This work presents, for the first time, that therapeutic hypothermia following hypoxia-ischemia in newborn pigs is also cardioprotective in our established newborn pig global HIE model. This global injury model of neonatal brain injury with multi-organ failure was used in neuroprotective cooling research (13
) and contributed towards successful clinical trials (3
). Increasingly, therapeutic hypothermia is becoming standard of care in treating HIE infants worldwide(25
). Hypothermia has proven to be neuroprotective, but there is no sufficient evidence demonstrating whether HT confers cardioprotection in intensive care patients. Some concern relates to work on adults where hypothermia treatment causes hypotension (26
). However, hypotension during 3 days of HT treatment was not found in 3 large neonatal clinical hypothermia trials with appropriate care (3
The significant heart rate reduction in the cooling group may confer reduced stress and be an indirect measure of reduced energy and metabolic demand in our study. Our study has followed a strict protocol to treat hypotension and to avoid other adverse incidents. We have introduced background sedation which reduce the stress level during HT treatment (13
). We have also previously shown that hypothermia and sedations per se
do not increase the apoptotic cell death in our model (27
). These may all contribute to hypothermic cardioprotection. We did not see the differences in MABP and in use of inotropics in the first 3 days between the two groups. There is however a trend of a lower MABP in the HT group. In this study, there was only one HT pig which had severe and also drug resistant hypotension during the whole experimental period. Despite this long lasting low MABP, this HT pig only had two ischemic lesions in the heart. The rest of the HT pigs all had MABP >40mmHg during the experiment. Clinically, maintained MABP and reduced use of inotropic support are often noted as signs of cardiac recovery. We did see reduced cTnI levels and improved pathology in the heart in the cooled group which indicated the hypothermic cardiac-protection. It is not possible to obtain the data on heart pathology clinically but cTnI can be used as an early marker to describe injury and cardiac recovery in asphyxia infants.
Cardiac troponin I as a cardiac biomarker is well established in adults and has been recognised as not only a diagnostic but prognostic marker (8
). However, its potential application in neonatal medicine has not been fully explored. Cord cTnI was suggested as a good early predicator of severity of HIE in term infants (11
). The levels of cTnI are barely detectable in healthy adults and increases in cTnI above the 99th
percentile reference limit are considered an indication of myocardial injury. This limit has been reported as low as 0.03ng/ml in adults (28
). Comparing cTnI in adults with infants, higher baseline levels of cTnI (>0.03ng/ml)are observed in the first 3 months of life (29
). According to our pilot study and the specific cTnI assay used, we have set the normal range of cTnI for a new born pig is below 0.2ng/ml.
In animal studies, it has been reported that the level of cTnI starts to increase by 30 min with a peak value at 3h after cardiac arrest in adult pigs (30
) and started to rise from 0.5-1h after induced cardiac injury and normalized within 48h after the insult in adult rodents (31
). Our data confirms this rapid release of cTnI after acute injury as we observed the first rise in cTnI at 1h after a 45-min global () insult with a peak around 3-6h in the pilot study. The time course of cTnI release in our normothermic animals is similar to that seen in older children after acute myocardial injury (32
). We have also shown the level of cTnI normalised in both treated groups 48 hours after the insult. Similar data for cooled/non-cooled newborns are only available at the age of 72 hours (33
) where there was also no difference observed between two groups. In this randomised study, we showed earlier time points after an acute injury. There was a nearly 9-fold cTnI increase 6h after HI and no increase in heart rate or cTnI in the NT pigs when intravenous anaesthesia was ended during the rewarming period. The findings from the HT group are different. In these anaesthetised cooled pigs, the cTnI was much lower in the cooled group compared to NT group. This suggests that hypothermia may provide cellular protection to the heart. Due to small numbers we did not reach significance when comparing the pathology results. Neonatal hearts, different from adult hearts, rely on anaerobic metabolism. They have high glycogen stores. Therefore, neonatal heart has increased tolerance to transient hypoxic-ischemic damage (34
). Cardiac troponin I is a more sensitive biomarker compared to cardiac pathology. We may see the transient increase in cTnI without permanent damage in the heart. Additionally, neonatal hearts is better at cellular repair than adult hearts. This may explain that we only see smaller lesions in a subset of animals but a huge increase of cTnI level soon after the insult. During fast rewarming (near 0.8°C/hr) in the HT group, the heart rate almost doubled and there was a significant increase in cTnI which may indicate cardiac stress. The NT pigs had longer durations of seizures. Seizure activity may also increase HR and cardiac stress. Slow rewarming rates may be beneficial for the heart.
In the normothermic newborn pigs after a HI insult, we find a strong relationship between myocardial damage and peak cTnI levels. Prospective clinical data is needed to examine whether cTnI is a valid predictor of neurological/cardiac outcome for HIE infants after therapeutic hypothermia.
There are some limitations to our study. The experimental insult was not carried out around the time of birth but within 24h of age. To introduce HI in our newborn pig model, pigs were ventilated and sedated with halothane for 45 minutes. Clinically, the fetus or newborn may have different types and degree of injury due to the time of the onset and the duration of the insult(s). Some infants may have one prolonged systemic HI injury as demonstrated in our model and others may have a few but shorter episodes of insults that may affect the heart differently. Most injuries were seen in the right ventricle which is a typical location in the newborn stressed heart (35
). We used 24 hours cooling treatment in this study which is different from 72 hours applied clinically. Different animal species require different effective cooling durations. We have previously shown that 24 hours cooling provides better neuroprotection than 12 hours cooling and significant neuroprotection than normothermia (36
) in this model. Therefore, we believe 24 hours cooling is the optimal cooling duration in newborn pigs.
In conclusion, mild immediate hypothermia as a post-insult neuroprotective intervention may also protect the heart, in particular if conducted with appropriate clinical management; a slow rewarming rate and with adequate sedation during cooling therapy.