Published online May 1, 2006
PEDIATRICS Vol. 117 No. 5 May 2006, pp. 1549-1559 (doi:10.1542/peds.2005-1649)
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Delayed Whole-Body Cooling to 33 or 35°C and the Development of Impaired Energy Generation Consequential to Transient Cerebral Hypoxia-Ischemia in the Newborn Piglet

Frances E. O'Brien, MRCPCHa, Osuke Iwata, MDa, John S. Thornton, PhDb, Enrico De Vita, PhDc, Mark W. Sellwood, MRCPCHa, Sachiko Iwata, MDa, Yasuko S. Sakata, MDa, Susan Charman, MScd, Roger Ordidge, PhDb, Ernest B. Cady, FInstPc, John S. Wyatt, FRCPCHa and Nicola J. Robertson, FRCPCH, PhDa

a Departments of Pediatrics and Child Health and
b Medical Physics and Bioengineering, University College London, London, United Kingdom
c Department of Medical Physics and Bioengineering
d Medical Statistics Unit, Research and Development Directorate, University College London National Health Service Foundation Trust, London, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
OBJECTIVES. Fundamental questions remain about the precise temperature providing optimal neuroprotection after perinatal hypoxia-ischemia (HI). Furthermore, if hypothermia delays the onset of the neurotoxic cascade and the secondary impairment in cerebral energy generation, the "latent phase" may be prolonged, thus extending the period when additional treatments may be effective. The aims of this study were to investigate the effects of delayed systemic cooling at either 33°C or 35°C on the following: (1) latent-phase duration, and (2) cerebral metabolism during secondary energy failure itself, in the 48-hour period after transient HI.

METHODS. Piglets were randomly assigned to the following: (1) HI-normothermic (HI-n) rectal temperature (Trectal; n = 12), (2) HI-Trectal 35°C (HI-35; n = 7), and (3) HI-Trectal 33°C (HI-33; n = 10). Groups were cooled to the target Trectal between 2 and 26 hours after HI. Serial magnetic resonance spectroscopy was performed over 48 hours. The effect of cooling on secondary energy failure severity (indexed by the nucleotide triphosphate/exchangeable phosphate pool [NTP/EPP] and phosphocreatine/inorganic phosphate [PCr/Pi] ratios) was assessed.

RESULTS. Compared with HI-n, HI-35 and HI-33 had a longer NTP/EPP latent phase and during the entire study duration had higher mean NTP/EPP and PCr/Pi. The latent phase (both PCr/Pi and NTP/EPP) and the whole-brain cerebral energetics were similar for HI-35 and HI-33. During the hypothermic period, compared with HI-n, PCr/Pi was preserved in the cooled groups, but this advantage was not maintained after rewarming. Compared with HI-n, HI-35 and HI-33 had higher NTP/EPP after rewarming.

CONCLUSIONS. Whole-body hypothermia for 24 hours at either 35 or 33°C, commenced 2 hours after resuscitation, prolonged the NTP/EPP latent phase and reduced the overall secondary falls in mean PCr/Pi and NTP/EPP during 48 hours after HI. Reducing the temperature from 35 to 33°C neither increased mean PCr/Pi and NTP/EPP nor further lengthened the latent phase.

Key Words: phosphorus magnetic resonance spectroscopy • hypoxic-ischemic encephalopathy • perinatal asphyxia • whole-body cooling • neuroprotection • hypothermia

Abbreviations: HI—hypoxia-ischemia • NE—neonatal encephalopathy • 31P—phosphorus • MRS—magnetic resonance spectroscopy • PCr—phosphocreatine • ATP—adenosine triphosphate • Pi—inorganic phosphate • FID—free induction decay • NTP—nucleotide triphosphate • EPP—exchangeable phosphate pool • Trectal—rectal temperature • FIO2—inspired oxygen fraction • AED—acute energy depletion • CI—confidence interval • ANOVA—analysis of variance • NAD—nicotinamide adenine dinucleotide • GM—gray matter

Perinatal cerebral hypoxia-ischemia (HI) is responsible for significant disability and death worldwide. Of the 4 million annual worldwide neonatal deaths, 23% are caused by perinatal asphyxia,1 and in the United Kingdom, hypoxic-ischemic injury leads to death or severe neurodisability in 1 to 2 per 1000 term infants.2 Within the last year, however, the clinical context has been transformed by the results of 3 randomized clinical trials of mild to moderate hypothermia in infants with neonatal encephalopathy (NE).3–5 These trials build on the impressive and consistent record of hypothermic neuroprotection seen in experimental models6–8 and demonstrate that cooling within the first 72 hours of life can improve long-term outcome in some infants. Despite the exciting prospect of an effective treatment for infants with NE, these trials raise important questions about the optimal mode, duration, and precise temperature providing the best neuroprotection. It is vital that these questions are addressed if the full neuroprotective potential of hypothermia is to be realized.

