Limited benefit of slow rewarming after cerebral hypothermia for global cerebral ischemia in near-term fetal sheep

Abstract

The optimal rate of rewarming after therapeutic hypothermia for neonatal hypoxic–ischemic encephalopathy is unknown, although it is widely suggested that slow rewarming is beneficial. Some preclinical studies suggest better outcomes with slower rewarming, but did not control for the duration of hypothermia. In this study, near-term fetal sheep (0.85 gestation) received 30 min cerebral ischemia followed by normothermia, 48 h hypothermia with rapid rewarming over 1 h, 48-h hypothermia with slow rewarming over 24 h, or 72-h hypothermia with rapid rewarming. Slow rewarming after 48 h of hypothermia improved recovery of EEG power compared with rapid rewarming (p < 0.05), but was not different from rapid rewarming after 72 h of hypothermia. At seven days recovery, neuronal survival was partially improved by both fast and slow rewarming after 48-h hypothermia, but less than 72-h hypothermia in the cortex and CA4 (p < 0.05). In conclusion, although electrographic recovery was partially improved by slow rewarming over 24 h following cerebral hypothermia for 48 h, optimal neuroprotection was seen with hypothermia for 72 h with rapid rewarming, suggesting that the overall duration of cooling was the critical determinant of outcomes after therapeutic hypothermia.

Introduction

There is now compelling clinical and experimental evidence that therapeutic hypothermia can reduce neuronal loss and improve neurological outcome after a severe hypoxic-ischemic (HI) insult in term infants.^1,2 Nevertheless, hypothermic neuroprotection remains partial and many infants still have adverse outcomes.² The optimal rate of rewarming after therapeutic hypothermia is highly controversial. The randomized clinical trials of therapeutic hypothermia for HIE rewarmed neonates at no more than 0.5℃ per h.³ This was largely based on case reports that fast rewarming might destabilize cardiovascular function,⁴ or trigger rebound seizures.⁵ However, there is no controlled human evidence for the optimal rate of rewarming.

There is sparse evidence from animal studies that rapid rewarming may reverse the depression of potential injurious processes such as oxidative stress or excitotoxin release,^6,7 and slow rewarming may improve neural outcomes. In neonatal piglets exposed to severe HI, hypothermia for 18 h followed by rewarming at 0.5℃/h was associated with less caspase-3 activation in the cerebral cortex and white matter tracts than with rewarming at 4℃/h.^8,9 Moreover, in adult gerbils subjected to transient forebrain ischemia, fast rewarming after hypothermia for 2 h was associated with transient uncoupling of cerebral blood flow and metabolism and loss of neuroprotection in the CA1 region of the hippocampus, which was prevented by slow or stepwise rewarming.¹⁰ However, these studies tested very short intervals of cooling that are likely to have been highly sub-optimal. There is now considerable evidence from a range of adult and neonatal studies that following delayed initiation of hypothermia, cooling needs to be continued for 48 to 72 h for optimal neuroprotection.^11,12 Thus, as the total duration of cooling was not controlled in those studies,^8,9 it is possible that the benefits of slow rewarming in those studies were simply related to a longer net duration of treatment.

Rapid, spontaneous rewarming after hypothermia for 72 h has been associated with a modest, transient increase in epileptiform events in near-term fetal sheep, although there was no correlation with neural outcomes.¹³ It has since been confirmed that infants occasionally develop rebound seizures during or shortly after rewarming even at 0.5℃ per h and that recooling can suppress these seizures.^5,14 However, the effect of the rate of rewarming on other outcomes is highly unclear.¹⁵

We have recently shown in near-term fetal sheep that 48 h of cerebral hypothermia, started 3 h after global cerebral ischemia, was associated with a striking deterioration in EEG power and significantly less neuroprotection than 72 h of hypothermia.¹² In that study, fetuses were allowed to rewarm spontaneously, typically reaching control temperatures in approximately 1 h.¹³ This finding suggested the hypothesis that rapid rewarming might have triggered secondary deterioration.

