and forced hypothermia groups (Table 3). Blood glucose in the HBN-1 group was significantly lower compared to the forced hypothermia group ( p = 0.003). Serum NSE was zero in all groups before cardiac arrest. The serum NSE level was lower in the HBN-1-induced hypothermia group (0.7 – 1.9 ng/mL) compared to the forced hypothermia group (6.0 – 1.6 ng/mL) at 180 minutes after ROSC ( p < 0.0001). Cerebral spinal fluid NSE was also lower in the HBN-1-induced hypothermia group (17.3 – 1.9 ng/mL) compared to the forced hypothermia group (49.7 – 32 ng/mL) at 180 minutes after resuscitation ( p = 0.02). There were no significant correlations between glucose and NSE for either the forced (sNSE r = 0.27, p = 0.56, csfNSE r = - 0.62, p = 0.14) or HBN-1 (sNSE r = - 0.41, p = 0.36, csfNSE r = 0.04, p = 0.94) groups. Blood alcohol levels averaged 322 – 48 mg/dL std dev (60 minutes) and 356 – 37 mg/dL (180 minutes) after ROSC in the HBN-1-induced hypothermia group. No animals in the HBN-1 group (0 of 7) shivered after resuscitation, whereas all animals (7 of 7) in the forced hypothermia group showed profound shivering after resuscitation ( p < 0.001). The onset of shivering averaged 22 – 3 minutes after ROSC and continued until sacrifice at 180 minutes after resuscitation. Discussion This feasibility study, using a translational large-animal model of asphyxial cardiac arrest, demonstrated that peripheral intravenous infusion of HBN-1 at room temperature in nonparalyzed animals was superior to iced saline vehicle infusion in lowering core temperature 5–45 minutes after resuscitation from cardiac arrest. The hypothermic response of HBN-1 compared to forced hypothermia also reduced the time to reach the target hypothermic temperature. Blood
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FIG. 1. Mean core (esophageal) temperature in swine treated with HBN-1 or forced hypothermia after resuscitation from asphyxial cardiac arrest. HBN-1 was infused at room temperature, and iced saline vehicle in the forced hypothermia group was infused at 4C starting 5 minutes after resuscitation. Both groups were externally cooled with gel pads at 4C applied 45 minutes after resuscitation. Error bars represent standard deviations.
glucose was significantly lower in the HBN-1 group. Moreover, surrogate biomarkers of brain injury observed during early reperfusion were significantly reduced in swine made hypothermic with HBN-1. The 5–45-minute time period was chosen for the initial rapid intravenous therapy, because it corresponds to a reasonable time and volume of fluid administered by Emergency Medical Services (EMS) before arriving in the Emergency Department (ED) (Bernard et al., 2003). HBN-1 may provide EMS with the potential to initiate induction of hypothermia before reaching the hospital, since it can be administered intravenously without any special equipment.
Currently, induction of hypothermia is often delayed until hospital arrival, since most external or endovascular cooling devices are large, heavy, and are not readily available until arrival in the ED. In addition, chemical paralysis, an intervention not available to many EMS services, is required for use with these cooling devices, since they trigger shivering and other thermoregulatory mechanisms that slow or prevent cooling without paralysis. Paralysis has the disadvantage of limiting the ability to follow progress of neurological changes during reperfusion. The HBN-1 group continued to cool rapidly and showed no evidence of shivering with the addition of external cooling. In
FIG. 2. Mean arterial pressure in swine treated with HBN-1 or forced hypothermia after resuscitation from asphyxial cardiac arrest. ROSC, return of spontaneous circulation.
FIG. 3. Mean cardiac output in swine treated with HBN-1 or forced hypothermia after resuscitation from asphyxial cardiac arrest.
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KATZ ET AL.
FIG. 4. Mean pulmonary capillary wedge pressure in swine treated with HBN-1 or forced hypothermia after resuscitation from asphyxial cardiac arrest. PCWP, pulmonary capillary wedge pressure. contrast, the forced hypothermia group continued to shiver with the addition of external cooling. The rapid cooling and lack of shivering observed with HBN-1 administration in the current study support our earlier work in rodents that HBN-1 may induce regulated hypothermia (Katz et al., 2012). These results are important, because they suggest that HBN-1 induces similar thermoregulatory responses in small and large mammals. Future studies in humans are needed to establish the translational value of these results. Long-term survival and improved cognitive function are the best measures of neuroprotective therapies. However, these outcome measures are complex and expensive to perform in large animals. Therefore, sNSE and csfNSE were used in this nonsurvival study to screen for surrogate markers of brain injury (Auer et al., 2006; Berger et al., 2010). NSE is a soluble protein enolase enzyme (2-phopho-D-glyceride hydrolase) of the glycolytic pathway found in high concentrations in neurons and released into the cerebral spinal fluid and Table 3. Biomarkers of Brain Injury Forced hypothermia
HBN-1 hypothermia
p-Value
0 ng/mL 0 ng/mL Baseline serum NSE Serum NSE 6.0 – 1.6 ng/mL 0.7 – 1.9 ng/mL < 0.0001 Cerebral spinal 49.7 – 32 ng/mL 17.3 – 1.9 ng/mL 0.02 fluid NSE Glucose 213 – 32 123 – 7 mg/dL 0.003 Shivering 7/7 0/7 < 0.0001 Baseline serum neuron-specific enolase obtained at normothermia before cardiac arrest. Serum NSE obtained 180 minutes after ROSC. Cerebral spinal fluid NSE obtained 180 minutes after ROSC. Serum glucose 180 minutes after ROSC. Incidence of shivering during the 180-minute period after ROSC. Values reported as means and standard deviations. NSE, neuron-specific enolase.
