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Influence of age on heart rate variability during therapeutic hypothermia in a rat model
Resuscitation 82 (2011) 1350–1354
Contents lists available at ScienceDirect
Resuscitation journal homepage: www.elsevier.com/locate/resuscitation
Experimental paper
Influence of age on heart rate variability during therapeutic hypothermia in a rat model夽 Yun-Te Chang a,d,∗ , Shue-Ren Wann a,b , Pei-Lin Wu a , Kai-Hseng Hsieh c , Chu-Chuan Lin c , Mu-Shun Huang d , Hong-Tai Chang a a
Department of Emergency Medicine, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan The Infectious Diseases Section, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan c Department of Pediatrics, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan d School of Medicine, National Yang-Ming University, Taiwan b
a r t i c l e
i n f o
Article history: Received 29 October 2010 Received in revised form 2 April 2011 Accepted 9 April 2011
Keywords: Heart rate variability Age Autonomic regulation Hypothermia Arrhythmia
a b s t r a c t Objectives: To evaluate the effect of age on heart rate variability (HRV) in a rat model of therapeutic hypothermia. Methods: Thirty-six male Sprague–Dawley rats (18 were 2 months old and 18 were 18 months old) were randomized into one of three groups: normothermia (37 ◦ C), mild hypothermia (34 ◦ C), and moderate hypothermia (31 ◦ C). An electrocardiogram (ECG) was recorded at baseline and continuously for 1 h once the target core body temperature was reached. Various heart rate variability measurements were calculated. Results: Significant effects of age were observed in respect to the ratio of standard deviation of all normal to normal R-R [NN] intervals (SDNN)/standard deviation of the differences between adjacent NN intervals (SD of delta NN) (P = 0.037), low frequency (LF) power, normalized units (nu, %) (P < 0.001), and the ratio of LF and high frequency (HF) (P < 0.001). Significant effects of temperature were found in LF power and a significant body-temperature interaction was found in HF power. HF power was significantly lower in the young rats at mild and moderate hypothermic conditions. For the LF/HF, the ratio was significantly lower in the young animals compared to the older animals at normal body temperatures and during mild hypothermia. LF/HF increased significantly at both 34 ◦ C and 31 ◦ C in the young rats compared to the young rats at 37 ◦ C. In contrast, LF/HF was significantly lower in the older group of rats at 34 ◦ C and 31 ◦ C compared to the older group of rats maintained under normothermic conditions. Conclusions: This study noted that autonomic regulation determined via HRV, primarily the ratio of LF to HF, was different between different age groups. Additional studies on this topic are needed to achieve a more detailed understanding of therapeutic hypothermia. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Therapeutic hypothermia is employed in the management of a variety of medical conditions. For example, therapeutic hypothermia is known to help improve neurological outcomes
Abbreviations: HRV, heart rate variability; ANS, autonomic nervous system; ECG, electrocardiogram; SDNN, standard deviation of all normal to normal R-R [NN] intervals; SD Delta NN, standard deviation of the differences between adjacent NN intervals; LF, low frequency power; HF, high frequency power. 夽 A Spanish translated version of the abstract of this article appears as Appendix in the final online version at doi:10.1016/j.resuscitation.2011.04.031. ∗ Corresponding author at: Department of Emergency Medicine, Kaohsiung Veterans General Hospital, 386, Ta-Chung 1st Rd, Kaohsiung City 813, Taiwan. Tel.: +886 7 346 8342; fax: +886 7 346 8343. E-mail addresses: [email protected], [email protected] (Y.-T. Chang). 0300-9572/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.resuscitation.2011.04.031
and decrease injury in patients with traumatic brain injury and stroke.1–3 It has also been studied in refractory tachyarrhythemias,4 during percutaneous coronary intervention for acute myocardial infarction,5 and during defibrillation and resuscitation.6–9 To date, two prospective, randomized studies assessing the impact of therapeutic hypothermia in patients with out-of-hospital cardiac arrest caused by ventricular fibrillation have been reported. Both indicated that therapeutic hypothermia decreases mortality and improves cerebral prognosis in these patients.10,11 Notwithstanding the number of reports extolling the virtues of therapeutic hypothermia, the procedure continues to be considered “alternative” likely because the exact mechanism(s) underlying the positive effect of the procedure remain to be elucidated. One method that can be used to assess patients during or after therapeutic hypothermia is by evaluating heart rate variability (HRV). This technique measures beat-to-beat fluctuations in the heart rate thought to arise due to changes in autonomic inputs to
Y.-T. Chang et al. / Resuscitation 82 (2011) 1350–1354
the heart. That is, HRV is used to assess the autonomic nervous system (ANS) and can indicate whether the ANS is making the necessary adjustments to maintain homeostasis.12 The Task Force for the European Society of Cardiology and North American Society of Pacing and Electrophysiology has noted that HRV can be used clinically to help adapt patient care. Depressed HRV is thought to be related to increased sympathetic excitation, decreased activity of the vagus nerve, and/or a reduction in how the sinus nodal cells respond to neural modulation.13 According to Gordon, aging is associated with pathophysiologic deficits that can alter physiologic processes or cause a patient to respond differently to stress, toxicants, and drugs. For example, aging is associated with an increased production of free radicals, a decline in cardiovascular function, an increased susceptibility to heat or cold stress, and altered drug metabolism.14 One study found that acute coronary ligation increased the occurrence of ventricular tachycardia and/or fibrillation in older rats compared with younger rats and that aged rats had altered regulation of visceral sympathetic nerve discharge responses to progressive hypothermia.15 Another study noted that both the cardiovascular and thermoregulatory systems are susceptible in the aging population and found that daytime core body temperature is significantly elevated in rats compared to younger animals.14 Thus, it is possible that patient age can impact the safety or efficacy of therapeutic hypothermia. The original goal of this study was to assess the impact of temperature on HRV in a rat model of therapeutic hypothermia. Preliminary results identified significant differences in HRV between young and old rats. Given these preliminary results and the knowledge that physiologic processes are not necessarily identical between the young and old, the purpose of this small, pilot study was therefore modified. To better understand therapeutic hypothermia, the effect of age on autonomic regulation determined via HRV was studied in a rat model using both mild and moderate hypothermic conditions. This information is anticipated to have important clinical contributions in that patient age is not often considered when utilizing therapeutic hypothermia. A more detailed understanding of therapeutic hypothermia may ultimately improve patient care and facilitate the integration of therapeutic hypothermia into standard treatment protocols rather than remaining an “alternative” therapy. 