In the present study, we therefore compared the impact of very slow rewarming (∼0.2℃/h) over 24 h with rapid spontaneous rewarming (∼5℃/h), after 48 h of cerebral hypothermia for cerebral ischemia in near-term fetal sheep, on recovery of EEG power and histological neuroprotection. This very slow rewarming allowed us to compare outcomes to 72 h of hypothermia followed by rapid rewarming, in order to control for the possibility that any effect of slow rewarming could have been mediated by the longer total duration of cooling.

Materials and methods

Fetal surgery

All procedures were approved by the Animal Ethics Committee of The University of Auckland under the New Zealand Animal Welfare Act, and the Code of Ethical Conduct for animals in research established by the Ministry of Primary Industries, Government of New Zealand. The experiment has been reported in compliance with the ARRIVE guidelines.¹⁶ In brief, 40 time-mated Romney/Suffolk fetal sheep were instrumented using sterile techniques at 124 ± 1 days' gestation (term is 145). Food, but not water was withdrawn 18 h before surgery. Ewes were given long-acting oxytetracycline (20 mg/kg, Phoenix Pharm, Auckland, New Zealand) i.m. 30 min before the start of surgery. Anesthesia was induced by intravenous injection of propofol (5 mg/kg, AstraZeneca Limited, Auckland, New Zealand) and maintained using 2–3% isoflurane in O₂. The depth of anesthesia, maternal heart rate and respiration was constantly monitored by trained anesthetic staff. Ewes received a constant infusion of isotonic saline drip (at an infusion rate of approximately 250 mL/h) to maintain the fluid balance.

Following a maternal midline abdominal incision, the fetus was exposed and both fetal brachial arteries were catheterized with polyvinyl catheters to measure mean arterial blood pressure (MAP) and allow fetal blood sampling. An amniotic catheter was secured to the fetal shoulder. Electrocardiogram (ECG) electrodes (AS633–3SSF; Cooner Wire Co., Chatsworth, California, USA) were sewn across the fetal chest to record fetal heart rate (FHR). The vertebral-occipital anastomoses were ligated and inflatable carotid occluder cuffs were placed around both the carotid arteries. A 3S transonic ultrasonic flow probe (Transonic systems, Ithaca, NY) was placed around the right carotid artery. Using a seven stranded stainless steel wire (AS633–7SSF; Cooner Wire Co.), two pairs of electroencephalogram (EEG) electrodes were placed on the dura over the parasagittal parietal cortex (10 mm and 20 mm anterior to bregma and 10 mm lateral) and secured with cyanoacrylate glue. A reference electrode was sewn over the occiput. A further two electrodes were sewn in the nuchal muscle to record electromyographic (EMG) activity as a measure of fetal movement. To measure cortical impedance, a third pair of electrodes (AS633-3SSF; Cooner Wire Co.) was placed over the dura 10 mm lateral to the EEG electrodes. A thermistor (Replacement Parts Industries, Inc.) was placed over the parasagittal dura 30 mm anterior to bregma to measure extradural temperature and a second thermistor was inserted into the esophagus to measure core temperature. A cooling cap made from silicon tubing (3 × 6 mm, Degania Silicone, Israel) was secured to the fetal head. The uterus was then closed and antibiotics (80 mg gentamicin, Pharmacia and Upjohn, Rydalmere, New South Wales, Australia) were administered into the amniotic sac. The maternal laparotomy skin incision was repaired and infiltrated with 10 ml 0.5% bupivacaine plus adrenaline (AstraZeneca Ltd, Auckland, New Zealand). All fetal catheters and leads were exteriorized through the maternal flank. The maternal long saphenous vein was catheterized to provide access for the post-operative maternal care and euthanasia.

Post-operative care

Sheep were housed together in separate metabolic cages with access to food and water ad libitum. They were kept in a temperature-controlled room (16 ± 1℃, humidity 50 ± 10%), in a 12-h light/dark cycle. Intravenous antibiotics were administered daily for four days to the ewe (600 mg benzylpenicillin sodium, Novartis Ltd, Auckland, New Zealand, and 80 mg gentamicin). Fetal catheters were maintained patent by the continuous infusion of heparinized saline (20 U/mL at 0.2 mL/h) and the maternal catheter maintained by the daily flushing.

