Hypothesis / aims of study
Animals models are fundamental for IC/BPS investigation. The water avoidance stress (WAS) test has been used with this purpose [1,2], but the influence of animal age was never evaluated despite the fact that IC/BPS phenotype is influenced by patients age, BPS prevailing in younger and IC in older women [3]. Here, we investigated the influence of rats age in the outcomes of several tests carried out in the WAS model.
Study design, materials and methods
Adult (6M), mature (15M) and old (23M) female Wistar rats (6 animals/group) were submitted to WAS, in which animals stand on a podium in a box full of water, 1h for 10d. Aged-matched naïve females standing in the podium in a waterless box were used as controls.
Before-after design was used to study pain (grimace and Von Frey test), and bladder function (voiding spot assay - VSA). Between-subjects design was used to evaluate anxiety/coping mechanism (marble burying) and bladder reflex activity (cystometry under anesthesia).
Data are presented as mean+/-sd or as the median (IQ1, IQ3). Paired t-test, unpaired t-test, Wilcoxon matched pairs test, Ordinary one-way ANOVA followed by uncorrected Fisher's LSD and Fisher's exact test were used.
Results
WAS increased the grimace score of adults, but not mature and old rats (Figure 1).
WAS caused sensitization of L6-S1 dermatomes only in the 6M group and did not affect L3-L5 dermatomes in any group.
WAS induced bladder hyperactivity in the 6M group when compared to controls (0.82+/-0.11 vs 0.48+/-0.13), but not in the 15M or 23M groups.
Compared to baseline WAS did not change the mean number of the small-volume voided spots (inferior to the baseline mean). However, there was a trend to an increase in the number of small spots in the 6M group.
After WAS, the 6M group, but not the 15M or 23M groups, buried more marbles than their controls (Figure 2).
Interpretation of results
Changes in the grimace score were only slightly visible in the 6M group (score closer to 0 than to 1). However, the before-after analysis allows individual changes to discriminate with a very large effect size (Cohen's d 1.71) suggesting that WAS induces pain behavior. The Von Frey test is very sensitive to WAS as the difference between before-after measurements was extremely large (Cliff’s d 7.10).
WAS induced bladder hyperactivity indicating that the model also reflets the urinary changes present in patients. Although showing a trend towards an increase in the number of urinary spots, the lack of statistical differences in the mean number of spots may be related to the short period of analysis (20 minutes instead of the 30-60 minutes described by others).
The increase in the number of marbles buried by females of the 6M group may indicate that WAS induces an anxious behavior or a coping mechanism due to pain and bladder hyperactivity.
The apparent lack of effect of WAS in females of 15M and 23M groups in all tests used may be associated with the limited influence of stress in older animals and should be taken in consideration when using this model.
Concluding message
WAS only reflected IC/BPS phenotype (pain, bladder activity and anxious behavior) in 6M female rats, suggesting that age related analysis may be better reflected by other models (e.g.: cyclophosphamide model).
Due to the slight variation in the grimace test, it remains unclear if this test is useful to study the pathophysiological mechanisms of IC/BPS or to investigate the analgesic effect using WAS as a paradigm. Also, a refinement of the VSA test, in particular the duration of the observation period, is necessary before using it with WAS paradigm.