Deleterious Effect of Crossfostering in Rat Pups on Hypoxic-Ischaemic Injury Tolerance and Hypothermic Neuroprotection

Abstract

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.

Restraint stress during neonatal hypoxia‐ischemia alters brain injury following normothermia and hypothermia

“…We controlled for this by matching the non‐probe controls to each skin and rectal probe. Moreover, 34% of the litters analyzed were crossfostered by an older dam, which we recently reported to exacerbate HI injury in a mild Vannucci model (Gundersen, Menassa, et al, 2021 ). However, in the current study we found no significant difference between crossfostered and non‐crossfostered rats, presumably due to the longer insult duration (90 min) diminishing this effect.…”

“…To achieve a sufficient sample size of probe rats, we collected data from 23 experiments previously published and conducted in the Oslo laboratory between 2013 and 2016 (Wood et al, 2020 ). Thirty‐four percent of the litters were crossfostered (Gundersen, Menassa, et al, 2021 ). Each experiment was conducted on a set of five or six litters of ten pups each exposed to hypoxia in one large chamber with 60 individual partitions.…”

“…Histopathology is the gold standard for assessing HI injury. The regional injury was assessed in the hippocampus (CA1‐CA4 and gyrus dentatus), cortex, thalamus and basal ganglia using a validated 9‐point (0.0–4.0) histopathology score as previously described (Gundersen, Menassa, et al, 2021 ; Thoresen, Bagenholm, et al, 1996 ). The grade was decided by regional morphological changes of necrotic neurons, complete or incomplete infarction and distribution of injury and tissue disintegration.…”