Data recording

Fetal MAP, corrected for maternal movement by subtraction of amniotic pressure (Novatrans II, MX860; Medex Inc., Hilliard, OH, USA), CaBF, ECG, EEG, EMG, temperatures and impedance were recorded from 24 h before the start of the experiment and continued for the remainder of the experiment. The raw ECG was analogue filtered between 0.05 and 100 Hz and digitized at 1024 Hz. The EEG signal was processed with a first-order high-pass filter at 1.6 Hz and a sixth-order Butterworth low-pass filter with the cut-off frequency at 256 Hz, and then digitally stored at a sampling rate of 512 Hz. EEG intensity (power) was derived from the power spectrum between 1 and 20 Hz and log transformed (decibels (dB), 20 × log (intensity)) as this transformation gives a better approximation of the normal distribution.¹⁷ Spectral edge was defined as the frequency below which 90% of total EEG power was present, as a measure of the relative frequency of the EEG. Impedance increases as the temperature of the medium, through which the signal passes, falls. Therefore, in each fetus, the slope of impedance change at the onset of hypothermia was used to correct the impedance signal: corrected impedance = impedance – (slope × Δtemperature).^18,19 Data were recorded and saved continuously to disk for off-line analysis using custom data acquisition programs (LabVIEW for Windows, National Instruments, Austin, Texas, USA).

Arterial blood samples were taken for pre-ductal pH, blood gas, base excess (ABL800 Flex Analyzer, Radiometer, Auckland, New Zealand), glucose and lactate measurements (YSI model 2300, Yellow Springs, Ohio, USA). All fetuses had normal biochemical variables for their gestational ages.

Experimental protocols

At 128 ± 1 day gestation, cerebral ischemia was induced by reversible inflation of the carotid occluder cuffs with sterile saline for 30 min. Successful occlusion was confirmed by the onset of an isoelectric EEG signal within 30 s of inflation. The carotid occluder cuffs were not inflated in sham control experiments. Fetal blood samples were drawn just before the occlusion and 2, 4 h and 6 h after occlusion followed by daily sampling for the remainder of the experiment.

Randomization was stratified by cohort to control for time of year. Within each cohort, fetuses were randomized to ischemia-normothermia (n = 8), ischemia-48-h hypothermia (n = 8), ischemia-48-h hypothermia-slow-rewarming (n = 7), ischemia-72-h hypothermia (n = 8) or sham control (n = 9) groups. Based on a pooled standard deviation of 20 for neuronal survival in the parasagittal cortex after cerebral ischemia, a group size of 8 was estimated to be needed to provide 80% power to detect a difference of 30 cells per field of view or more between groups.

Cooling was started 3 h after release of carotid occlusion and continued until either 48 or 72 h after the onset of ischemia, followed by spontaneous rewarming in the ischemia-48-h hypothermia and ischemia-72-h hypothermia groups and slow rewarming over 24 h in the ischemia-48-h hypothermia-slow-rewarming group. Cooling was performed by linking the cooling coil over the fetal scalp with a pump (TX150 Heating circulator, Grant Instruments Ltd, Cambridge, England) in a cooled water bath and circulating cold water through the cooling coil. Consistent with previous studies in near-term fetal sheep, the initial target extradural temperature was set to between 31 and 33℃.^18,20,21 In the ischemia-normothermia and sham control groups, the water was not circulated and the cooling coil remained in equilibrium with fetal temperature.

At the end of the cooling period, the water pump was switched off and fetuses were allowed to spontaneously rewarm over approximately 60 min in the spontaneous rewarming groups, while fetuses in the slow-rewarming group received computer-controlled graded rewarming from the treatment temperature up to 39℃ over 24 h.^13,18 Ewes and fetuses were killed seven days after ischemia with an overdose of sodium pentobarbitone (9 g intravenous to the ewe; Pentobarb 300; Chemstock, Christchurch, New Zealand) for immunohistochemistry.

