Study design, materials and methods
MDM was induced on female C57BL/6 by separating pups from mother and from littermates, for 1h, from P2 to P15 (MDM WT). Each separated animal was maintained on a heat blancket. Experiments were performed at P156. Non separated pups (wildtype, WT) were used as controls.
Mechanical pain threshold was analysed at P156. Briefly, animals were placed in individual chambers (23.0 × 17.0 × 14.0 cm3) with a wire mesh floor and allowed to acclimate. This test was performed in the lower abdomen and consists in touching this region with one of a series of Von Frey monofilaments (rated at 0.008, 0.02, 0.04, 0.07, 0.16 g, 0.40 g). Filaments were applied perpendicularly with enough strength to cause the monofilament to slightly bend. Each monofilament was tested five times, 5 s interval between each application. It was considered a positive response when the animal reacted to the filament (abdomen retraction or jump) in at least three of the five filament applications. In case of no response to the filament, the next-stronger monofilament was applied within an interval of 30 s.
At P157, animals were anaesthetised with urethane (1.2g/kg body weight) laid on a heating pad with a rectal probe underneath their body to measure body temperature in order to maintain it at 37 ºC. The bladder was then exposed through a low abdominal incision. A needle was inserted in the bladder dome and saline was infused (1.8 ml/h). After a stabilization period of 30 minutes, the cystometrograms were recorded for 10 minutes. Cystometrograms were analysed and bladder reflex activity was expressed as the number of bladder contractions per minute.
Afterwards, the bladders were harvested, fixed, dehydrated, embedded in paraffin and sectioned at 10 micrometres. Bladder sections were stained with Hematoxylin-eosin to analyse urothelium integrity and with Toluidine blue to analyse mast cell presence.
MDM was repeated on female TRPV1 knockout (KO) mice (MDM TRPV1 KO) to investigate the role of TRPV1 nociceptors in this model. Non separated TRPV1 KO pups (TRPV1 KO) were used as controls.
Wildtype (WT) mice had a lower abdominal pain threshold of 0.08±0.01g. MDM WT animals had a decrease in their threshold to 0.02±0.01 (P=0.0002). TRPV1 KO and MDM TRPV1 KO mice had similar lower abdominal pain threshold (0.13±0.18g and 0.28±0.14g respectively; P=0.2).
WT mice presented 0.48±0.15 bladder contractions/minute while MDM WT had 1.05±0.35 contractions/minute (P=0.04). TRPV1 KO presented 0.50±0.12 bladder contractions/minute and MDM TRPV1 KO mice had 0.50±0.08 bladder contractions/minute.
The bladder of WT, TRPV1 KO and MDM TRPV1 KO mice presented normal urothelium. However, MDM WT animals presented urothelial disruption in 7% of the urothelial surface.
In the present experiment, the bladder wall of WT, MDM, TRPV1 KO and MDM TRPV1 KO mice did not present mastocystosis.
Interpretation of results
The results of the present work showed that MDM is a good model to study some aspects of BPS/IC, as MDM WT mice showed some of the bladder changes observed in BPS/IC, such as pain signs, increase number of voiding contractions and a mild urothelial disruption.
Also, since MDM TRPV1 KO mice did not present the changes observed in MDM WT mice, the results indicated a role for TRPV1-expressing nociceptive fibres in the development of the observed pain and bladder changes.