2. Materials and methods 2.1. Animals Eighteen male 2-month-old Sprague–Dawley rats (Rattus Norvegicus) and 18 male Sprague–Dawley rats aged 18 months were included in this study. The old and young rats were divided into one of three groups: the normothermia group (37 ◦ C, n = 12), the mild hypothermia group (34 ◦ C, n = 12), or the moderate hypothermia group (31 ◦ C, n = 12). All animals received humane care in compliance with the principles enunciated in the “Guide for the Care and Use of Laboratory Animals” and local ethics approval was granted (IACUC approval number VGHKS96A0008). 2.2. Animal preparation All rats were fasted overnight but had ad libitum access to water. Animals were anesthetized by an intraperitoneal injection of 50 mg kg−1 pentobarbital sodium. Additional doses of 10 mg kg−1 were administered at hourly intervals to maintain light anesthesia. Animals were secured to a surgical board in a supine position with the extremities immobilized. A 14-gauge cannula (AbbocathT, Abbott Hospital Division, North Chicago, IL) mounted on a blunt
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needle with a 145◦ angled tip was used as an endoctracheal tube. Animals breathed spontaneously. Core body temperature was measured with a thermocouple microprobe measuring 10 cm in length and 0.5 mm in diameter (#9030-12-34, Columbus Instruments, Columbus, OH). The microprobe was advanced through the left femoral artery into the descending thoracic aorta. An electrocardiogram (ECG) was recorded continuously during experiment to analyze HRV as a measure of autonomic regulation; A PC-based data acquisition system (58321, PowerLab, Illinois, USA) supported by their software (56402, PowerLab, Illinois, USA) was used with a sampling rate of 1000s−1 . 2.3. Experimental procedure Baseline measurements were recorded for 10 min prior to adjusting core body temperature by adding submerging the rats in ice water to achieve the desired temperature (i.e., 34 ◦ C or 31 ◦ C). Normothermic rats were not submerged in ice water but were placed on the board under lights to maintain a core body temperature of 37 ◦ C. Once the target core body temperature was reached, ECGs were recorded for 1 h. All animals were subsequently euthanized. 2.4. Heart rate variability Both time domain measurements and spectral analysis or R-R intervals were calculated in this study as described previously.13 These included: spatial SDNN (standard deviation of all normal to normal R-R [NN] intervals), SD Delta NN (standard deviation of the differences between adjacent NN intervals), ratio of LF/HF, LF (low frequency power [0.04 and 0.15 Hz] derived from both vagal and sympathetic activity hypothesized to reflect the delay in the baroreceptor loop), and HF (high frequency power [0.15 and 0.4 Hz] driven by respiration mainly via vagal activity). The spectral components of heart rate variability were analyzed as absolute units (ms2 ), and the LF and HF components also as normalized units (nu, %). Normalized units were calculated using the following: LFnu =
LF power × 100; and total power − VLF power
HFnu =
HF power × 100. total power − VLF power
2.5. Statistical analysis Descriptive results regarding continuous variables are presented as the mean ± standard deviation (SD). Data were analyzed with a two-way of analysis of variance (ANOVA) to determine significant effects of age, body temperature, and interaction of age and temperature on the HRV parameters. For subgroup analysis, data were compared by one-way ANOVA or independent two sample t test. When a significant between groups was apparent, multiple comparisons of means were performed using the Bonferroni procedure with type-I error adjustment. All statistical assessments were two-sided and considered significant when P < 0.05. Statistical analyses were performed using SPSS 15.0 statistical software (SPSS Inc., Chicago, IL). 3. Results The mean (±standard deviation) length of time required for the rats to reach the target core body temperatures of 34 ◦ C and 31 ◦ C in the young rats was 34 ± 6 min and 49 ± 11 min, respectively. In the older groups of rats, the mean time needed to reach core body
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Table 1 Two-way ANOVA results for various heart rate variability measurements. P value Total beats Age Temperature Age × temperature Length, s Age Temperature Age × temperature Heart rate Age Temperature Age × temperature SDNN Age Temperature Age × temperature SD Delta NN Age Temperature Age × temperature Ratio [SDNN/(SD of delta NN)] Age Temperature Age × temperature VLF power, ms Age Temperature Age × temperature LF power, ms Age Temperature Age × temperature LF power, nu Age Temperature Age × temperature HF power, ms Age Temperature Age × temperature HF power, nu Age Temperature Age × temperature LF/HF Age Temperature Age × temperature
0.516 0.589 0.716 0.591 0.407 0.696 0.457 0.133 0.388 0.226 0.121 0.060 0.114 0.091 0.076 0.037* 0.296 0.856 0.939 0.405 0.939 0.273 0.293 0.281 <0.001* 0.040* 0.204 0.241 0.254 0.251 0.334 <0.001* 0.002* <0.001* 0.135 <0.001*
temperatures of 34 ◦ C and 31 ◦ C were 38 ± 7 min and 53 ± 6 min. No statistical difference between the two age groups to reach the target body temperatures existed. As indicated in Table 1, there were no significant main effects (age and temperature) and interaction effect of age and temperature in total beats, length, heart rate, SDNN, SD Delta NN, VLF power, LF power, ms, and HF power, ms. In contrast, significant effects of age were observed for the ratio of SDNN/(SD of delta NN) (P = 0.037), LF power, nu (P < 0.001) and the ratio of LF and HF (P < 0.001). Significant effects of temperature were found in LF power, nu (P = 0.040), HF power, nu (P = 0.002) and a significant body-temperature interaction were found in HF power (P = 0.002), nu and LF/HF (P < 0.001). Additional analyses revealed significant differences in HF power (nu) and LF/HF in the young age rats and significant differences in LF/HF in the groups of older rats at the three different temperatures (Table 2). As illustrated in Fig. 1, HF power was highest in the young rats maintained at 37 ◦ C, but HF power was higher in the old rats subjected to therapeutic hypothermia. In addition, HF power was significantly lower in the young rats at mild and moderate hypothermic conditions. In the older group of rats, no change in HF was noted upon cooling (Fig. 1).