Immunohistochemistry

Fetal brains were perfusion fixed with 10% phosphate-buffered formalin. Coronal slices (10 µm thick) were cut using a microtome (Leica Jung RM2035, Wetzlar, Germany) starting at the level of the dorsal hippocampus. Slides were dewaxed in xylene and rehydrated in decreasing concentrations of ethanol, then washed in 0.1 mol/L phosphate-buffered saline (PBS). Antigen retrieval was performed using the pressure cooker method (2100 Antigen Retriever, Aptum, Southampton, England) in citrate buffer followed by incubation in 1% H₂O₂ in methanol to block endogenous peroxidase activity. Blocking was performed in 3% normal goat serum (NGS), for 1 h at room temperature. Sections were labelled with 1:200 rabbit anti-neuronal nuclei monoclonal antibody (NeuN, Abcam, Cambridge, England) overnight at 4℃. Sections were incubated for 3 h in biotin-conjugated 1:200 anti-rabbit IgG antibody (Vector Laboratories, Burlingame, USA) in 3% NGS. Slides were then incubated in ExtrAvidin® (1:200, Sigma-Aldrich Pty. Ltd, St Louis, USA) in PBS for 2 h at room temperature and then reacted in diaminobenzidine tetrachloride (Sigma-Aldrich Pty. Ltd). The reaction was stopped by washing in PBS. Sections were dehydrated in increasing concentrations of alcohol and mounted.

Neurons in the cortex of the first and second parasagittal gyri, CA1/2, CA3, CA4 and dentate gyrus of the hippocampus were counted using light microscopy (Nikon eclipse 80i, Scitech Ltd, Preston, Victoria, Australia) at 20 × magnification (image size 512 µm × 384 µm) by an investigator masked to the treatment group by separate coding of the slides. Normal-appearing NeuN-positive neurons were identified morphologically by the presence of typical nuclei; cells showing condensed nuclei or fragmented appearance were not counted.²² Images were all auto-contrasted using ImageJ (National Institutes of Health, Bethesda, USA) to make all individual cells easier to identify.

Sleep state cycling

The return of sleep state cycling was visually quantified from the 1 min averaged EEG recording by the presence of established, well-defined alternating patterns of low voltage, high frequency and high voltage, low frequency EEG activity lasting approximately 20 min each. Fetuses that did not resume sleep state cycling within the seven-day recovery period, were assigned a value of 168 h.

Seizure burden

Seizure activity was defined as previously described,²³ as sudden repetitive, evolving stereotypical waveforms lasting at least 10 s with an amplitude > 20 µV. Seizure burden was quantified as the total duration of seizures.

Statistical analysis

Data were analyzed by an investigator who was not masked to the treatment group. Normally distributed data were analyzed using ANOVA or repeated measures ANOVA, followed by the Tukey post hoc test when a significant difference was found. The proportion of fetuses having seizures between 48 and 96 h was analyzed by Chi-squared test. The median duration of the return of sleep state cycle and the duration of seizures between 48 and 96 h were analyzed with the Kruskal–Wallis test. Statistical significance was accepted when p < 0.05.

Results

Sex distribution, body and brain weight at post-mortem

There were no significant differences in the sex distribution or post-mortem body weight between groups (Supplementary Table 1). Ischemia-normothermia was associated with reduced post-mortem brain weight (40.1 ± 1.5 g) compared to sham controls (48.9 ± 1.9 g). The 48-h cooling groups also showed reduced brain weight: ischemia-48-h hypothermia (41.9 ± 1.7 g, p < 0.05 vs. sham control) and ischemia-48-h hypothermia-slow-rewarming (43.0 ± 1.4 g, p < 0.05 vs. sham control), and were not significantly different from ischemia-normothermia. By contrast, ischemia-72-h hypothermia was associated with significantly increased brain weight compared to ischemia-normothermia (44.9 ± 0.9 g, p < 0.05).

Blood gas, glucose and lactate measurements

There were no significant differences in baseline blood gas, pH, glucose or lactate measurements between the groups (Supplementary Table 2). Ischemia was associated with transiently increased plasma lactate and glucose concentrations (p < 0.05), which resolved to baseline levels by day 3.

