Habituation and housing interact to shape the magnitude and consistency of post-operative recovery after kidney imaging window implantation in mice

Abstract Abdominal imaging windows enable longitudinal intravital microscopy but require surgical implantation, which induces physiological stress and may influence experimental outcomes. Although pre-operative habituation is widely used as a refinement strategy, its effects on post-operative physiological responses remain incompletely understood. Here, male and female BALB/c mice underwent kidney abdominal imaging window implantation following either a two-week handling habituation protocol or standard husbandry and were housed either in groups or individually after surgery. Post-operative recovery was assessed using clinical health scores, body weight, peripheral leukocyte counts, urinary corticosterone, and renal immunohistochemistry. Habituation altered both the magnitude and the consistency of post-operative physiological responses. Mean effects were primarily observed in males, including greater transient post-operative weight loss, delayed body weight recovery, and reduced urinary corticosterone concentrations. Across immune and endocrine endpoints, however, the predominant effect was a change in inter-individual variability. Under group-housed conditions, habituated mice showed reduced variation in leukocyte counts, urinary corticosterone concentrations, and renal CD68-positive cell infiltration, whereas these effects were absent or reversed in individually housed animals. Females were affected predominantly through changes in inter-individual variability. These findings demonstrate that pre-operative habituation reshapes the physiological response to surgery in a manner that depends on both post-operative housing and sex. More broadly, they identify inter-individual variability as a biologically meaningful outcome of peri-operative refinement, suggesting that refinement strategies should be evaluated not only by their effects on average physiological responses but also by their ability to promote more consistent responses across individual animals.