Fig. 1. Comparison of high frequency (HF) power (nu) in old and young rats at different core body temperatures. a Indicates a statistically significant difference between young and old age groups. * Indicates a statistically significant difference between the indicated group and the normothermia in both the young and old age group. Pairwise multiple comparisons between groups were determined using Bonferroni’s test with ˛ = 0.017 adjustment.
The LF/HF was significantly lower in the young animals compared to the older animals at normal body temperatures and during mild hypothermia, but not in the moderate hypothermia groups. LF/HF increased significantly at both 34 ◦ C and 31 ◦ C in the young rats compared to the young rats at 37 ◦ C. In contrast, LF/HF was significantly lower in the older group of rats at 34 ◦ C and 31 ◦ C compared to the older group of rats maintained under normothermic conditions (Fig. 2). 4. Discussion HRV is a tool used to evaluate beat-to-beat fluctuations in heart rate that are thought to arise due to changes in autonomic inputs to the heart.19 Both time and spectral domain analysis can be performed to determine HRV. In the spectral domain, HF and LF powers are commonly calculated.17 HF cyclic fluctuations are reportedly modulated by ventilation and are mediated entirely via vagal out-
Fig. 2. Comparison of the ratio of lower frequency (LF) and high frequency (HF) power in old and young rats at different core body temperatures. a Indicates a statistically significant difference between young and old age groups. * Indicates a statistically significant difference between the indicated group and the normothermia in both the young and old age group. Pair-wise multiple comparisons between groups were determined using Bonferroni’s test with ˛ = 0.017 adjustment.
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Table 2 Measurement of various heart rate variability parameters at different core body temperatures determined based on age of the rats. Data are presented as mean ± standard deviation. Normothermia 37 ◦ C (n = 6)
Mild hypothermia 34 ◦ C (n = 6)
Moderate hypothermia 31 ◦ C (n = 6)
P value
Young rats Total beats Length, s Heart rate SDNN SD Delta NN Ratio VLF upper, Hz VLF power, ms LF upper, Hz LF power, ms LF power, nu HF upper, Hz HF power, ms HF power, nu LF/HF
1335 232 348 30.34 42.77 0.78 0.04 37.41 0.15 208.86 6.53 0.40 343.91 13.33 0.49
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
809 143 33 41.75 61.29 0.08 0.00 68.63 0.00 448.13 1.72 0.00 701.41 2.89 0.08
2064 332 382 1.67 0.93 1.97 0.04 0.28 0.15 0.02 3.86 0.40 0.01 2.32 1.76
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1262 281 25 1.00 0.27 1.50 0.00 0.23 0.00 0.01 2.62 0.00 0.01 1.60 0.77
2632 545 326 2.32 2.23 1.14 0.04 0.56 0.15 0.05 2.75 0.40 0.02 1.32 1.75
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2023 514 51 1.74 2.10 0.65 0.00 0.71 0.00 0.08 3.36 0.00 0.03 1.23 0.68
0.385 0.310 0.063 0.095 0.099 0.116 1.000 0.209 1.000 0.301 0.069 1.000 0.267 <0.001* 0.003*
Old rats Total beats Length, s Heart rate SDNN SD Delta NN Ratio VLF upper, Hz VLF power, ms LF upper, Hz LF power, ms LF power, nu HF upper, Hz HF power, ms HF power, nu LF/HF
2319 390 359 2.80 1.17 2.67 0.04 1.62 0.15 0.33 34.24 0.40 0.07 7.55 4.79
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1641 259 32 2.28 0.60 1.86 0.00 1.29 0.00 0.17 19.48 0.00 0.03 4.71 0.96
2366 445 341 4.26 1.13 4.01 0.04 2.38 0.15 0.21 19.66 0.40 0.07 6.95 2.71
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2228 488 51 5.31 0.40 4.84 0.00 1.85 0.00 0.20 13.44 0.00 0.07 4.38 0.66
2473 468 321 5.76 2.87 2.21 0.04 37.12 0.15 4.03 14.35 0.40 1.39 6.08 2.37
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1603 299 73 5.80 2.79 1.37 0.00 59.19 0.00 6.35 12.99 0.00 2.17 5.22 0.72*
0.989 0.928 0.501 0.567 0.150 0.590 1.000 0.156 1.000 0.156 0.106 1.000 0.145 0.867 <0.001*
*
Significant difference between the three different temperatures.
