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Background: Hypothermia is neuroprotective after neonatal hypoxic-ischaemic brain injury. However, systemic cooling to hypothermic temperatures is a stressor and may reduce neuroprotection in awake pigs. We compared two experiments of global hypoxic-ischaemic injury in newborn pigs, in which one group received propofol-remifentanil and the other remained awake during post-insult hypothermia treatment., Methods: In both studies, newborn pigs were anaesthetised using halothane during a 45-min global hypoxic-ischaemic insult induced by reducing F io 2 and graded hypotension until a low-voltage <7 μV electroencephalogram was achieved. On reoxygenation, the pigs were randomly allocated to receive 24 h of normothermia or hypothermia. In the first study ( n =18) anaesthesia was discontinued and the pigs' tracheas were extubated. In the second study ( n =14) anaesthesia was continued using propofol and remifentanil. Brain injury was assessed after 72 h by classical global histopathology, Purkinje cell count, and apoptotic cell counts in the hippocampus and cerebellum., Results: Global injury was nearly 10-fold greater in the awake group compared with the anaesthetised group ( P =0.021). Hypothermia was neuroprotective in the anaesthetised pigs but not the awake pigs. In the hippocampus, the density of cleaved caspase-3-positive cells was increased in awake compared with anaesthetised pigs in normothermia. In the cerebellum, Purkinje cell density was reduced in the awake pigs irrespective of treatment, and the number of cleaved caspase-3-positive Purkinje cells was greatly increased in hypothermic awake pigs. We detected no difference in cleaved caspase-3 in the granular cell layer or microglial reactivity across the groups., Conclusions: Our study provides novel insights into the significance of anaesthesia/sedation during hypothermia for achieving optimal neuroprotection., (© 2024 The Authors.)

We study the effect of hypothermia (HT) following hypoxic-ischaemic (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 dams 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 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 50 min of hypoxia (8% O2, 36°C). Immediately after this mild insult, the pups were randomized to post-insult normothermia or HT treatment. Pups were culled at P14. Injury was assessed by area loss of the ipsilateral hemisphere and histopathology scoring of the hippocampus, cortex, thalamus, and basal ganglia. Crossfostered pups had double the injury compared to non-crossfostered pups irrespective of the 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 was 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., (© 2021 The Author(s). Published by S. Karger AG, Basel.)