blood during acute brain injury (Persson et al., 1987). NSE measured early after resuscitation from cardiac arrest in humans is considered to be a biomarker of hypoxic brain injury and a two- to nine fold higher NSE level was predictive of poor outcome (death or persistent coma) compared to the neurologically intact group in humans (Daubin et al., 2011). The relative lower NSE levels with HBN-1 versus the forced hypothermia group in our study were comparable to the differences predictive of good neurological outcome in the human study. Despite the encouraging results of lower sNSE and csfNSE levels with HBN-1-induced hypothermia, the biological significance of lower NSE levels requires long-term survival and neurocognitive function studies to adequately evaluate the neuroprotective potential of HBN-1 after acute brain injury. Elevated glucose is associated with worsened neurological outcome after acute brain injury (Warner et al., 1992). Glucose levels were unexpectedly lower in the HBN-1 group compared to the forced hypothermia group during reperfusion from cardiac arrest. The reason for this finding is unclear, but HBN-1 may reduce the stress response and insulin resistance induced by forced hypothermia (Macho et al., 2003; Polderman, 2009). There is an association between elevated glucose and serum NSE levels in stroke patients (Pandey et al., 2011). However, there were no significant correlations between the glucose and NSE levels in our study. There are no practical pharmacological methods to induce therapeutic hypothermia after resuscitation from cardiac arrest. General anesthetics such as isoflurane and barbiturates can lower body temperature, but do not induce regulated hypothermia and are not practical for Prehospital or Emergency Department administration, because it must be carefully titrated by an experienced anesthesiologists and can cause respiratory arrest and hypotension (Sessler et al., 1991). Hydrogen sulfide can lower body temperature in rodents, but failed to lower body temperature in anesthetized and paralyzed pigs, presumably due to failure to reduce metabolism (Li et al., 2008; Drabek et al., 2011). The combination of meperidine, buspirone, granisetron, and magnesium has been used in attempts to induce hypothermia in humans (Testori et al., 2011). Despite the addition of external cooling during the combination drug therapy, therapeutic levels of hypothermia could not be consistently obtained. A number of drug strategies have been developed to attenuate or eliminate shivering during forced cooling, but these drugs have the potential for respiratory depression and hypotension (Sessler, 2009). However, despite eliminating shivering during cooling, none of these pharmacological strategies reach therapeutic hypothermia temperatures without paralysis or the addition of forced cooling methods. HBN-1-induced hypothermia holds the advantage of being given as a single agent through one peripheral intravenous line, requires no paralysis, and does not cause respiratory depression or hypotension. In addition, HBN-1 promotes heat loss and reduces heat production (metabolism) consistent with regulated hypothermia demonstrated in earlier work (Katz et al., 2012). The individual drugs in HBN-1 are FDA approved and used in clinical practice. Ethanol has a well-defined toxicity, safety margin, and pharmacology in humans and other mammals. Ethanol induces a regulated hypothermia by lowering the thermoregulatory setpoint (Gordon and Stead, 1986). Recent human studies have shown that ethanol elicits a
DRUG-INDUCED THERAPEUTIC HYPOTHERMIA IN SWINE heat-dissipating response, characterized by sweating, peripheral vasodilation, and preference for cool ambient temperatures (Yoda et al., 2005). Ethanol alone is unable to sustain a long-term hypothermia, because hypothermia tolerance rapidly develops (Froehlich et al., 2001). The addition of vasopressin and lidocaine to ethanol used to formulate HBN-1 provides prolonged hypothermia by minimizing ethanol hypothermia tolerance, but the mechanism for this attenuated tolerance is unknown (Katz et al., 2012). The vasopressin component of HBN-1 also increases blood pressure during shock and may improve cerebral blood flow, but is not known to lower body temperature (Banet and Wieland, 1985; Wenzel et al., 1999). Lidocaine raises ventricular fibrillation threshold, but also does not lower body temperature (Harris et al., 1989). The combination of ethanol, vasopressin, and lidocaine in HBN-1 holds the potential for adverse affects, including sedation, respiratory depression, hypertension, hypotension, and lowering of the seizure threshold. However, none of these adverse affects were noted during this study. Future studies are required to determine the safety profile of HBN-1 in humans, especially when considered for use in ambulances and other clinical settings. Limitations This study has several limitations. The study utilized an assigned block design rather than a randomized protocol. This was due to a need to conduct pilot experiments to adjust the HBN-1 dosing while performing concurrent forced hypothermia experiments. However, the duration of