It is likely that hypothermia exerts a significant neuroprotective effect as a consequence of interaction at a number of levels within the pathophysiological cascade leading to cell death. Its mechanisms of action may include attenuation of metabolic rate,9 downregulation of glutamate and N-methyl-d-aspartate receptors,10 and reduction of nitric oxide and oxygen radicals and their derivatives.11,12 However, hypothermia may also have adverse effects, such as reduced cardiac contractility, reduced cerebral blood flow, sympathetic and neuroendocrine stimulation, and increased blood viscosity.13,14 It is likely, therefore, that an effective temperature range exists below which hypothermic neuroprotection is lost.

Phosphorus (31P) magnetic resonance spectroscopy (MRS) studies of infants with NE performed 15 to 20 years ago typically demonstrated normal brain energetics during the first few hours after resuscitation; by 8 to 24 hours, however, there were progressive declines in cerebral phosphocreatine (PCr) and adenosine triphosphate (ATP) and increased inorganic phosphate (Pi) despite adequate oxygenation and circulation.15–17 This sequence of events was termed "secondary energy failure." A close relationship between the magnitude of these secondary changes and the severity of subsequent neurodevelopmental impairment has been described.15,18 Using serial 31P MRS, our group and others have observed a biphasic pattern of impaired cerebral energy metabolism in a newborn piglet model of transient cerebral HI.19,20 Between HI and the appearance of the secondary energy failure, it is possible that the neurotoxic cascade is largely inhibited ("latent phase") and that this period provides a "therapeutic window." Mild hypothermia of 35°C commenced immediately after HI ameliorated the delayed fall in PCr/Pi and ATP21 and reduced the number of cells undergoing apoptosis,22 suggesting that intervention before the onset of secondary energy failure may be neuroprotective. We have also previously reported a reduction in regional neuronal injury after delayed cooling in the same experimental subjects as in this current study; 33°C provided better neuroprotection in the cortex, and 35°C provided better neuroprotection in the deep gray matter.23

In addition to any direct cerebroprotective property, if hypothermia delays the onset of secondary energy failure, the therapeutic window may be prolonged, thus extending the period when additional treatments may be effective.24,25 The aims of this study were to investigate the effect of delayed systemic cooling at either 33 or 35°C on (1) latent-phase duration and (2) cerebral metabolism during secondary energy failure itself in the 48-hour period after transient HI. Cooling was commenced 2 hours after resuscitation to replicate the clinical situation in which immediate cooling is impracticable. The systemic cooling temperatures, 33 and 35°C, were similar to those used in the clinical trials of combined selective and mild whole-body cooling4 and moderate whole-body cooling;3,5 however, because of the higher normal temperature of the newborn piglet (38.5–39°C), it should be noted that this incurred up to 2°C greater temperature reduction than in the human studies.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Experiments were performed according to United Kingdom guidelines under licenses issued by the Home Office under the Animal (Scientific Procedures) Act (United Kingdom), 1986. We studied 42 healthy large white piglets of either gender within 24 hours of birth. They were born at term (115 ± 2 days) and weighed 1495 ± 170 g (mean ± SD).

Surgical Preparation
Piglets were anesthetized and received continuous physiologic monitoring and intensive life support throughout the experimental procedure as described previously.19 Briefly, piglets were sedated with intramuscular midazolam (0.2 mg/kg), and the arterial oxygen saturation was monitored (8600 FO, Nonin Medical, Plymouth, MN). Isoflurane was given initially at 5% through a facial mask during the insertion of a tracheostomy tube. After mechanical ventilation was commenced, anesthesia was maintained with isoflurane <2%, nitrous oxide and a continuous infusion of morphine (0.05 mg/kg per hour) through an umbilical venous catheter. This catheter was also used for the continuous infusion of maintenance fluids (10% dextrose, 60 mL/kg per day) and intravenous injections of antibiotics (benzylpenicillin, 50 mg/kg, and gentamicin, 2.5 mg/kg, every 12 hours). An umbilical arterial catheter was also inserted to monitor the heart rate and blood pressure continuously and also to measure the arterial blood gas. Temperature-corrected arterial oxygen and carbon dioxide tensions were repeatedly measured, and the ventilator settings were adjusted to maintain the normal levels (8–13 and 4.5–6.5 kPa, respectively). A mean arterial blood pressure >40 mmHg was maintained with bolus infusions of colloid (Gelofusin, Braun Medical, Emmenbrucke, Switzerland) and dopamine infusions (5–15 µg/kg per minute) as required.