Temperature changes

Head cooling was associated with a fall in extradural temperature to similar nadirs of 32.3 ± 0.3℃ in the ischemia-48-h hypothermia group, 32.3 ± 0.3℃ in the ischemia-48-h hypothermia-slow-rewarming group and 31.3 ± 0.2℃ in the ischemia-72-h hypothermia group (N.S.), compared to 39.5 ± 0.1℃ in the ischemia-normothermia group (p < 0.05, Figure 1). Esophageal temperatures fell to between 37 and 38℃ during cooling (p < 0.05 vs. ischemia-normothermia). During the cooling period, extradural temperatures gradually rose but remained consistently well below values in the ischemia-normothermia group. After the end of hypothermia, extradural and esophageal temperature returned to baseline values after approximately 1 h in the ischemia-48-h hypothermia group and the ischemia-72-h hypothermia group (equivalent to a mean of 5℃/h). In the ischemia-48-h hypothermia-slow-rewarming group, extradural temperatures increased gradually at 0.2 ºC/h. Overshoot hyperthermia was not seen in any group.

Figure 1.

Change in extradural temperature, esophageal temperature and carotid artery blood flow before, during and after 30 min of global cerebral ischemia (time zero) in the near-term fetal sheep, showing the ischemia-normothermia (n = 8), ischemia-48-h hypothermia (n = 8), ischemia-48-h hypothermia-slow rewarming (n = 7) and ischemia-72-h hypothermia (n = 8) groups. Data are mean±SEM, *p < 0.05 ischemia-72-h hypothermia vs. ischemia-normothermia, ^#p < 0.05 ischemia-48-h hypothermia vs. ischemia-normothermia, ^ap < 0.05 ischemia-48-h hypothermia-slow-rewarming vs. ischemia-normothermia.

Neurophysiology

Cerebral ischemia was associated with rapid, profound suppression of EEG power (Figure 2). After release of occlusion, EEG was initially suppressed, followed by intense seizure activity from ∼8 to 48 h after ischemia. After seizures resolved, total EEG power fell below baseline levels in the ischemia-normothermia group. In contrast, the ischemia-48-h hypothermia, ischemia-48-h hypothermia-slow rewarming and ischemia-72-h hypothermia groups showed substantially greater recovery of EEG power than ischemia-normothermia from 24 h until the end of the experiment (p < 0.05). Slow rewarming over 24 h after 48 h of hypothermia prevented the progressive deterioration in EEG power seen with spontaneous rewarming in the ischemia-48-h hypothermia group. EEG power remained significantly higher in both the ischemia-48-h hypothermia-slow-rewarming and ischemia-72-h hypothermia groups compared to the ischemia-48-h hypothermia group for the remainder of the experiment (p < 0.05).

Figure 2.

Changes in EEG power, spectral edge frequency and impedance before, during and after 30 min of global cerebral ischemia (time zero) in the ischemia-normothermia (n = 8), ischemia-48-h hypothermia (n = 8), ischemia-48-h hypothermia-slow-rewarming (n = 7) and ischemia-72-h hypothermia (n = 8) groups. Data are mean±SEM, *p < 0.05 ischemia-72-h hypothermia vs. ischemia-normothermia group, ^#p < 0.05 ischemia-48-h hypothermia group vs. ischemia-normothermia group, ^ap < 0.05 ischemia-48-h hypothermia-slow-rewarming vs. ischemia-normothermia group, ^bp < 0.05 ischemia-72-h hypothermia vs. ischemia-48-h hypothermia group.

Ischemia was associated with rapid suppression of EEG spectral edge frequency, which remained below baseline until the end of the experiment in the ischemia-normothermia group. In contrast, all the three hypothermia groups showed significantly higher spectral edge frequency from 6 to 24 h and from 48 h until the end of the experiment (Figure 2, p < 0.05). There was no significant difference between the hypothermia groups (N.S.).