flow. The slower LF cyclic fluctuations are thought to occur due to barorelexes or thermoregulation.17 In the study described herein, only HF and LF/HF were significantly impacted by age and temperature. In young animals, there was a decrease in HRV assessed via HF power with decreasing core body temperature. In the older group of rats, this pattern was not observed. Instead, no change in HF was noted at any of the three core body temperatures. The HF power was significantly lower in the older group of rats at normal body temperatures and under mild hypothermic conditions, but not under moderate hypothermic conditions. This indicates that older rats have a decreased HRV compared to younger animals. Just as an increase in HF is indicative of decreased autonomic nervous system activity, an increase in the LF/HF ratio also indicates less HRV. At all temperatures, the ratio of LF and HF was higher in the old rats compared to the young rats, but this difference only reached statistical significance in the normothermic and mildly hypothermic groups. In the young group of rats, LF/HF was significantly increased in rats with core body temperatures below normal. In the older group of rats, however, a significant decrease in LF/HF was noted in the rats subjected to both mild and moderate hypothermia. Together, these data suggest that patient age should be considered when using therapeutic hypothermia in a clinical setting. Presently, patient age is not a major consideration and a heightened attention to age could ultimately improve outcomes when using therapeutic hypothermia in a clinical setting. While HRV during therapeutic hypothermia does not appear to have been previously been addressed per se (based on a MEDLINE search), some studies have reported changes in HR and HRV in the aged. For example, Tasaki et al. reported that HRV represented by LF/HF
significantly decreased in aged patients compared to the measurements obtained in the same patients 15 years earlier.18 This finding is contradictory to the results generated in this study where LF/HF was significantly increased in the older rats compared to the young rats. It is likely that additional parameters such as Tc thresholds for vasoconstriction, heat production, and plasma norepinephrine concentrations in addition to HRV may provide a more comprehensive picture of the physiologic differences that occur in young and aged patients during cooling. Indeed, Frank et al. reported that elderly patients had decreased vasomotor responses and subjective sensory thermal perception than their younger counterparts.19 Some limitations of this study worthy of mention largely relate to the use of a rodent model. HRV is known to be challenging to measure in small rodents consistently due to the rapid heart rate and frequent artifacts.12 In addition, only 1 h of data (ECG recording) was collected for each rat. Some human studies report 24-h mean HRV (Tasaki et al., for example), which is likely a more accurate estimation or representation of HRV.18 Another limitation is the fact that the rats were anesthetized rather than conscious. Barbiturates such as pentobarbital are frequently chosen for anesthesia in rats because of ease of administration and minimal cardiovascular depression. It is possible that HRV could be impacted by the pentobarbital; however, a continuous intravenous administration of pentobarbital reportedly causes only mild hypotension and tachycardia accompanied by a dose-related depression of both mean arterial blood pressure and heart rate. Finally, the study design (i.e., submerging the rats in cold water to achieve hypothermia) could have potentially impacted various physiologic parameters in rats such as the dive reflex. Additional studies addressing these limitations are needed to more comprehensively explore the impact of
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age on HRV as this was only a small pilot study. Ultimately, this may enhance the use of therapeutic hypothermia in the clinical setting and help design specific treatment protocols tailored to patients based on their age and underlying medical condition. 5. Conclusion This small, preliminary study on HRV and age during therapeutic hypothermia noted that autonomic regulation determined via HRV, primarily the ratio of LF to HF, was different between different age groups. For the developing therapeutic hypothermia technique, additional studies are needed to define optimal conditions considering that patient age appears to impact HRV during therapeutic hypothermia and could be an important factor to consider. Additional experimentation considering disease, comorbidities, and medications will also be useful to design protocols for therapeutic hypothermia in the clinical setting. Conflict of interest statement The authors have no conflict of interest to declare. Acknowledgements The authors would like to thank Huei-Ying Hsu for assisting with animal care and laboratory examinations. Funding: This study was funded by a grant from the Kaohsiung Veterans General Hospital (VGHKS97-117). References 1. Marion DW, Obrist WD, Carlier PM, Penrod LE, Darby JM. The use of moderate therapeutic hypothermia for patients with severe head injuries: a preliminary report. J Neurosurg 1993;79:354–62. 2. Schwab S, Schwarz S, Spranger M, Keller E, Bertram M, Hacke W. Moderate hypothermia in the treatment of patients with severe middle cerebral artery infarction. Stroke 1998;29:2461–6.
3. Fritz HG, Bauer R. Secondary injuries in brain trauma: effects of hypothermia. J Neurosurg Anesthesiol 2004;16:43–52. 4. Balaji S, Sullivan I, Deanfield J, James I. Moderate hypothermia in the management of resistant automatic tachycardias in children. Br Heart J 1991;66: 221–4. 5. Dixon SR, Whitbourn RJ, Dae MW, et al. Induction of mild systemic hypothermia with endovascular cooling during primary percutaneous coronary intervention for acute myocardial infarction. J Am Coll Cardiol 2002;40:1928–34. 6. Rhee BJ, Zhang Y, Boddicker KA, Davies LR, Kerber RE. Effect of hypothermia on transthoracic defibrillation in a swine model. Resuscitation 2005;65: 79–85. 7. Boddicker KA, Zhang Y, Zimmerman MB, Davies LR, Kerber RE. Hypothermia improves defibrillation success and resuscitation outcomes from ventricular fibrillation. Circulation 2005;111:3195–201. 8. Belliard G, Catez E, Charron C, et al. Efficacy of therapeutic hypothermia after out-of-hospital cardiac arrest due to ventricular fibrillation. Resuscitation 2007;75:252–9. 9. Kim F, Olsufka M, Longstreth Jr WT. Pilot randomized clinical trial of prehospital induction of mild hypothermia in out-of-hospital cardiac arrest patients with a rapid infusion of 4 degrees C normal saline. Circulation 2007;115:3064–70. 10. Hypothermia after Cardiac Arrest Study Group. Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. N Engl J Med 2002;346:549–56. 11. Bernard SA, Gray TW, Buist MD, et al. Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med 2002;346:557–63. 12. Thireau J, Zhang BL, Poisson D, Babuty D. Heart rate variability in mice: a theoretical and practical guide. Exp Physiol 2007;93:83–94. 13. Task Force of the European Society of Cardiology, the North American Society of Pacing, Electrophysiology. Heart rate variability: standards of measurement, physiological interpretation and clinical use. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Circulation 1996;93:1043–65. 14. Gordon CJ. Cardiac and thermal homeostasis in the aging Brown Norway rat. J Gerontol A Biol Sci Med Sci 2008;63:1307–13. 15. Helwig BG, Parimi S, Ganta CK, Cober R, Fels RJ, Kenney MJ. Aging alters regulation of visceral sympathetic nerve responses to acute hypothermia. Am J Physiol Regul Integr Comp Physiol 2006;291:R573–9. 17. Kleiger RE, Stein PK, Bigger Jr JT. Heart rate variability: measurement and clinical utility. Ann Noninvasive Electrocardiol 2005;10:88–101. 18. Tasaki H, Serita T, Ueyama C, Kitano K, Seto S, Yano K. Long-term follow-up of the circadian rhythm of heart rate and heart rate variability in healthy elderly patients. Circ J 2006;70:889–95. 19. Frank SM, Raja SN, Bulcao C, Goldstein DS. Age-related thermoregulatory differences during core cooling in humans. Am J Physiol Regul Integr Comp Physiol 2000;279:R349–54.