Both common carotid arteries were surgically isolated at the level of the fourth cervical vertebra and encircled by remotely controlled inflatable vascular occluders (OC2A, In Vivo Metric, Healdsburg, CA). After the surgical procedures, the piglet was positioned prone within a plastic cylindrical pod, the scalp was immobilized below the 31P MRS surface coil, and then the pod was inserted into the magnet bore of the MRS system.

31P MRS
Spectra (resonance frequency: 121.6 MHz) were acquired by using a 7-T Biospec spectrometer (Bruker Medizintechnik, Karlsruhe, Germany). An elliptical surface coil (6.5 x 5.5 cm) was centered on the intact scalp so as to collect spectra from the whole brain. Acquisition conditions were: single-pulse acquire; repetition time, 10 seconds; quadrature data points, 2048; and spectral width, 14286 Hz. A long repetition time was used to enable the acquisition of "fully relaxed" spectra, and the peak-area ratios were, thus, directly related to metabolite concentration ratios. Free induction decays (FIDs) were summed before HI (192 FIDs), during HI and resuscitation (24 FIDs), after recovery from HI (384 FIDs), and also for sham-operated animals (384 FIDs for all acquisitions) to evaluate the nucleotide triphosphate ([NTP] mainly ATP; quantified using the ß resonance) and the high-energy exchangeable phosphate pool ([EPP] = Pi + PCr + {alpha}–NTP + ß–NTP + {gamma}–NTP).

In HI animals, spectra were obtained approximately every 4 hours until 48 hours after resuscitation. In sham animals, spectra were acquired every 4 hours until 48 hours after the baseline acquisition.

Experimental Groups
After acquisition of baseline data, piglets were randomly assigned using sealed envelopes into 5 groups: (1) sham operation and normothermia at rectal temperature (Trectal) 38.5 to 39°C (Sham-n, n = 10), (2) sham operation and transient hypothermia, Trectal 33°C (Sham-33, n = 3), (3) HI and normothermia (HI-n, n = 12), (4) HI and delayed transient hypothermia at Trectal 35°C (HI-35, n = 7), and (5) HI and delayed transient hypothermia at Trectal 33°C (HI-33, n = 10). The unequal group numbers result from the randomization process, which continued, for statistical power considerations, until there were ≥7 piglets in each group apart from Sham-33.

Acute Cerebral HI
After randomization, HI-n, HI-35, and HI-33 piglets were exposed to transient HI. The inspired oxygen fraction (FIO2) was reduced to 0.10 to 0.12, and, simultaneously, the carotid artery occluders were inflated. During HI, 31P spectra were continuously acquired every 4 minutes, and the degree of cerebral energy impairment was assessed using the amplitude of the ß-NTP peak. After the ß-NTP peak had decreased on visual assessment to ~30% of the baseline amplitude, this level was maintained for 20 minutes by titrating FIO2. Resuscitation was commenced immediately after the ongoing spectrum acquisition was finished (thereby allowing up to 4 additional minutes of HI) by deflating the carotid artery occluders and increasing FIO2 to achieve normal oxygen saturation. The acute energy depletion (AED), the time integral of NTP/EPP depletion relative to mean baseline during transient HI and the first hour of resuscitation (Fig 1), were measured to quantify acute insult severity. AED was so defined on the basis that the degree of cellular injury is related not only to the duration and amount of NTP depletion during transient HI but also to that during resuscitation when energy generation is recovering.19 The latent phase was defined as the period between PCr/Pi and NTP/EPP recovering to within their respective baseline 99% confidence intervals (CI) after HI and their falling below again indicating the development of secondary energy failure (see Fig 1).


Figure 1
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  		FIGURE 1 Illustrative NTP/EPP reduction during HI, AED, apparent recovery of NTP/EPP to baseline levels during resuscitation, and its secondary decline (secondary energy failure) thereafter. AED was quantitated as the time integral of NTP/EPP depletion relative to mean baseline during transient HI and the first 60 minutes of resuscitation. The latent phase was defined as the period after HI but before secondary energy failure when PCr/Pi and NTP/EPP were above the lower 99% CI of the respective baseline mean.

