Water Avoidance Stress induces changes in hippocampal areas involved in pain perception

Santos-Pereira M1, Silva S1, Santos-Leite L1, Monteiro F2, Summavielle T3, Meijlink J4, Treede R5, Nagel J6, Vicent K7, Cruz F8, Charrua A8, on behalve of IMI PainCare group .9

Research Type

Pure and Applied Science / Translational

Abstract Category

Pelvic Pain Syndromes

Abstract 132
Science 1 - Pelvic Pain
Scientific Podium Short Oral Session 11
Thursday 18th September 2025
17:22 - 17:30
Parallel Hall 4
Animal Study Basic Science Painful Bladder Syndrome/Interstitial Cystitis (IC) Outcomes Research Methods
1. FMUP, 2. FMUP and I3S, 3. I3S, 4. International Painful Bladder Foundation, 5. Medical Faculty Mannheim of University of Heidelberg, 6. Merz, 7. Oxford University, 8. FMUP, RISE-HEALTH, 9. EU
Presenter
Links

Abstract

Hypothesis / aims of study
IC/BPS patients present changes in different areas of the brain, including increased neuronal activity in the hippocampus [1]. The hippocampus, particularly the CA1 subregion, has been implicated in the affective and motivational dimensions of pain perception [2].
In this study, we aimed to investigate whether the Water Avoidance Stress (WAS) model induces hippocampal changes that resemble those observed in IC/BPS patients.
Study design, materials and methods
Adult female Wistar rats (6 months old; n=6/group) underwent the WAS protocol, in which animals stood on a podium in a box filled with water for 1 hour per day over 10 consecutive days. Age-matched naïve females standing on a podium in a dry box served as controls.
A before-after design was employed to assess mechanical (Von Frey test) and thermal (Hargreaves test) pain thresholds, which were evaluated on day 11. 
Brain tissue was harvested on day 15 for RNA and protein analyses. A between-subjects design was used to analyze hippocampal mRNA expression of various markers (multi-plex real-time PCR; neuronal markers: c-fos, Pdyn, and Sst; glial markers: Iba-1, CD68, TNFα, Olig2; inflammatory marker: TNFα), as well as the morphology and activity (CD68+) of microglial (Iba1+) cells in the CA1 subregion (LASAF and ImageJ software).
Data are presented as mean ± SD [confidence interval]. 
Paired t-test, one-sample t-test, Log-rank (Mantel-Cox) test, and two-way ANOVA followed by Fisher's LSD were used for statistical analysis.
Results
Adult female Wistar rats exposed to WAS exhibited mechanical hyperalgesia (1.16 ± 0.50 vs. 0.66 ± 0.50, [95% CI: 0.44 – 1.69]; Figure 1A). Additionally, they exhibited significant thermal hyperalgesia, as evidenced by a reduction in withdrawal latency in the L6-S1 dermatome in the Hargreaves test (Log-rank test: χ² = 8.741, p = 0.0031; Figure 1B).

Animals subjected to WAS (n=3/group, preliminary data) showed elevated mRNA ex-pression of c-fos but no significant differences in mRNA levels of the interneuron markers Pdyn (p = 0.30) and Sst (p = 0.18) compared to controls, as determined by a one-sample t-test (Figure 2A). Furthermore, they exhibited increased levels of the microglial marker CCR2 but no significant differences in the mRNA levels of other microglial markers (Iba1: p = 0.20; CD68: p = 0.16), the astrocyte marker GFAP (p = 0.68), the oligodendrocyte marker Olig2 (p = 0.78), or the inflammatory mediator TNFα (p = 0.66) compared to con-trols (Figure 2 B, C).

WAS did not induce changes in the number of glial cells in the CA1 region (Figure 3A). However, CD68 expression showed a slight increase compared to controls (Figure 3B). Although the soma size remained similar to controls (Figure 3C), WAS induced a reor-ganization of microglial microfilaments (Figure 3D, grey line), but without significantly altering their terminal number or the area they occupied (Figure 3 E, F).
Interpretation of results
The sensitization of the L6-S1 dermatome suggests that the neural pathways conveying bladder sensory input to the central nervous system are also sensitized or more active.
In the hippocampus of WAS-exposed animals, increased neuronal activity occurred alongside microglial recruitment through a CCL2/MCP1-dependent pathway, suggesting that this brain area became more active following model induction.
The observed rearrangement of microfilaments and the slight increase in the active microglial cells suggest that these cells are in a primed state, making them more reactive to subsequent stimuli.
Concluding message
WAS induces mechanical and thermal hyperalgesia. This process is accompanied by alterations in the phenotype and activity of microglia in the CA1 region, raising the hypothesis that circuits involved in the affective-cognitive dimension of pain may be altered. Altogether, these data suggest that the WAS model partially reflects the sensitization observed in IC/BPS patients.
Figure 1 Outcomes of Von Frey test (A) and Hargreaves test (B) performed before and after the induction of WAS model in adult female rats.
Figure 2 Relative expression of genes classified as neuronal markers (A), glial cell markers (B), and inflammatory mediators (C) in the hippocampus of control vs. WAS animals, normalized by 2^−ΔΔCt.
Figure 3 Quantification of microglial morphology and activation in the CA1 region of the hippocampus in control and WAS animals.
References
  1. PMID: 34992450
  2. PMID: 19784080
Disclosures
Funding This project received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No. 777500. This joint undertaking receives support from the European Union’s Horizon2020 research and innovation program and EFPIA. The statements and opinions presented here reflect the authors’ views. IMI, the European Union, EFPIA, and any associated partners are not responsible for any use that may be made of the information contained herein. www.imi.europa.eu www.imi-paincare.eu Clinical Trial No Subjects Animal Species Rats Ethics Committee ORBEA-FMUP
03/07/2025 12:11:21