Contents lists available at ScienceDirect
Resuscitation journal homepage: www.elsevier.com/locate/resuscitation
Experimental paper
Influence of age on heart rate variability during therapeutic hypothermia in a rat model夽 Yun-Te Chang a,d,∗ , Shue-Ren Wann a,b , Pei-Lin Wu a , Kai-Hseng Hsieh c , Chu-Chuan Lin c , Mu-Shun Huang d , Hong-Tai Chang a a
Department of Emergency Medicine, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan The Infectious Diseases Section, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan c Department of Pediatrics, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan d School of Medicine, National Yang-Ming University, Taiwan b
a r t i c l e
i n f o
Article history: Received 29 October 2010 Received in revised form 2 April 2011 Accepted 9 April 2011
Keywords: Heart rate variability Age Autonomic regulation Hypothermia Arrhythmia
a b s t r a c t Objectives: To evaluate the effect of age on heart rate variability (HRV) in a rat model of therapeutic hypothermia. Methods: Thirty-six male Sprague–Dawley rats (18 were 2 months old and 18 were 18 months old) were randomized into one of three groups: normothermia (37 ◦ C), mild hypothermia (34 ◦ C), and moderate hypothermia (31 ◦ C). An electrocardiogram (ECG) was recorded at baseline and continuously for 1 h once the target core body temperature was reached. Various heart rate variability measurements were calculated. Results: Significant effects of age were observed in respect to the ratio of standard deviation of all normal to normal R-R [NN] intervals (SDNN)/standard deviation of the differences between adjacent NN intervals (SD of delta NN) (P = 0.037), low frequency (LF) power, normalized units (nu, %) (P < 0.001), and the ratio of LF and high frequency (HF) (P < 0.001). Significant effects of temperature were found in LF power and a significant body-temperature interaction was found in HF power. HF power was significantly lower in the young rats at mild and moderate hypothermic conditions. For the LF/HF, the ratio was significantly lower in the young animals compared to the older animals at normal body temperatures and during mild hypothermia. LF/HF increased significantly at both 34 ◦ C and 31 ◦ C in the young rats compared to the young rats at 37 ◦ C. In contrast, LF/HF was significantly lower in the older group of rats at 34 ◦ C and 31 ◦ C compared to the older group of rats maintained under normothermic conditions. Conclusions: This study noted that autonomic regulation determined via HRV, primarily the ratio of LF to HF, was different between different age groups. Additional studies on this topic are needed to achieve a more detailed understanding of therapeutic hypothermia. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Therapeutic hypothermia is employed in the management of a variety of medical conditions. For example, therapeutic hypothermia is known to help improve neurological outcomes
Abbreviations: HRV, heart rate variability; ANS, autonomic nervous system; ECG, electrocardiogram; SDNN, standard deviation of all normal to normal R-R [NN] intervals; SD Delta NN, standard deviation of the differences between adjacent NN intervals; LF, low frequency power; HF, high frequency power. 夽 A Spanish translated version of the abstract of this article appears as Appendix in the final online version at doi:10.1016/j.resuscitation.2011.04.031. ∗ Corresponding author at: Department of Emergency Medicine, Kaohsiung Veterans General Hospital, 386, Ta-Chung 1st Rd, Kaohsiung City 813, Taiwan. Tel.: +886 7 346 8342; fax: +886 7 346 8343. E-mail addresses: [email protected], [email protected] (Y.-T. Chang). 0300-9572/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.resuscitation.2011.04.031
and decrease injury in patients with traumatic brain injury and stroke.1–3 It has also been studied in refractory tachyarrhythemias,4 during percutaneous coronary intervention for acute myocardial infarction,5 and during defibrillation and resuscitation.6–9 To date, two prospective, randomized studies assessing the impact of therapeutic hypothermia in patients with out-of-hospital cardiac arrest caused by ventricular fibrillation have been reported. Both indicated that therapeutic hypothermia decreases mortality and improves cerebral prognosis in these patients.10,11 Notwithstanding the number of reports extolling the virtues of therapeutic hypothermia, the procedure continues to be considered “alternative” likely because the exact mechanism(s) underlying the positive effect of the procedure remain to be elucidated. One method that can be used to assess patients during or after therapeutic hypothermia is by evaluating heart rate variability (HRV). This technique measures beat-to-beat fluctuations in the heart rate thought to arise due to changes in autonomic inputs to
Y.-T. Chang et al. / Resuscitation 82 (2011) 1350–1354
the heart. That is, HRV is used to assess the autonomic nervous system (ANS) and can indicate whether the ANS is making the necessary adjustments to maintain homeostasis.12 The Task Force for the European Society of Cardiology and North American Society of Pacing and Electrophysiology has noted that HRV can be used clinically to help adapt patient care. Depressed HRV is thought to be related to increased sympathetic excitation, decreased activity of the vagus nerve, and/or a reduction in how the sinus nodal cells respond to neural modulation.13 According to Gordon, aging is associated with pathophysiologic deficits that can alter physiologic processes or cause a patient to respond differently to stress, toxicants, and drugs. For example, aging is associated with an increased production of free radicals, a decline in cardiovascular function, an increased susceptibility to heat or cold stress, and altered drug metabolism.14 One study found that acute coronary ligation increased the occurrence of ventricular tachycardia and/or fibrillation in older rats compared with younger rats and that aged rats had altered regulation of visceral sympathetic nerve discharge responses to progressive hypothermia.15 Another study noted that both the cardiovascular and thermoregulatory systems are susceptible in the aging population and found that daytime core body temperature is significantly elevated in rats compared to younger animals.14 Thus, it is possible that patient age can impact the safety or efficacy of therapeutic hypothermia. The original goal of this study was to assess the impact of temperature on HRV in a rat model of therapeutic hypothermia. Preliminary results identified significant differences in HRV between young and old rats. Given these preliminary results and the knowledge that physiologic processes are not necessarily identical between the young and old, the purpose of this small, pilot study was therefore modified. To better understand therapeutic hypothermia, the effect of age on autonomic regulation determined via HRV was studied in a rat model using both mild and moderate hypothermic conditions. This information is anticipated to have important clinical contributions in that patient age is not often considered when utilizing therapeutic hypothermia. A more detailed understanding of therapeutic hypothermia may ultimately improve patient care and facilitate the integration of therapeutic hypothermia into standard treatment protocols rather than remaining an “alternative” therapy. 