 
Temperature Regulation and Hypothermia
Trectal was continuously monitored throughout the experimental procedures using a thermistor probe (Arbo N44-91, Kendall, Powell, TN). Surgery was performed on a heated table under an overhead heater to maintain the normal newborn piglet body temperature of 38.5 to 39.0°C. In the MRS system, the core temperature was maintained using a temperature-adjustable, water-filled mattress, which covered the ventral half of the piglet’s trunk and limbs. In groups HI-35 and HI-33, whole-body cooling was commenced 2 hours after resuscitation, whereas in Sham-33, cooling was commenced after the completion of the baseline acquisition. To induce whole-body hypothermia, the mattress water temperature was reduced until the required Trectal was achieved. Twenty-four hours after the commencement of hypothermia, animals were slowly rewarmed at a rate not exceeding 1°C/hour and then maintained normothermic until the termination of the experiment.

Data Analyses
Physiologic data were inspected for normality and equality of variance, and the HI groups were compared by using either parametric (1-way analysis of variance [ANOVA] and Tukey or Bonferroni) or nonparametric (Kruskal-Wallis ANOVA on ranks and Dunn's) tests, as appropriate.

In all parts of the analysis described below, a random-effects model was used. The model included a random effect for each subject, which allows appropriate analyses (eg, comparison of means or slopes) while allowing for correlation in the repeated measures within each subject. First, overall mean NTP/EPP and PCr/Pi (from 2–48 hours after resuscitation) were compared among the HI-n, HI-35, and HI-33 groups. Second, to examine the effect of cooling and rewarming on brain energy metabolism more precisely, the mean values and the rates of change of PCr/Pi and NTP/EPP in the HI groups were compared during (1) the period with temperature modification (2–36 hours after resuscitation including 24 hours of cooling and ~8–10 hours of rewarming) and (2) normothermia after rewarming (36–48 hours after resuscitation). Intergroup rate-of-change comparisons were made by incorporating a slope in the random-effects model and using Wald tests. Unadjusted P values, means, and mean rates of change are reported with 95% CI.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Two studies were terminated 2 hours after resuscitation, because high-energy phosphates did not recover to the baseline values (1 from HI-n and 1 HI-33); 3 studies were terminated because of equipment problems (1 Sham-n and 2 HI-n). An additional 3 piglets were excluded from the analysis, because they died as a consequence of HI (2 HI-n at 18 and 28 hours and 1 HI-33 at 20 hours after resuscitation). All of the other piglets survived to the end of the experiment; in 1 HI-33, piglet data were not collected beyond 36 hours because of spectrometer failure, and this piglet was excluded from the 36- to 48-hour analysis. Thus, the numbers of piglets available for analysis were as follows: 9 Sham-n, 3 Sham-33, 7 HI-n, 7 HI-35, and 8 HI-33.

Temperature
After the commencement of cooling, Trectal reached the protocol target ranges of 35.0 ± 0.5°C and 33.0 ± 0.5°C by 1.9 ± 0.4 hours (HI-35), 3.7 ± 0.6 hours (Sham-33), and 2.2 ± 0.9 hours (HI-33; Fig 2).


Figure 2
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  		FIGURE 2 In group HI-n, Trectal was maintained normothermic throughout the experiment, whereas in groups HI-35 and HI-33, whole-body cooling was commenced 2 hours after resuscitation. Twenty-four hours after the commencement of hypothermia, animals were rewarmed at ~1°C per hour and then maintained normothermic until the termination of the experiment.

 
Physiologic Measurements
There were no significant differences between study groups in body weight, age, mean arterial blood pressure, heart rate, and other physiologic and biochemical measures at the time of baseline MRS (Table 1). To maintain normal blood pressure, 10 piglets, including 1 piglet in the Sham-n group, required a colloid infusion (2 from HI-n, 4 HI-35, 3 HI-33, and 1 Sham-n); an additional 1 piglet from each HI group required a dopamine infusion presumably because of physiologic stress, such as surgical procedures and prolonged anesthetic, as well as the consequence of HI. There were no significant differences in acute HI duration or PaO2 at the end of acute HI between the HI groups (Table 1).


