Handling mice before surgery sounds like an obvious way to make recovery gentler. But in this study, habituation sometimes increased weight loss—and its biggest effect was not the average mouse, but how different the mice were from one another.

Intravital microscopy enables real-time, high-resolution imaging of cellular and molecular processes in living organisms under physiological conditions. Advances in chronic imaging window technology have transformed intravital microscopy into a powerful approach for repeated, longitudinal observation of the same tissue over days to weeks.

Abdominal imaging windows enable longitudinal visualization of internal organs, but they require surgical access to the peritoneal cavity and placement of the window in direct contact with visceral tissues. For the kidney abdominal imaging window, implantation involves opening the peritoneal cavity and positioning the window against the renal capsule.

The invasiveness of this procedure raises important questions about post-operative recovery and physiological stress. Surgical procedures activate the hypothalamic–pituitary–adrenal axis and the sympathetic nervous system, leading to glucocorticoid and catecholamine release with direct consequences for immune function, leukocyte trafficking, and tissue inflammation.

Acute pre-operative stress has been shown to increase leukocyte infiltration at surgical implant sites by two hundred to three hundred percent. Uncontrolled surgery-related stress and the resulting immune activation can compromise the validity of data collected using these models.

Habituation is repeated, non-threatening pre-exposure to handling, and it is a well-established refinement strategy that lowers baseline stress and anxiety in laboratory mice. Repeatedly handled animals show attenuated behavioral and physiological stress responses, lower corticosterone levels, and reduced discomfort during experimental procedures compared to non-habituated animals.

It remained unclear whether pre-operative habituation extends its beneficial effects to the post-operative period following invasive procedures such as abdominal imaging window implantation. Post-operative housing condition represents an additional factor that may influence recovery and experimental outcomes.

Social housing is generally considered beneficial for laboratory mice and has been associated with improved welfare following standard surgical interventions. Single housing may protect the implant but impose isolation stress, while group housing may support welfare but risk mechanical complications.

Biological sex is an important determinant of stress responsiveness, hypothalamic–pituitary–adrenal axis activity, and immune regulation, with documented differences between males and females in glucocorticoid responses and leukocyte dynamics. The study included both male and female mice in equal numbers and performed sex-stratified analyses throughout.

Despite the growing use of chronic imaging windows, relatively little systematic information is available on post-operative health and recovery in these models. Studies have focused primarily on surgical feasibility, technical optimization, and imaging performance, with only incidental or qualitative observations of post-operative animal condition.

Studies have rarely characterized post-operative welfare trajectories, quantified endocrine and immune responses to implantation, or evaluated how peri-operative management influences recovery. The study evaluated pre-operative handling habituation and post-operative housing condition, individually and in combination, across both sexes, in BALB/c mice following kidney abdominal imaging window implantation.

The assessed outcomes were body weight, clinical health scores, peripheral leukocyte counts, urinary corticosterone levels, and renal immune cell infiltration over eight post-operative days. Sex-stratified analyses were performed throughout to determine whether responses to habituation and housing differed between male and female mice.

Twenty-four BALB/cAnNCrl mice of both sexes and eight weeks of age were used, group-housed in threes, and allowed one week of acclimatization without handling before experimental procedures. Following surgery, half of the mice were housed individually, while the remaining mice were returned to their home cages and maintained in groups of three.

Mice were observed and scored twice daily for the first three post-operative days, then once daily thereafter, and were weighed daily for five days and on day eight after surgery. Two females were excluded from all analyses because of surgical complications, so subsequent analyses used twenty-three animals per habituation group and twenty-three per housing group.

The study used four experimental groups crossing habituated or non-habituated status with individually or group-housed conditions, multiple biological endpoints, and repeated measurements per animal. The study was powered to detect effects of biological and practical relevance—effects large enough to justify implementing these welfare measures in a research setting.

An effect size of zero point three, a two-sided significance level of zero point zero five, and eighty percent power were selected for the calculation. This yielded a minimum of six to nine animals per group and supported the use of twelve animals per group, forty-eight animals in total, balanced for sex across all groups.

Mice were anesthetized with two to three percent Isoflurane, and the surgical site was disinfected with chlorhexidine. A small opening was made in the peritoneum, the kidney was gently exteriorized, and a thin ring of gel cyanoacrylate adhesive outlined the imaging area.

The abdominal imaging window was placed onto the kidney and held firmly for approximately sixty seconds until the adhesive had cured. Buprenorphine was administered immediately before surgery for preemptive analgesia and repeated on postoperative days one and two. Forty-eight BALB/c mice, twenty-four males and twenty-four females, were allocated to four experimental groups in a two-by-two design crossing habituation status with post-operative housing condition.

