Therapeutic hypothermia (HT) is standard care for term infants with hypoxic-ischaemic (HI) encephalopathy. However, the efficacy of HT in preclinical models, such as the Vannucci model of unilateral HI in the newborn rat, is often greater than that reported from clinical trials. Here, we report a meta-analysis of data from every experiment in a single laboratory, including pilot data, examining the effect of HT in the Vannucci model. Across 21 experiments using 106 litters, median (95% CI) hemispheric area loss was 50.1% (46.0-51.9%; n = 305) in the normothermia group, and 41.3% (35.1-44.9%; n = 317) in the HT group, with a bimodal injury distribution. Median neuroprotection by HT was 17.6% (6.8-28.3%), including in severe injury, but was highly-variable across experiments. Neuroprotection was significant in females (p < 0.001), with a non-significant benefit in males (p = 0.07). Animals representing the median injury in each group within each litter (n = 277, 44.5%) were also analysed using formal neuropathology, which showed neuroprotection by HT throughout the brain, particularly in females. Our results suggest an inherent variability and sex-dependence of the neuroprotective response to HT, with the majority of studies in the Vannucci model vastly underpowered to detect true treatment effects due to the distribution of injury. The use of animal research has dramatically advanced human knowledge of physiology, pathology, and sciencebased medicine 1. However, despite centuries of improvement in the methodological and ethical approaches to experiments involving animals 1 , preclinical research across all fields of medicine has also significantly underperformed with regards to the translation of robust treatments for complex diseases in humans 2. For perinatal asphyxia and subsequent hypoxic-ischaemic encephalopathy (HIE), just one treatment so far, therapeutic hypothermia (HT), has emerged from the preclinical research to provide a robust treatment effect in term newborns with moderate-to-severe neurological injury 3. A recent meta-analysis of those clinical trials found that the numbers needed to treat (NNT) were 11 to reduce mortality, and 8 to reduce major neurodevelopmental disability 3. However, HT is not universally neuroprotective. Meta-analyses of the original large clinical trials of HT found that 40-50% of treated infants still experienced a poor outcome (death or severe disability) 4 , though more recent trials suggest it is now around 30% 5. In the most widely-researched model of neonatal hypoxic-ischaemic (HI) brain injury, the Vannucci model of unilateral HI, our group and others have traditionally found that HT provides around 40% reduction in neuropathology score and tissue loss 6-9. This neuroprotective effect of HT was corroborated in larger animal models 10-12 ,

Background: Hypothermia-treated and intubated infants with moderate or severe hypoxic-ischemic encephalopathy (HIE) usually receive morphine for sedation and analgesia (SA) during therapeutic hypothermia (TH) and endotracheal ventilation. Altered drug pharmacokinetics in this population increases the risk of drug accumulation. Opioids are neurotoxic in preterm infants. In term infants undergoing TH, the long-term effects of morphine exposure are unknown. We examined the effect of opioid administration during TH on neurodevelopmental outcome and time to extubation after sedation ended. Methods: In this prospectively collected population-based cohort of 282 infants with HIE treated with TH (2007À2017), the cumulative opioid dose of morphine and equipotent fentanyl (10À60 mg/kg/h) administered during the first week of life was calculated. Clinical outcomes and concomitant medications were also collected. Of 258 survivors, 229 underwent Bayley-3 neurodevelopmental assessments of cognition, language and motor function at 18À24 months. Multivariate stepwise linear regression analysis was used to examine the relation between cumulative opioid dose and Bayley-3 scores. Three severity-groups (mildmoderate-severe) were stratified by early (<6 h) amplitude-integrated electroencephalography (aEEG) patterns. Findings:The cumulative dose of opioid administered as SA during TH was median (IQR) 2121 mg/kg (1343, 2741). Time to extubation was independent of SA dose (p > 0.2). There was no significant association between cumulative SA dose and any of the Bayley-3 domains when analysing the entire cohort or any of the aEEG severity groups. Interpretation: Higher cumulative opioid doses in TH-treated infants with HIE was not associated with worse Bayley-3 scores at 18À24 months of age. Funding: The Bristol cooling program was funded by the Children's Medical Research Charity SPARKS managing donations for our research from the UK and US, the UK Moulton Foundation, the Laerdal Foundation for Acute Medicine in Norway and the Norwegian Research Council (JKG).

We study the effect of hypothermia (HT) following hypoxic-ischemic (HI) brain injury in postnatal day 7 (P7) rats. In 2015, new European Union animal transport regulations prompted a change in practice at the breeding facility, which henceforth crossfostered P3 litters to P8 older lactating dam prior to transportation. It is generally assumed that crossfostering does not significantly affect the experimental results. The aim of this study was to examine whether crossfostering affects our model consistency by modifying injury susceptibility and hypothermic neuroprotection. We analysed 219 pups (56 litters) from 11 experiments conducted between 2013 and 2015: 73 non-crossfostered and 146 crossfostered pups. At P7, all pups underwent unilateral common carotid artery ligation followed by 50min of hypoxia (8% O2, 36°C). Immediately after this mild insult, the pups were randomised to post-insult normothermia (NT) or HT treatment. Pups were culled at P14. Injury was assessed by area loss of the ipsilateral hemisphere and histopathology scoring of hippocampus, cortex, thalamus, and basal ganglia. Crossfostered pups had double the injury compared to non-crossfostered pups irrespective of treatment group. Hypothermic neuroprotection was statistically significant, but with a smaller and less consistent effect in crossfostered pups (relative neuroprotection 16% vs. 31% in non-crossfostered). These results demonstrate hypothermic neuroprotection following a mild HI insult. A representative subset of 41 animals were also assessed for evidence of microglial reactivity, however no detectable difference in microglial reactivity was observed between any of the groups. In conclusion, crossfostering alters outcomes in our established model through reduced insult tolerance and variable neuroprotection. Crossfostering as a common breeding practice is a largely unexplored variable in animal research that may result in invalid research conclusions if inadequately adjusted for by larger group sizes. As a result, crossfostering is likely to be inconsistent with the principles of replacement, reduction, and refinement.