Sleep state cycling was initially lost after cerebral ischemia. In the ischemia-normothermia group, sleep state cycling took an average of approximately 131 h to return, although it should be noted that 3/7 animals did not resume sleep state cycling within the seven-day recovery period and so were assigned a value of 168 h (Figure 3). In contrast, all hypothermia protocols were associated with a significant reduction in the time taken for sleep state cycling to resume, and recovery of sleep state cycling with the seven-day recovery period. In the ischemia-48-h hypothermia group, two animals showed return of sleep state cycling, followed by a transient loss of sleep state cycling, before sleep state cycling became properly established. This pattern was never seen in any other group.

Figure 3.

Top: Box plot (the line represents the median, the box represents the 25th and 75th percentiles and the whiskers show minimum to maximum) showing time taken for sleep state cycling (SSC) to resume after 30 min of global cerebral ischemia in the ischemia-normothermia (n = 8), ischemia-48-h hypothermia (n = 8), ischemia-48-h hypothermia-slow-rewarming (n = 7) and ischemia-72-h hypothermia (n = 8) groups. *p < 0.05 vs. ischemia-normothermia. Bottom: Correlation between the number of NeuN-positive neurons in the cortex and the time until sleep state cycling resumed after cerebral ischemia.

No seizures were seen in sham control animals. In the ischemia-normothermia group, stereotypic evolving seizures occurred, the great majority between 6 and 48 h (Figure 4). Occasional seizures were seen in 6/8 of the ischemia-normothermia group between 48 and 96 h (Table 1). All hypothermia protocols were associated with a significant reduction in seizure burden between 18 and 36 h compared to the ischemia-normothermia group (p < 0.05). There was no evidence of rebound seizures after rewarming in the hypothermia groups and there was no significant difference in the number of fetuses having seizures or the seizure burden from 48 to 96 h between the hypothermia protocols. Representative examples of the pattern of EEG recovery over the first 96 h are shown in Figure 5.

Figure 4.

Total seizure burden (min/h) during 96 h of recovery after 30 min of global cerebral ischemia in the ischemia-normothermia (n = 8), ischemia-48-h hypothermia (n = 8), ischemia-48-h hypothermia-slow-rewarming (n = 7) and ischemia-72-h hypothermia (n = 8) groups.

Table 1.

Stereotypic evolving seizures between 48 and 96 h after 30 min of global cerebral ischemia: fetuses developing seizures and total duration of seizure activity.

Group	Fetuses developing seizures (N)	Total duration (s, median (range))
Ischemia-normothermia	5/8	89 (6912)
Ischemia-48-h hypothermia	6/8	316.5 (1961)
Ischemia-48-h hypothermia- slow-rewarming	3/7	0 (1897)
Ischemia-72-h hypothermia	2/8	0 (941)

Figure 5.

Representative examples showing EEG power with 24 h of baseline, 30 min of global cerebral ischemia and 96 h of recovery in a fetus from each of the ischemia-normothermia, ischemia-48-h hypothermia, ischemia-48-h hypothermia-slow-rewarming and ischemia-72-h hypothermia groups. Data are 1 min averages.

Ischemia-normothermia was associated with a secondary increase in cortical impedance between 24 and 72 h after ischemia compared to sham controls, ischemia-48-h hypothermia, ischemia-48-h hypothermia-slow-rewarming and ischemia-72-h hypothermia (p < 0.05, Figure 2). Impedance was significantly reduced in the ischemia-normothermia group compared to all three hypothermia groups from 120 h onward and in the ischemia-48 h hypothermia group compared to ischemia-48 h hypothermia-slow rewarming and ischemia-72 h hypothermia from 144 h onwards (p < 0.05).

Physiological parameters

Carotid artery blood flow (CaBF) showed a delayed increase in the ischemia-normothermia group between 6 and 36 h after ischemia, which was suppressed in all hypothermia groups (Figure 1, p < 0.05 vs. ischemia-normothermia). There were no significant differences in mean arterial pressure, FHR or nuchal EMG between groups (data not shown).