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  		TABLE 1 Physiological Measurements for Piglets in Each Group

 
Changes in MRS-Measurable Values
In the groups Sham-n and Sham-33, PCr/Pi and NTP/EPP showed no changes throughout each experiment. Figure 3 shows the biological variability in the temporal evolution of PCr/Pi and NTP/EPP for each piglet in each HI group. Normal baseline PCr/Pi and NTP/EPP were 1.23 (0.17) and 0.160 (0.010), respectively (mean [99% CI]). These CIs were used to define the latent phase.


Figure 3
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  		FIGURE 3 Cerebral PCr/Pi (A, C, and E) and NTP/EPP (B, D, and F) for each piglet at baseline, during transient HI, and for 48 hours after resuscitation in the HI-n (A and B), HI-35 (C and D), and HI-33 (E and F) groups. In 1 HI-33 piglet, no data were acquired beyond 36 hours because of spectrometer failure. Graphs demonstrate significant intrinsic biological variability despite similar HI insult severities. PCr/Pi tended to be preserved in cooled piglets. A significant decline in NTP/EPP was only seen in the HI-n group.

 
Latent-Phase Duration
After resuscitation, PCr/Pi and NTP/EPP rose to baseline levels, and a period with apparently normal energetic status was observed (the latent phase). When combining data from all of the HI groups, the NTP/EPP latent phase was significantly longer than PCr/Pi latent phase: 32 ± 13 hours for NTP/EPP and 27 ±15 hours for PCr/Pi (P = .039); individual HI group analysis demonstrated a longer NTP/EPP than PCr/Pi latent phase in HI-n (P = .027) and HI-35 (P = .022) but not the HI-33 groups. Comparing the normothermic and cooled HI groups, the NTP/EPP latent phase was longer in the HI-35 (42 ± 6 hours) and HI-33 (35 ± 13 hours) groups than in HI-n (21 ± 12 hours) with P = .004 and .041, respectively (Fig 4A); there was no difference between the HI-33 and HI-35 groups. There were no significant differences in the PCr/Pi latent-phase duration in the normothermic and cooled HI groups (Fig 4B).


Figure 4
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  		FIGURE 4 Duration of latent phase measured by (A) NTP/EPP and (B) PCr/Pi in the HI-n, HI-35, and HI-33 groups. (Latent phase is the period after transient HI and resuscitation when NTP/EPP or PCr/Pi are above the lower 99% CI of their respective mean baseline values). Duration of the NTP/EPP latent phase was significantly longer in the cooled groups. There was no difference in latent-phase duration between the cooled groups. There was no significant difference in the PCr/Pi latent-phase duration between the HI groups. aP < .01; bP < .05, compared with HI-n.

 
Energy Metabolism During Entire Study Duration (2–48 Hours)
Over the 2- to 48-hour period, 325 spectra were obtained from 22 piglets in the HI groups. During this period, cooled groups had significantly higher mean PCr/Pi (P = .019 for HI-35; P = .001 for HI-33) and higher NTP/EPP (P = .032 for HI-35; P = .044 for HI-33) than the HI-n group; however, there was no significant difference between the HI-35 and HI-33 groups.

Energy Metabolism During Temperature Modification (2–36 Hours)
During this period, 260 spectra were collected from 22 piglets. Cooled groups had significantly higher mean PCr/Pi (P = .017 for HI-35; P = .003 for HI-33); there was no significant difference between the HI-35 and HI-33 groups; mean NTP/EPP did not show any significant difference between groups (Table 2). Mean PCr/Pi declined in all of the HI groups; the fall was slower in HI-33 compared with HI-35 (P = .041; Table 3). There were no significant differences between HI-33 and HI-n or HI-35 and HI-n; a decline in NTP/EPP was observed only in HI-n (Table 3).


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  		TABLE 2 Mean PCr/Pi and NTP/EPP Values for the 2- to 36-Hour and 36- to 48-Hour Post-HI Period

 

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  		TABLE 3 Mean Rates of Change in PCr/Pi and NTP/EPP for the 2- to 36-Hour and 36- to 48-Hour Post-HI Period