2. Materials and methods 2.1. Animals Eighteen male 2-month-old Sprague–Dawley rats (Rattus Norvegicus) and 18 male Sprague–Dawley rats aged 18 months were included in this study. The old and young rats were divided into one of three groups: the normothermia group (37 ◦ C, n = 12), the mild hypothermia group (34 ◦ C, n = 12), or the moderate hypothermia group (31 ◦ C, n = 12). All animals received humane care in compliance with the principles enunciated in the “Guide for the Care and Use of Laboratory Animals” and local ethics approval was granted (IACUC approval number VGHKS96A0008). 2.2. Animal preparation All rats were fasted overnight but had ad libitum access to water. Animals were anesthetized by an intraperitoneal injection of 50 mg kg−1 pentobarbital sodium. Additional doses of 10 mg kg−1 were administered at hourly intervals to maintain light anesthesia. Animals were secured to a surgical board in a supine position with the extremities immobilized. A 14-gauge cannula (AbbocathT, Abbott Hospital Division, North Chicago, IL) mounted on a blunt
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needle with a 145◦ angled tip was used as an endoctracheal tube. Animals breathed spontaneously. Core body temperature was measured with a thermocouple microprobe measuring 10 cm in length and 0.5 mm in diameter (#9030-12-34, Columbus Instruments, Columbus, OH). The microprobe was advanced through the left femoral artery into the descending thoracic aorta. An electrocardiogram (ECG) was recorded continuously during experiment to analyze HRV as a measure of autonomic regulation; A PC-based data acquisition system (58321, PowerLab, Illinois, USA) supported by their software (56402, PowerLab, Illinois, USA) was used with a sampling rate of 1000s−1 . 2.3. Experimental procedure Baseline measurements were recorded for 10 min prior to adjusting core body temperature by adding submerging the rats in ice water to achieve the desired temperature (i.e., 34 ◦ C or 31 ◦ C). Normothermic rats were not submerged in ice water but were placed on the board under lights to maintain a core body temperature of 37 ◦ C. Once the target core body temperature was reached, ECGs were recorded for 1 h. All animals were subsequently euthanized. 2.4. Heart rate variability Both time domain measurements and spectral analysis or R-R intervals were calculated in this study as described previously.13 These included: spatial SDNN (standard deviation of all normal to normal R-R [NN] intervals), SD Delta NN (standard deviation of the differences between adjacent NN intervals), ratio of LF/HF, LF (low frequency power [0.04 and 0.15 Hz] derived from both vagal and sympathetic activity hypothesized to reflect the delay in the baroreceptor loop), and HF (high frequency power [0.15 and 0.4 Hz] driven by respiration mainly via vagal activity). The spectral components of heart rate variability were analyzed as absolute units (ms2 ), and the LF and HF components also as normalized units (nu, %). Normalized units were calculated using the following: LFnu =
LF power × 100; and total power − VLF power
HFnu =
HF power × 100. total power − VLF power
2.5. Statistical analysis Descriptive results regarding continuous variables are presented as the mean ± standard deviation (SD). Data were analyzed with a two-way of analysis of variance (ANOVA) to determine significant effects of age, body temperature, and interaction of age and temperature on the HRV parameters. For subgroup analysis, data were compared by one-way ANOVA or independent two sample t test. When a significant between groups was apparent, multiple comparisons of means were performed using the Bonferroni procedure with type-I error adjustment. All statistical assessments were two-sided and considered significant when P < 0.05. Statistical analyses were performed using SPSS 15.0 statistical software (SPSS Inc., Chicago, IL). 3. Results The mean (±standard deviation) length of time required for the rats to reach the target core body temperatures of 34 ◦ C and 31 ◦ C in the young rats was 34 ± 6 min and 49 ± 11 min, respectively. In the older groups of rats, the mean time needed to reach core body
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Table 1 Two-way ANOVA results for various heart rate variability measurements. P value Total beats Age Temperature Age × temperature Length, s Age Temperature Age × temperature Heart rate Age Temperature Age × temperature SDNN Age Temperature Age × temperature SD Delta NN Age Temperature Age × temperature Ratio [SDNN/(SD of delta NN)] Age Temperature Age × temperature VLF power, ms Age Temperature Age × temperature LF power, ms Age Temperature Age × temperature LF power, nu Age Temperature Age × temperature HF power, ms Age Temperature Age × temperature HF power, nu Age Temperature Age × temperature LF/HF Age Temperature Age × temperature
0.516 0.589 0.716 0.591 0.407 0.696 0.457 0.133 0.388 0.226 0.121 0.060 0.114 0.091 0.076 0.037* 0.296 0.856 0.939 0.405 0.939 0.273 0.293 0.281 <0.001* 0.040* 0.204 0.241 0.254 0.251 0.334 <0.001* 0.002* <0.001* 0.135 <0.001*
temperatures of 34 ◦ C and 31 ◦ C were 38 ± 7 min and 53 ± 6 min. No statistical difference between the two age groups to reach the target body temperatures existed. As indicated in Table 1, there were no significant main effects (age and temperature) and interaction effect of age and temperature in total beats, length, heart rate, SDNN, SD Delta NN, VLF power, LF power, ms, and HF power, ms. In contrast, significant effects of age were observed for the ratio of SDNN/(SD of delta NN) (P = 0.037), LF power, nu (P < 0.001) and the ratio of LF and HF (P < 0.001). Significant effects of temperature were found in LF power, nu (P = 0.040), HF power, nu (P = 0.002) and a significant body-temperature interaction were found in HF power (P = 0.002), nu and LF/HF (P < 0.001). Additional analyses revealed significant differences in HF power (nu) and LF/HF in the young age rats and significant differences in LF/HF in the groups of older rats at the three different temperatures (Table 2). As illustrated in Fig. 1, HF power was highest in the young rats maintained at 37 ◦ C, but HF power was higher in the old rats subjected to therapeutic hypothermia. In addition, HF power was significantly lower in the young rats at mild and moderate hypothermic conditions. In the older group of rats, no change in HF was noted upon cooling (Fig. 1).