Immunohistochemistry

Ischemia was associated with reduced numbers of surviving neurons in the cortex, CA1/2, CA3, CA4 and dentate gyrus of the hippocampus compared to sham controls (p < 0.05, Figure 6). All hypothermia groups showed significantly increased overall neuronal survival compared to ischemia-normothermia (p < 0.05). Neuronal survival in the ischemia-48-h hypothermia-slow-rewarming group was significantly greater in the cortex and dentate gyrus compared to ischemia-normothermia and was not significantly from ischemia-48-h hypothermia group in any region. Ischemia-72-h hypothermia was associated with greater neuronal survival in the cortex and CA4 compared to both ischemia-48 h hypothermia and ischemia-48 h hypothermia-slow-rewarming groups (p < 0.05), and showed similar overall neuronal survival to sham controls; post hoc tests suggested a small deficit in CA1/2 and CA3 compared to sham controls (p < 0.05).

Figure 6.

Panel (a). Neuronal survival seven days after 30 min of global cerebral ischemia in the sham control (n = 9), ischemia-normothermia (n = 8), ischemia-48-h hypothermia (n = 8), ischemia-48-h hypothermia-slow-rewarming (n = 7) and ischemia-72-h hypothermia (n = 8) groups. *p < 0.05 vs. sham control, ^#p < 0.05 vs. ischemia-normothermia, ^ap < 0.05 vs. ischemia-72-h hypothermia. Panel (b). Photomicrographs showing NeuN-positive staining in the cortex (a, f, k, p, u), CA1 (b, g, l, q, w), CA3 (c, h, m, r, x), CA4 (d, i, n, s, y), and dentate gyrus (e, j, o, t, z) in the sham control (a–e), ischemia-normothermia (f–j), ischemia-48-h hypothermia (k–o), ischemia-48-h hypothermia-slow-rewarming (p–t) and the ischemia-72-h hypothermia (u–z) groups. Scale bar 200 µm.

There was a significant correlation between the time taken for sleep state cycling to resume and the number of NeuN-positive neurons in the cortex (Figure 3, p < 0.05, R² = 0.30) as well as the CA1/2, CA3, CA4, and dentate gyrus (R² = 0.14, 0.27, 0.21 and 0.17, respectively, data not shown).

Discussion

The optimal speed of rewarming after therapeutic hypothermia is controversial. The randomized clinical trials of therapeutic cooling for HIE rewarmed neonates at no more than 0.5℃ per h.³ However, this was based on empiric concerns that fast rewarming might destabilize cardiovascular or neuronal function,^4,5 or that it might reverse the depression of potential injurious processes such as oxidative stress or excitotoxin release.^6,7 There is surprisingly little direct clinical or preclinical evidence that speed of rewarming affects outcomes separately from the effective increase in the duration of cooling.^24,25

We have recently shown that rapid spontaneous rewarming at 48 h, but not at 72 h, was associated with a striking deterioration in EEG power, over approximately 12 h.¹² In the current study, slow rewarming over 24 h prevented the deterioration in EEG power compared to rapid warming after hypothermia for 48 h. Indeed, this very slow rewarming protocol restored recovery of EEG power to that seen with 72 h of hypothermia. However, neuronal survival was substantially less than after rapid spontaneous rewarming from 72 h of hypothermia. These data suggest that the improvements seen in the slow rewarming group were mainly mediated by the longer total duration of hypothermia rather than the rate of rewarming per se.

Further, all hypothermia protocols were associated with a significant reduction in the time taken for sleep state cycling to resume compared to the ischemia-normothermia group. The emergence of sleep-wake cycling is a useful clinical prognostic factor in neonates with hypoxic–ischemic encephalopathy.²⁶ Interestingly, two animals in the ischemia-48-h hypothermia group showed transient return of sleep state cycling, interspersed by a secondary loss of sleep state cycling, before ultimate recovery. This transient loss most likely reflected the secondary deterioration of EEG power in this group.