 
Energy Metabolism After Reestablishment of Normothermia (36–48 Hours)
During this period, 65 spectra were acquired from 21 piglets. No difference was seen in mean PCr/Pi between groups; cooled groups had significantly higher NTP/EPP (P = .014 for HI-35; P = .028 for HI-33) than the HI-n group; there was no significant difference in NTP/EPP between the HI-35 and HI-33 groups (Table 2). The HI-33 group had a significantly steeper decline in PCr/Pi when compared with the HI-n and HI-35 groups (P < .001 and P = .038, respectively; Table 3). HI-35 PCr/Pi fell at a similar rate to HI-n. The rates of change in NTP/EPP were similar in all of the HI groups.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Whole-body hypothermia for 24 hours at either 35°C or 33°C commenced 2 hours after resuscitation prolonged the latent phase and reduced the overall secondary falls in mean PCr/Pi and NTP/EPP during 48 hours after HI. When the study period was divided into 2 parts to examine the effect of cooling and rewarming on brain energy metabolism more precisely, PCr/Pi was significantly higher in the hypothermic groups during the cooled period, whereas there was no observed difference in NTP/EPP. After rewarming for between 36 and 48 hours after HI, PCr/Pi declined more rapidly in the HI-33 group than the other groups resulting in there being no difference in PCr/Pi between groups. Conversely, NTP/EPP was higher in cooled groups compared with the HI-n group during this period after rewarming. Reducing the temperature from 35°C to 33°C neither increased mean PCr/Pi and NTP/EPP nor further lengthened the latent phase.

Strengths and Limitations of the Study
For practical and ethical reasons, it was not possible to maintain our experimental model for more than a total of 60 hours. The duration of cooling was, therefore, limited to 24 hours, with a rewarming period lasting 8 to 10 hours. These considerations necessarily limited the period of normothermia after rewarming before the termination of the experiment. As a consequence, our study did not definitively demonstrate that hypothermia led to a degree of permanent neuroprotection rather than a temporary limitation of the cerebral metabolic response to transient HI, although several long-term studies in other experimental models have previously demonstrated a permanent protective effect.6,26,27 A particular strength of our study, however, was the ability to provide detailed information that showed that delayed hypothermia extended the period before significant cerebral energy decline (latent phase) in the critically important period.

Clinical experience in infants with encephalopathy as a consequence of perinatal HI reveals a wide range of outcome severities presumably related to insults of varying, but unknown, morbidity acting concomitantly with other predisposing biological factors.28 However, in our study, we quantified the insult severity using the acute cerebral energy deficit during HI and for 60 minutes thereafter (AED; Table 1) and assessed secondary energy failure quantitatively (Fig 3) thereby avoiding many problems associated with biological variability.

Effect of Delayed Hypothermia on Cerebral Energetics and the Latent Phase
Previous studies using this model have also demonstrated that immediate cooling after HI was associated with apparent preservation of high-energy phosphates.21 However, it is possible that immediate cooling might modify the recovery from the primary energy depletion associated with HI rather than influence secondary energy failure. Furthermore, immediate hypothermia is impractical in the current clinical setting. In this study, we have demonstrated that whole-body cooling, initiated at 2 hours after resuscitation, to 33°C and 35°C for 24 hours, lengthened the latent phase and improved cerebral high-energy phosphate metabolic equilibria for 48 hours after HI.

There is accumulating evidence that, to achieve significant neuroprotection, therapeutic interventions should be initiated before overt secondary energy failure and the irreversible neurotoxic cascade associated with it.29 Our results suggest that, in addition to a direct neuroprotective effect, hypothermia commenced at 2 hours can lengthen the latent phase. If processes leading to eventual neuronal injury can be inhibited during the latent phase, hypothermia may considerably extend the therapeutic window to allow detailed assessment, transfer to major neonatal centers, and the introduction of additional treatments.

Relationship Between Measures of Cerebral Energy Metabolism
When the study period was divided into hypothermic and normothermic phases, PCr/Pi was higher in the cooled groups during the hypothermic phase; however, during the normothermic phase after rewarming, there was no observed difference in PCr/Pi between groups. Conversely, there was no difference in NTP/EPP during the hypothermic phase; however, during the normothermic phase after rewarming, NTP/EPP was significantly higher in the cooled groups. This may be explained by the individual physiologic profiles of PCr/Pi and NTP/EPP, which correlate with cerebral energy availability in different ways. Before cellular membrane integrity is impaired, NTP/EPP is closely related to the cerebral ATP concentration, whereas PCr/Pi is affected by many factors, including the flux within the mitochondrial electron transport chain, the activity of the creatine kinase enzyme, and even creatine supplementation.29–31 Furthermore, PCr/Pi can fall substantially before ATP levels decline, because, provided substrate delivery is unimpaired, the latter can be maintained by anaerobic glycolysis, and a major role of PCr is to rephosphorylate adenosine diphosphate (creatine kinase reaction) to maintain ATP levels.