cardiac arrest and resuscitation was similar between groups before initiation of therapeutic interventions. In addition, the observer who recorded cardiovascular, shivering, and temperature data was blinded to intervention and did not participate in surgical preparation. There was no normothermia group or HBN-1 group maintained normothermic in this study because of cost limitations; therefore, normothermic cardiopulmonary, glucose, sNSE, and csfNSE values during normothermic reperfusion in this cardiac arrest model in our laboratory are unknown. Since forced hypothermia can lower NSE levels after cardiac arrest (Tiainen et al., 2003; Sun et al., 2012), there is a value in demonstrating that HBN-1 did not raise NSE or glucose levels compared to forced hypothermia, but interpretation of the glucose and NSE values are limited without a normothermic control group. However, the focus of this feasibility study was to compare different methods of inducing hypothermia in large mammals after resuscitation from cardiac arrest in swine. Spontaneous hypothermia can occur in normothermic rats after cardiac arrest, but is unlikely to occur early after cardiac arrest in swine because of the relatively large thermal inertia compared to rodents. The study design was unable to determine whether HBN-1 alone can lower core temperature to target hypothermia, and future studies are needed to determine the efficiency of HBN1 alone to induce therapeutic hypothermia. A cooling device (external cooling pad) was added to both groups 40 minutes after initiation of infusion therapy to simulate the current clinical practice of inducing hypothermia in many EDs with cooling devices. Future studies with HBN-1 are planned to evaluate the ability to induce target hypothermia rapidly without cooling devices.
181 The cerebral insult for this model was mild with only 10 minutes of asphyxia (*3 minutes of no flow) and an average of 4 minutes of low flow (CPR). Previous work with asphyxial cardiac arrest has demonstrated that asphyxial cardiac arrest causes significant permanent brain injury, which may be relatively more severe compared to ventricular fibrillation cardiac arrest of the same duration (Vaagenes et al., 1997). Since the primary outcome of this study was comparing different methods of inducing hypothermia after cardiac arrest, a more severe insult may have reduced the chances of resuscitation and cardiovascular stability during reperfusion. These potential confounding variables could limit the ability to compare the different methods of inducing hypothermia. Animals were not paralyzed after cardiac arrest, although they all received anesthesia as is appropriate for the humane treatment of experimental animals. Anesthesia can affect thermoregulation, so extrapolation of these results to humans without anesthesia should be approached with caution (Farber et al., 1994). Lastly, the short duration of reperfusion in this study was inadequate to allow maturation of brain injury; thus, the study cannot adequately assess the neuroprotective effects of HBN-1 (Pulsinelli et al., 1982; Kirino et al., 1984; Katz et al., 1995). HBN-1-induced hypothermia was associated with a shortened time to hypothermia and decreased sNSE and csfNSE levels during early reperfusion when compared with forced hypothermia. These results are encouraging, but a normothermic control group and longer reperfusion times are needed to determine the values of NSE for predicting brain damage during early reperfusion in this model before drawing any conclusions regarding the potential neuroprotective properties of HBN-1. In addition, long-term survival studies will be needed to determine whether HBN-1 or the shortened time to hypothermia induced by HBN-1 provides neuroprotection. Conclusions Administration of intravenous HBN-1 in a translational swine model of cardiac arrest lowered core temperature and shortened the time to reach target hypothermia compared to the forced hypothermia method. HBN-1 prevented shivering and reduced blood glucose during induction of hypothermia. Surrogate biomarkers of brain injury sNSE and csfNSE were attenuated with HBN-1-induced hypothermia as compared to forced hypothermia. While HBN-1 induced hypothermia showed promise for being neuroprotective, survival studies are needed to confirm these preliminary findings. Acknowledgments The study was funded by a grant from the Laerdal Foundation for Acute Medicine. We thank Bonita L. Marks, PhD., for her review of the manuscript. We would also like to thank Jonathan E. Frank, Shane McCurdy, and Chad Spruill for their technical support. Disclosure Statement Dr. Katz submitted a patent entitled Methods and Compositions for the Induction of Hypothermia with the University of North Carolina at Chapel Hill. Dr. Katz owns stock in the University of North Carolina spin-off company Hibernaid. No other author has a conflict of interest.
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Address correspondence to: Laurence M. Katz, M.D. Department Emergency Medicine University of North Carolina School of Medicine 170 Manning Drive CB 7594 Chapel Hill, NC 27599 E-mail: [email protected]