An approach test showed that habituated mice approached the stationary experimenter's hand more frequently and had a shorter latency to first approach than non-habituated mice. Individually housed mice showed higher accumulated health scores than group-housed mice and also exhibited greater variance, indicating a higher overall post-operative burden and increased inter-individual variability.

Females exhibited greater variance in accumulated health scores than males despite similar mean values, while increased score variability was observed in individually housed animals of both sexes. Although no overall effect of habituation was detected, sex-stratified analysis revealed a clear sex-specific response in post-operative body weight trajectory.

In females, weight trajectories were comparable irrespective of habituation or housing condition. Habituated males exhibited greater post-operative weight loss than non-habituated males, with differences emerging on postoperative days three to five in single-housed animals and on days three to four in group-housed animals.

Habituation did not affect weight regain in females, whereas habituated group-housed males regained body weight later than non-habituated group-housed males. Together, these findings indicate that pre-operative habituation delayed post-operative weight recovery specifically in males.

Before surgery, peripheral blood leukocyte counts did not differ between habituated and non-habituated mice, indicating that the two-week habituation protocol did not alter baseline peripheral leukocyte counts. After surgery, group-housed mice showed a decrease in peripheral leukocyte counts on postoperative day two followed by recovery toward baseline by day eight.

In contrast, habituated single-housed mice showed no detectable postoperative decrease in leukocyte counts, while inter-individual variability on postoperative day two was markedly increased. Following normalization to each animal's own pre-surgical baseline, habituation had little effect on mean postoperative leukocyte counts.

In group-housed animals, habituation reduced inter-individual variability on postoperative day two, whereas in single-housed animals it increased inter-individual variability on day two and, in females, also on day eight. In the normalized plots, the clearest variance contrast appears in the leftmost all-mouse panel: the day-two error bars are much tighter for the non-habituated group than for the habituated group.

The female panel shows a similar spread difference, while the male panel has broad error bars in both groups, making the comparison less visually distinct. Urinary corticosterone levels, normalized to creatinine, were measured one day before surgery and on postoperative days two and seven.

Females exhibited higher baseline corticosterone levels and greater inter-individual variability than males. Habituation primarily affected inter-individual variability rather than mean corticosterone levels. At baseline, habituation increased variability in females and reduced it in males without affecting mean levels.

In panel A, the error bars show the spread changing in opposite directions: females expand from roughly six hundred to two thousand six hundred, while males contract from about one thousand two hundred to three hundred. Panel B shows the sex contrast visually: the female mean is near nine hundred in non-habituated animals and about one thousand five hundred in habituated animals, versus male means near one hundred.

Pre-operative habituation modified post-operative immune and endocrine responses in a sex- and housing-dependent manner, affecting both response magnitude and inter-individual variability. Peri-operative management altered endocrine and immune responses in a manner that depended on both sex and the post-operative housing environment, rather than producing a uniform improvement in post-operative outcomes.

Across the different physiological endpoints examined, the most consistent effect was a change in inter-individual variability rather than a shift in average responses, particularly in peripheral leukocyte counts and urinary corticosterone. Peri-operative refinement should therefore be considered not only as a means of improving animal welfare but also as an experimental factor that shapes endocrine and immune responses measured in intravital microscopy studies.

Across nearly all endpoints examined, including post-operative health scores, peripheral leukocyte counts, urinary corticosterone, and renal CD68-positive cell infiltration, the most consistent effect of habituation was a change in inter-individual variability rather than group means.

Under group-housed conditions, habituated mice exhibited more homogeneous post-operative leukocyte and corticosterone responses, together with reduced variability in CD68-positive cell infiltration of the operated kidney. Thus, habituation did not primarily alter the average physiological response to surgery.

In the top row, the CD68 male panel shows a marked left–right separation for non-habituated mice, with the operated-kidney point near twenty-five objects per square millimetre versus roughly three on the contralateral side. In the bottom row, the CD74 male panel shows overlapping left and right values, with a grey p-value of zero point five one one zero.

All experiments were performed in BALB/c mice, an inbred strain with a comparatively hyperreactive hypothalamic–pituitary–adrenal axis, so the magnitude and potentially the direction of the observed effects may differ in other strains such as C57BL/6. The observation period was limited to eight post-operative days, and it remains unknown whether the variance-reducing effects of habituation persist over longer periods.

Renal immunohistochemistry was based on serial tissue sections from a limited number of animals rather than whole-organ imaging, and mechanistic analyses such as cytokine profiling or flow cytometry were not performed. Some sex-stratified comparisons were based on relatively small subgroup sizes and should therefore be interpreted with appropriate caution.

The central lesson is that habituation did not produce one universal recovery benefit. Its effects depended on sex and housing, and it most consistently changed inter-individual variability—an outcome that matters for both welfare and reproducible imaging experiments.