Our current study showed that, in the HI-n group, NTP/EPP latent phase was significantly longer than PCr/Pi latent phase; the decline in PCr/Pi was less steep during the period from 36 to 48 hours compared with the previous period; the decline in NTP/EPP was similar throughout the 2 study periods. These results suggest the slower evolution and establishment of secondary energy failure reflected by NTP/EPP compared with PCr/Pi. When cooling was introduced, the NTP/EPP latent phase was further prolonged; the decline in PCr/Pi was delayed until 36 to 48 hours (especially in HI-33); the decline in NTP/EPP was delayed, and only observed after rewarming. These observations support the effect of hypothermia to delay both the evolution and establishment of secondary energy failure. Given that energetic markers are still changing in cooled groups after rewarming, it was impossible to fully characterize the effect of hypothermia on the evolution of secondary energy failure with our limited study duration; it is scientifically very important to address whether the preservation of NTP/EPP observed during the period from 36 to 48 hours is maintained longer than 48 hours.

Apart from the different timing in the evolution and establishment of secondary energy failure for PCr/Pi and NTP/EPP, it is important to understand why PCr/Pi was not preserved after rewarming. One possibility may be that the protective effect derived from cooling for 24 hours might be insufficient to give a long-lasting energy preservation, given the relationship between the duration of cooling and the amount of neuroprotection shown in other experimental studies.32 Although we have seen a significant increase in neuronal viability on histologic analysis in the groups cooled for 24 hours in a previous publication of similar subjects,23 it will be important to investigate the effects of different durations of cooling on cerebral energetics and neuronal viability in the future.

Secondary Energy Failure, Mitochondrial Function, and Hypothermia
Although there are close associations between the extent of the energy depletion at the nadir of secondary energy failure and histologic damage,33 the precise mechanisms leading to secondary energy failure are unclear. Indeed, there is controversy as to whether secondary energy failure itself leads to cellular mortality or whether secondary energy failure is merely a symptom of cells that are in the process of dying as a result of covert irreversible injury. Vannucci et al34 demonstrated recently that declines in PCr and ATP concentration during secondary energy failure were accompanied by concomitant falls in total creatine and adenine nucleotide concentrations, respectively. Their study also demonstrated that these reductions were accompanied by the appearance of early histologic markers of neuronal injury. In newborn infants with NE, absolute quantitation studies using proton and 31P MRS early after birth have demonstrated reductions in total creatine35 PCr, ATP, and total mobile phosphate.16,36 Thus, the combined current evidence suggests that secondary energy failure is accompanied by loss of high-energy phosphates concomitant with destruction and loss of brain tissue. In our current study, PCr/Pi and NTP/EPP were used as surrogate markers of cerebral vitality; absolute PCr and NTP concentrations were not measured because of the greater acquisition time and calibration complexity. Given the reduction of EPP and Pi concentration during secondary energy failure, it should be noted that PCr/Pi and NTP/EPP do not reflect the energetic status of some cellular populations in which the membrane integrity and, therefore, cellular phosphate pool are already lost.

An understanding of the factors influencing the secondary decline in brain energetics is crucial for optimal application and refinement of neuroprotective agents in the newborn. Several groups have observed the phenomenon "secondary energy failure" occurring after a latent phase; in these studies, the secondary decline in cerebral energy metabolism occurred on a background of normal arterial oxygen tension, blood glucose concentration, and cerebral blood flow.19,37,38 Although there remains some possibility that arteriovenous shunts, microvascular failure, or cytotoxic edema may impair oxygen uptake to injured tissue despite sufficient oxygen and glucose supply to the major vessels,39 a substantial amount of evidence suggests that mitochondrial impairment leads to or is a consequence of secondary energy failure after HI.

Disruption of oxidative phosphorylation during acute HI leads to dysfunction of energy-dependent cellular processes, such as the maintenance of membrane potential, leading to the influx and accumulation of calcium into the cytoplasm and mitochondria.31 After resuscitation, mitochondria may gradually restore function and increase high-energy phosphates back to baseline levels; however, oxygen-free radicals, high Pi concentrations, and calcium deposition in the mitochondria may open mitochondrial permeability transition pores in the inner membrane.40 Once mitochondrial permeability transition pores are open, the membrane potential is disrupted, and both inner and outer membranes are irreversibly damaged. This results in the loss of crucial and toxic enzymes, such as cytochrome c, into the cytoplasm, leading to the terminal impairment of mitochondrial respiration and activation of apoptogenic proteins, such as caspase-3, caspase-9, and apoptosis-inducing factor.31,41