All hypothermia protocols completely abolished the secondary rise in impedance, which is a measure of cell swelling,²⁷ and significantly reduced the occurrence of stereotypic evolving seizures during hypothermia. Rewarming at 48 h in the present study was not associated with rebound cytotoxic edema or a significant increase in the number of animals having stereotypic evolving seizures or the median duration of seizure activity between 48 and 96 h. This suggests that influx of ions, such as sodium and calcium, into cells and excessive release of glutamate were not important triggers of further injury. However, both the ischemia-normothermia and ischemia-48 h hypothermia groups showed a progressive decrease in cortical impedance from 120 and 144 h onwards, respectively, compared to ischemia-72-h hypothermia or ischemia-48-h hypothermia-slow rewarming. This late reduction in impedance to below baseline levels is believed to reflect greater total cell loss leading to reduced impedance to the flow of current through the extracellular space.^27,28

In contrast with the improved electrophysiological recovery, slow rewarming after 48 h of hypothermia was not associated with a significant improvement of neuronal survival compared to spontaneous rewarming after 48 h of hypothermia. Strikingly, there was less improvement in neuronal survival than with spontaneous rewarming after 72 h of hypothermia. The reasons for this discrepancy are not known. The severity of neuronal loss and final cortical EEG power show a highly non-linear relationship in the same experimental paradigm as the present study.^28,29 In the present study, although there was a significant correlation between the time taken for sleep state cycling to resume and neuronal survival in the cortex and hippocampus, there was considerable residual variance in this relationship. The correlation between neuron number and brain activity may be influenced by dendritic spine density,^30,31 neuronal plasticity,³² as well as survival and function of other cell types, such as astrocytes and oligodendrocytes.²⁹

Previous studies report less caspase-3 activation in the cerebral cortex of neonatal swine subjected to HI with slower rewarming (0.5℃ vs. 4℃ per h), and greater hippocampal neuroprotection in adult gerbils recovering from transient forebrain ischemia.^8,10 However, those studies tested highly sub-optimal durations of cooling (18 and 2 h, respectively). The present study addressed whether slow rewarming could enhance neuroprotection after a sub-optimal duration of hypothermia (48 h) compared with the known optimal duration of 72 h in this experimental protocol.^12,20,21 Our findings that rapid rewarming after hypothermia for 72 h provided better overall neuroprotection than slow rewarming for 24 h after hypothermia for 48 h strongly suggests that the improved outcomes with slow rewarming were primarily a function of extending the total duration of therapeutic hypothermia.

Some limitations of the present study should be considered. It is not known how the rate of rewarming after 72 h of hypothermia affects neuronal survival and recovery of EEG power. This will be an important future study. We found no apparent effect of speed of rewarming on cardiovascular stability. However, our fetal protocol does not cause cardiac injury and these experiments were undertaken in the fetus rather than the newborn. It is possible that these differences would reduce the impact of rapid rewarming on cardiovascular adaptation. Further, the present study did not examine cell survival in other brain regions, such as the basal ganglia, thalamus and white matter tracts, which may be injured in this paradigm. Finally, the long-term impact of the rate of rewarming on chronic inflammation,³³ and neurological recovery at full term,³⁴ should be addressed in future studies.

Conclusion

These data demonstrate that slow rewarming over 24 h after 48 h of hypothermia can prevent the deterioration in EEG power observed with early termination of hypothermia. However, slow rewarming had little effect on neuronal survival compared to 48 h of hypothermia and was significantly worse than simply continuing hypothermia to 72 h and then allowing rapid, spontaneous rewarming. These data suggest that the total duration of hypothermia has a greater influence on neuroprotection than the rate of rewarming after hypothermia, and strongly reinforce that for optimal neuroprotection, therapeutic hypothermia must be continued for 72 h.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by the Health Research Council of New Zealand (17/601) and the Neurological Foundation (NF1715-PG).

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors' contributions

Joanne Davidson, Laura Bennet and Alistair J Gunn conceptualized and designed the study. Joanne Davidson, Simerdeep Dhillon and Guido Wassink undertook experiments and analyzed data. Vittoria Draghi and Guido Wassink undertook immunohistochemistry, cell quantification, analysis and preparation of figures. AJ Gunn provided overall oversight of the research. All authors critically reviewed the manuscript and approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

Supplementary material

Supplementary material for this paper can be found at the journal website: http://journals.sagepub.com/home/jcb