The deleterious effect of crossfostering in rat pups on hypoxic-ischemic injury tolerance and hypothermic neuroprotection

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.

Integrating Human and Non-Human Primate Data to Estimate Human Tolerances for Traumatic Brain Injury

Traumatic brain injury (TBI) contributes to a significant portion of the injuries resulting from motor vehicle crashes, falls, and sports collisions. The development of advanced countermeasures to mitigate these injuries requires a complete understanding of the tolerance of the human brain to injury. In this study, we developed a new method to establish human injury tolerance levels using an integrated database of reconstructed football impacts, sub-injurious human volunteer data, and non-human primate data. The human tolerance levels were analyzed using tissue-level metrics determined using harmonized species-specific finite element brain models. Kinematics-based metrics involving complete characterization of angular motion (e.g., DAMAGE) showed better power of predicting tissue-level deformation in a variety of impact conditions and were subsequently used to characterize injury tolerance. The proposed human brain tolerances for mild and severe TBI were estimated and presented in the form of injury risk curves based on selected tissue-level and kinematics-based injury metrics. The application of the estimated injury tolerances was finally demonstrated using real-world automotive crash data.

Effect of Ethylene-Insensitive Mutation etr2b on Postharvest Chilling Injury in Zucchini Fruit

Zucchini is a vegetable fruit that is very susceptible to postharvest chilling injury, and fruit ethylene production is correlated with chilling injury sensitivity, such that the more tolerant the cultivar, the lower is its ethylene production. It is expected that zucchini fruit with reduced sensitivity to ethylene would have a higher chilling injury tolerance. In this study, we compared the postharvest fruit quality of wild type and ethylene-insensitive mutant etr2b, in which a mutation was identified in the coding region of the ethylene receptor gene CpETR2B. Flowers from homozygous WT (wt/wt), mutant plants in homozygous (etr2b/etr2b) and heterozygous (wt/etr2b) were hand-pollinated, and all fruits were harvested with the same length, at about 8 days after pollination. After harvesting, fruit of each genotype was randomly divided in 3 batches of 12 fruits each (four replications with three fruits each), and then stored at 4 °C and 95% RH. At 0, 7, and 14 days after cold storage, each batch was used to assess ethylene production, respiration rate, weight and firmness loss, chilling injury, and oxidative stress metabolites. The results showed a lower chilling injury associated with lower cold-induced ethylene production in the mutant fruit, in comparison with the WT fruit. These data demonstrated that the ethylene-insensitive etr2b mutant fruit was more tolerant to chilling injury, confirming that basal ethylene in the still undamaged fruit could function as a modulator of post-harvest chilling injury. Moreover, the higher chilling tolerance of the etr2b mutant fruit was not associated with MDA content, but was concomitant with a reduction in the accumulation of hydrogen peroxide in the refrigerated mutant fruit.