Fig. 1. Comparison of high frequency (HF) power (nu) in old and young rats at different core body temperatures. a Indicates a statistically significant difference between young and old age groups. * Indicates a statistically significant difference between the indicated group and the normothermia in both the young and old age group. Pairwise multiple comparisons between groups were determined using Bonferroni’s test with ˛ = 0.017 adjustment.
The LF/HF was significantly lower in the young animals compared to the older animals at normal body temperatures and during mild hypothermia, but not in the moderate hypothermia groups. LF/HF increased significantly at both 34 ◦ C and 31 ◦ C in the young rats compared to the young rats at 37 ◦ C. In contrast, LF/HF was significantly lower in the older group of rats at 34 ◦ C and 31 ◦ C compared to the older group of rats maintained under normothermic conditions (Fig. 2). 4. Discussion HRV is a tool used to evaluate beat-to-beat fluctuations in heart rate that are thought to arise due to changes in autonomic inputs to the heart.19 Both time and spectral domain analysis can be performed to determine HRV. In the spectral domain, HF and LF powers are commonly calculated.17 HF cyclic fluctuations are reportedly modulated by ventilation and are mediated entirely via vagal out-
Fig. 2. Comparison of the ratio of lower frequency (LF) and high frequency (HF) power in old and young rats at different core body temperatures. a Indicates a statistically significant difference between young and old age groups. * Indicates a statistically significant difference between the indicated group and the normothermia in both the young and old age group. Pair-wise multiple comparisons between groups were determined using Bonferroni’s test with ˛ = 0.017 adjustment.
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Table 2 Measurement of various heart rate variability parameters at different core body temperatures determined based on age of the rats. Data are presented as mean ± standard deviation. Normothermia 37 ◦ C (n = 6)
Mild hypothermia 34 ◦ C (n = 6)
Moderate hypothermia 31 ◦ C (n = 6)
P value
Young rats Total beats Length, s Heart rate SDNN SD Delta NN Ratio VLF upper, Hz VLF power, ms LF upper, Hz LF power, ms LF power, nu HF upper, Hz HF power, ms HF power, nu LF/HF
1335 232 348 30.34 42.77 0.78 0.04 37.41 0.15 208.86 6.53 0.40 343.91 13.33 0.49
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
809 143 33 41.75 61.29 0.08 0.00 68.63 0.00 448.13 1.72 0.00 701.41 2.89 0.08
2064 332 382 1.67 0.93 1.97 0.04 0.28 0.15 0.02 3.86 0.40 0.01 2.32 1.76
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1262 281 25 1.00 0.27 1.50 0.00 0.23 0.00 0.01 2.62 0.00 0.01 1.60 0.77
2632 545 326 2.32 2.23 1.14 0.04 0.56 0.15 0.05 2.75 0.40 0.02 1.32 1.75
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2023 514 51 1.74 2.10 0.65 0.00 0.71 0.00 0.08 3.36 0.00 0.03 1.23 0.68
0.385 0.310 0.063 0.095 0.099 0.116 1.000 0.209 1.000 0.301 0.069 1.000 0.267 <0.001* 0.003*
Old rats Total beats Length, s Heart rate SDNN SD Delta NN Ratio VLF upper, Hz VLF power, ms LF upper, Hz LF power, ms LF power, nu HF upper, Hz HF power, ms HF power, nu LF/HF
2319 390 359 2.80 1.17 2.67 0.04 1.62 0.15 0.33 34.24 0.40 0.07 7.55 4.79
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1641 259 32 2.28 0.60 1.86 0.00 1.29 0.00 0.17 19.48 0.00 0.03 4.71 0.96
2366 445 341 4.26 1.13 4.01 0.04 2.38 0.15 0.21 19.66 0.40 0.07 6.95 2.71
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2228 488 51 5.31 0.40 4.84 0.00 1.85 0.00 0.20 13.44 0.00 0.07 4.38 0.66
2473 468 321 5.76 2.87 2.21 0.04 37.12 0.15 4.03 14.35 0.40 1.39 6.08 2.37
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1603 299 73 5.80 2.79 1.37 0.00 59.19 0.00 6.35 12.99 0.00 2.17 5.22 0.72*
0.989 0.928 0.501 0.567 0.150 0.590 1.000 0.156 1.000 0.156 0.106 1.000 0.145 0.867 <0.001*
*
Significant difference between the three different temperatures.