Another possible explanation for the secondary mitochondrial failure is shortage of nicotinamide adenine dinucleotide (NAD), because this enzyme is converted into poly(adenosine diphosphate-ribose) polymerase, which is used to repair the damaged DNA strands.42 Because NAD is a crucial mitochondrial respiratory chain enzyme, NAD shortage may directly lead to a reduction in ATP generation. It is very likely that the magnitude of secondary energy failure and subsequent neuronal injury is not determined by a single simple cytotoxic process, but by numerous cascades, which work simultaneously leading to mitochondrial dysfunction, and providing many targets for neuroprotective interventions.

Regional Variation of Neuronal Injury and Global Energetic Status
We have recently reported data on regional patterns of cerebral injury in the same experimental subjects.23 Whole-body cooling to either 35 or 33°C led to different patterns of neuroprotection in cortical and deep gray matter (GM): compared with normothermia, cooling to 35 and 33°C led to 25% and 55% more neuronal viability in the cortical GM, respectively, whereas in the deep GM, more viable neurons (39%) were seen only at 35°C. Although the lower normal temperature in the human infant compared with the piglet has to be considered, these data are important, because they suggest that different temperatures provide optimal neuroprotection in different brain regions, and, thus, specific cooling strategies tailored to the precise pattern of HI injury may be necessary to provide optimal neuroprotection. In addition, our neuropathological study underlines the probability that the overall neuroprotective effect is determined by the balance of protective and harmful factors, as well as local tissue susceptibility, and suggests that overzealous cooling may be detrimental to some parts of the brain, in particular, the deep GM.

In our current analyses, there was no significant difference in the degree of global energetic derangement between the 2 cooled groups. However, unlike our histologic investigations on the same subjects,23 it was impossible to elucidate regional variations in the differential temperature response in the 31P MRS study presented here, because the MRS surface coil used in our study probed mean energetics over a large part of the brain. Technical developments in this experimental model have recently enabled the simultaneous collection of 31P MRS imaging (8 x 8 central coronal matrix) within the brain, enabling detailed assessment of regional energy metabolism. This is an important area of future hypothermia research and requires a model that allows assessment of the complex relationship between regional vulnerability and neuroprotection.


   CONCLUSIONS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Our current study confirmed that systemic hypothermia at 35 and 33°C commenced 2 hours after resuscitation from HI is associated with a significant preservation of 31P MRS indices of cerebral energetics over the subsequent 48 hours and approximately doubled the duration of the latent phase (Fig 4). Reducing the temperature from 35 to 33°C neither increased mean PCr/Pi and NTP/EPP nor further lengthened the latent phase. In the future, localized 31P and proton MRS must be applied to this experimental model to enable direct comparison of local brain temperature, regional cerebral energy metabolism, and regional histologic evidence of neuronal injury after both selective and whole-body hypothermia.43 Such experimentation will establish the precise hypothermia "dose-response" relationship for different brain regions so that optimal neuroprotective treatment strategies can be refined and designed for specific injuries.


   ACKNOWLEDGMENTS
 
This work was supported by Action Medical Research, Engineering and Physical Sciences Research Council, the Medical Research Council, Sir Halley Stewart Trust, the Wellcome Trust, University College London National Health Service Foundation Trust, and the Daiwa Anglo-Japanese Foundation.

We thank Drs Philip Amess, Keith Brooks, Matthew Clemence, Quyen Nguyen, Martina Noone, Nicola Parker, Donald Peebles, Gennadij Raivich, Roger Springett, and Marzena Wylezinska for their input to the data collection for this study and Alison Skinner and Dr Michael Broadhead for their technical assistance.


   FOOTNOTES
 
Accepted Oct 7, 2005.

Address correspondence to Nicola Jayne Robertson, FRCPCH, PhD, Department of Obstetrics and Gynecology, University College London, 86-96 Chenies Mews, London WC1E 6HX, United Kingdom. E-mail: n.robertson{at}ucl.ac.uk

Drs O'Brien and Iwata contributed equally to this work.

The authors have indicated they have no financial relationships relevant to this article to disclose.


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O. Iwata, S. Iwata, A. Bainbridge, E. De Vita, T. Matsuishi, E. B. Cady, and N. J. Robertson
Supra- and sub-baseline phosphocreatine recovery in developing brain after transient hypoxia-ischaemia: relation to baseline energetics, insult severity and outcome
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