A large proportion of IC/BPS patients complain of pain in the pelvic region and extra pelvic areas of the body [1]. The stimulation of T12-L2 and S4-S5 dermatomes during quantitative sensory testing (QST) revealed that patients may present different sensory responses to mechanical and thermal stimulation, varying from gain of response to loss of response [3,4]. Gain of function suggests sensitisation of peripheral nociceptors and/or central pain pathways modification. In addition, patients with widespread forms of pain present more depression traits, anxiety, and insomnia [2]. Also, urinary frequency is a characteristic symptom of many IC/BPS patients. The existence of different phenotypes among IC/BPS patients generates obvious difficulties in the choice of animal models and the interpretation of the findings. In short fine-tuning of available animal models is necessary.
In the present work, we aim to (1) understand to which extent animals submitted to the IC/BPS, non-bladder centric, stress models mimic IC/BPS symptoms and (2) if individual observation of animals submitted to IC/BPS complex models reproduces the phenotypes observed in patients.

Naïve adult female Wistar rats (Control group), adult female Wistar rats submitted to Water Avoidance Stress (WAS group), and adult female Wistar rats submitted to Maternal Deprivation Model (MDM) were used (6 animals/group). The WAS was induced by putting the adult animal on the top of a pedestal standing in the middle of a box full of water, for one hour, for 10 consecutive days. The MDM was induced by separating the pups from their mother and littermates, for 1h, from P2 to P15.
Animals from all groups were tested for mechanical and thermal sensitivity and depression-related cognitive impairment at the age of 6-7 months. 
Mechanical sensitivity was assessed using the Von Frey up-and-down paradigm. The sensitivity in the L3-L5 (extra pelvic sensitization) and the L6-S1 dermatomes (pelvic sensitization) was tested. The outcome measurement was the mechanical sensitivity threshold inducing a response in 50% of the animals (MT50, g).
Thermal sensitivity was estimated using the Hargreaves test. The outcome measurement was the latency (time in seconds, LT) of response to the thermal stimulus.
To determine the presence of depression-related cognitive impairment, the recognition index (RI – depression-triggering cognitive dysfunction, %) was evaluated through the Novel Object Recognition (NOR) test.
Data are presented as mean +/- standard deviation (normal distributed population -Shapiro-Wilk test; one-way ANOVA followed by Holm-Šídák's multiple comparisons test was used to compare groups) or as the median and interquartile range (IQ1, IQ3) (not normal distributed population -Shapiro-Wilk test; Kruskal-Wallis test followed by Dunn's multiple comparisons test was used to compare groups).
For individual analysis of females from the WAS and MDM groups, their outcome was considered normal if within the minimal and maximal values of the control group outcome. If not, the outcome from the individual females from WAS and MDM groups was considered as the gain or loss of function.

When the L6-S1 dermatomes were mechanically stimulated, the median MT50 of the control, WAS and MDM groups were 60 (37,60) g, 5.5 (0.53, 8.5) g and 7.7 (1.9, 29) g respectively. The MT50 of the WAS group, but not of the MDM group (p=0.1372), was statistically different from the control (p=0.0082). Upon thermal stimulation in the L6-S1 dermatomes, the media LT of the control, WAS and MDM groups were 16 (15, 17) seconds, 12 (11, 16) seconds and 7.9 (5.3, 16) seconds, respectively. The LT of the MDM group, but not of the WAS group (p=0.2202), was statistically different from the LT of the control group (p=0.0344).
When mechanical stimulation of the L3-L5 dermatomes was performed, the mean MT50 of the control, WAS and MDM groups were 19 +/- 8.0 g, 14 +/- 9.9 g and 21 +/- 12 g, respectively. Neither group MT50 differed from the control group (p=0.6942 and p=0.7579, respectively). The thermal stimulation of L3-L5 dermatomes revealed that the control, WAS and MDM groups had a median LT of 18 (14,18) seconds, 17 (16,18) seconds and 9.8 (15,16) seconds respectively. Neither group's median LT value differed from the LT of the control group (p > 0.9999 and p = 0.0831, respectively).
Individual analysis of animals from the WAS group revealed that, of the 6 animals tested, two (33%) showed pelvic mechanical hypersensitivity, one (16.5%) showed pelvic thermal and mechanical hypersensitivity, two (33%) presented pelvic thermal and mechanical hypersensitivity and extra pelvic mechanical hypersensitivity, and one (16.5%) showed pelvic thermal hypersensitivity and extra pelvic mechanical hyposensitivity.
Individual analysis of animals from the MDM group revealed that of the 6 animals tested,  two (33%) had pelvic thermal and mechanical hypersensitivity, one (16.75%) had pelvic thermal and mechanical hypersensitivity and extra pelvic mechanical hypersensitivity, one (16.75%) showed pelvic mechanical hypersensitivity and extra pelvic thermal hypersensitivity, one (16.75%) had extra pelvic thermal hypersensitivity, and one (16.75%) had pelvic thermal hypersensitivity and extra pelvic mechanical hyposensitivity. 
When in the NOR arena, the novel object RI of the control, WAS and MDM groups were 57 +/- 16 %, 41 +/- 15 % and 56 +/- 9.3 %, respectively. The RI of the WAS and MDM groups did not differ from the control group (p=0.1656 and p=0.9095, respectively). 
Individual analysis of animals from the WAS group revealed that, of the 5 animals tested, 3 animals had lower RI than the controls. Individual analysis of animals from the MDM group revealed that, of the 6 animals tested, all had an RI similar to the control group.

Like in patients, WAS and MDM rats show phenotypes that may be blurred when analysed as a group. The WAS and MDM groups did not show mechanical and thermal sensitization when the L3-L5 dermatomes were stimulated. When the L6-S1 dermatomes were stimulated, the WAS group showed mechanical sensitisation and the MDM group showed thermal sensitisation.
The analysis of each individual in the WAS and MDM groups revealed that animals presented multiple mechanical and thermal sensitive phenotypes. These identified phenotypes might be relevant for the grouping of experimental animals in future pharmacological studies, which might provide additional information concerning the response to drugs.
The mechanical hyposensitivity and hypersensitivity found in L3-L5 dermatomes of some females raise the question of how QST analysis should be performed in patients. In some QST paradigms, an extra pelvic region is used as the control for the responses in the pelvic region of an individual. If patients have hyposensitivity and hypersensitivity in L3-L5 dermatomes, this may lead to false positive and false negative responses in the tested dermatomes, respectively.
The depression-related cognitive impairment observed in some animals was not associated with a specific mechanical or thermal threshold, showing that these two traits are not correlated.

Female rats submitted to WAS and MDM present different phenotypes resembling the heterogeneous phenotypes found among IC/BPS patients. Those phenotypes might be overlooked if group analysis is the only method used. The characterization of individual animals and their subdivision into specific subgroups should be used in future drug/biomarker/pathophysiological studies. The future determination of the studies’ sample size should take into consideration the existence of such phenotypes as they might influence the robustness of the outcomes.

[1] PMID: 22633363
[2] PMID: 25092637 [3] PMID: 28628232
[4] PMID: 37289573

Continence 12S (2024) 101492
DOI: 10.1016/j.cont.2024.101492