flow. The slower LF cyclic fluctuations are thought to occur due to barorelexes or thermoregulation.17 In the study described herein, only HF and LF/HF were significantly impacted by age and temperature. In young animals, there was a decrease in HRV assessed via HF power with decreasing core body temperature. In the older group of rats, this pattern was not observed. Instead, no change in HF was noted at any of the three core body temperatures. The HF power was significantly lower in the older group of rats at normal body temperatures and under mild hypothermic conditions, but not under moderate hypothermic conditions. This indicates that older rats have a decreased HRV compared to younger animals. Just as an increase in HF is indicative of decreased autonomic nervous system activity, an increase in the LF/HF ratio also indicates less HRV. At all temperatures, the ratio of LF and HF was higher in the old rats compared to the young rats, but this difference only reached statistical significance in the normothermic and mildly hypothermic groups. In the young group of rats, LF/HF was significantly increased in rats with core body temperatures below normal. In the older group of rats, however, a significant decrease in LF/HF was noted in the rats subjected to both mild and moderate hypothermia. Together, these data suggest that patient age should be considered when using therapeutic hypothermia in a clinical setting. Presently, patient age is not a major consideration and a heightened attention to age could ultimately improve outcomes when using therapeutic hypothermia in a clinical setting. While HRV during therapeutic hypothermia does not appear to have been previously been addressed per se (based on a MEDLINE search), some studies have reported changes in HR and HRV in the aged. For example, Tasaki et al. reported that HRV represented by LF/HF
significantly decreased in aged patients compared to the measurements obtained in the same patients 15 years earlier.18 This finding is contradictory to the results generated in this study where LF/HF was significantly increased in the older rats compared to the young rats. It is likely that additional parameters such as Tc thresholds for vasoconstriction, heat production, and plasma norepinephrine concentrations in addition to HRV may provide a more comprehensive picture of the physiologic differences that occur in young and aged patients during cooling. Indeed, Frank et al. reported that elderly patients had decreased vasomotor responses and subjective sensory thermal perception than their younger counterparts.19 Some limitations of this study worthy of mention largely relate to the use of a rodent model. HRV is known to be challenging to measure in small rodents consistently due to the rapid heart rate and frequent artifacts.12 In addition, only 1 h of data (ECG recording) was collected for each rat. Some human studies report 24-h mean HRV (Tasaki et al., for example), which is likely a more accurate estimation or representation of HRV.18 Another limitation is the fact that the rats were anesthetized rather than conscious. Barbiturates such as pentobarbital are frequently chosen for anesthesia in rats because of ease of administration and minimal cardiovascular depression. It is possible that HRV could be impacted by the pentobarbital; however, a continuous intravenous administration of pentobarbital reportedly causes only mild hypotension and tachycardia accompanied by a dose-related depression of both mean arterial blood pressure and heart rate. Finally, the study design (i.e., submerging the rats in cold water to achieve hypothermia) could have potentially impacted various physiologic parameters in rats such as the dive reflex. Additional studies addressing these limitations are needed to more comprehensively explore the impact of
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age on HRV as this was only a small pilot study. Ultimately, this may enhance the use of therapeutic hypothermia in the clinical setting and help design specific treatment protocols tailored to patients based on their age and underlying medical condition. 5. Conclusion This small, preliminary study on HRV and age during therapeutic hypothermia noted that autonomic regulation determined via HRV, primarily the ratio of LF to HF, was different between different age groups. For the developing therapeutic hypothermia technique, additional studies are needed to define optimal conditions considering that patient age appears to impact HRV during therapeutic hypothermia and could be an important factor to consider. Additional experimentation considering disease, comorbidities, and medications will also be useful to design protocols for therapeutic hypothermia in the clinical setting. Conflict of interest statement The authors have no conflict of interest to declare. Acknowledgements The authors would like to thank Huei-Ying Hsu for assisting with animal care and laboratory examinations. Funding: This study was funded by a grant from the Kaohsiung Veterans General Hospital (VGHKS97-117). References 1. Marion DW, Obrist WD, Carlier PM, Penrod LE, Darby JM. The use of moderate therapeutic hypothermia for patients with severe head injuries: a preliminary report. J Neurosurg 1993;79:354–62. 2. Schwab S, Schwarz S, Spranger M, Keller E, Bertram M, Hacke W. Moderate hypothermia in the treatment of patients with severe middle cerebral artery infarction. Stroke 1998;29:2461–6.
3. Fritz HG, Bauer R. Secondary injuries in brain trauma: effects of hypothermia. J Neurosurg Anesthesiol 2004;16:43–52. 4. Balaji S, Sullivan I, Deanfield J, James I. Moderate hypothermia in the management of resistant automatic tachycardias in children. Br Heart J 1991;66: 221–4. 5. Dixon SR, Whitbourn RJ, Dae MW, et al. Induction of mild systemic hypothermia with endovascular cooling during primary percutaneous coronary intervention for acute myocardial infarction. J Am Coll Cardiol 2002;40:1928–34. 6. Rhee BJ, Zhang Y, Boddicker KA, Davies LR, Kerber RE. Effect of hypothermia on transthoracic defibrillation in a swine model. Resuscitation 2005;65: 79–85. 7. Boddicker KA, Zhang Y, Zimmerman MB, Davies LR, Kerber RE. Hypothermia improves defibrillation success and resuscitation outcomes from ventricular fibrillation. Circulation 2005;111:3195–201. 8. Belliard G, Catez E, Charron C, et al. Efficacy of therapeutic hypothermia after out-of-hospital cardiac arrest due to ventricular fibrillation. Resuscitation 2007;75:252–9. 9. Kim F, Olsufka M, Longstreth Jr WT. Pilot randomized clinical trial of prehospital induction of mild hypothermia in out-of-hospital cardiac arrest patients with a rapid infusion of 4 degrees C normal saline. Circulation 2007;115:3064–70. 10. Hypothermia after Cardiac Arrest Study Group. Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. N Engl J Med 2002;346:549–56. 11. Bernard SA, Gray TW, Buist MD, et al. Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med 2002;346:557–63. 12. Thireau J, Zhang BL, Poisson D, Babuty D. Heart rate variability in mice: a theoretical and practical guide. Exp Physiol 2007;93:83–94. 13. Task Force of the European Society of Cardiology, the North American Society of Pacing, Electrophysiology. Heart rate variability: standards of measurement, physiological interpretation and clinical use. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Circulation 1996;93:1043–65. 14. Gordon CJ. Cardiac and thermal homeostasis in the aging Brown Norway rat. J Gerontol A Biol Sci Med Sci 2008;63:1307–13. 15. Helwig BG, Parimi S, Ganta CK, Cober R, Fels RJ, Kenney MJ. Aging alters regulation of visceral sympathetic nerve responses to acute hypothermia. Am J Physiol Regul Integr Comp Physiol 2006;291:R573–9. 17. Kleiger RE, Stein PK, Bigger Jr JT. Heart rate variability: measurement and clinical utility. Ann Noninvasive Electrocardiol 2005;10:88–101. 18. Tasaki H, Serita T, Ueyama C, Kitano K, Seto S, Yano K. Long-term follow-up of the circadian rhythm of heart rate and heart rate variability in healthy elderly patients. Circ J 2006;70:889–95. 19. Frank SM, Raja SN, Bulcao C, Goldstein DS. Age-related thermoregulatory differences during core cooling in humans. Am J Physiol Regul Integr Comp Physiol 2000;279:R349–54.