Can the adrenergic system be implicated in the pathophysiology of bladder pain syndrome/interstitial cystitis? A clinical and experimental study

Abstract

Aims: To evaluate sympathetic system activity in bladder pain syndrome/interstitial cystitis (BPS/IC) patients and to investigate if chronic adrenergic stimulation in intact rats induces BPS/IC-like bladder modifications.

Methods: Clinical study--In BPS/IC patients and aged and body mass index matched volunteers TILT test was undertaken and catecholamines were measured in plasma and 24 hr urine samples. Experimental study--Phenylephrine was injected subcutaneously (14 days) to female Wistar rats. Pain behavior, spinal Fos expression, urinary spotting, number of fecal pellets expelled, frequency of reflex bladder contractions, and urothelial height were analyzed. Urothelium permeability was investigated by trypan blue staining. Immunoreactivity against caspase 3 and bax were studied in the urothelium and against alpha-1-adrenoreceptor and TRPV1 in suburothelial nerves. Mast cell number was determined in the sub-urothelium. In rats with lipopolysaccharide-induced cystitis, urinary catecholamines, and Vesicular Monoamine Transporter 2 (VMAT2) expression in bladder nerves were analyzed.

Results: The TILT test showed an increase of sympathetic activity. Noradrenaline levels in blood at resting conditions and in 24-hr urine samples were higher in BPS/IC patients. Phenylephrine administration increased visceral pain, spinal Fos expression, bladder reflex activity, urinary spotting and the number of expelled fecal pellets. The mucosa showed urothelial thinning and increased immunoreactivity for caspase 3 and bax. Trypan blue staining was only observed in phenylephrine treated animals. Suburothelial nerves co-expressed alpha1 and TRPV1. Mastocytosis was present in the suburothelium. Cystitis increased sympathetic nerve density and urinary noradrenaline levels.

Conclusions: Excessive adrenergic stimulation of the bladder may contribute to the pathophysiological mechanisms of BPS/IC.

Keywords: bladder pain syndrome/interstitial cystitis; primary afferents; sympathetic system; urothelium.

Fig 1

(A) Graph bar showing test 1 mean score. After chronic adrenergic stimulation with 2.5 mg of PHE/kg body weight, animals presented a decrease in breathing rate, in eye aperture and rounded-back posture characteristic of pain-related behaviour. Pre-treatment with capsaicin abolished visceral pain behavior (*** P < 0.001 comparing day 8 or day 14 with day 0, Kruskal-Wallis test followed by Dunn’s test). (B) Graph bar showing test 2 mean score. After chronic adrenergic stimulation with 2.5 mg of PHE/kg body weight, animals presented strong piloerection, heavy breathing and abdomen stretched characteristic of visceral pain crisis. Pre-treatment with capsaicin abolished visceral pain crisis (P>0.05, comparing day 8 and day 14 in both tests, P>0.05 comparing day 0 with day 8 and day 14 of animals pre-treated with capsaicin, *** P < 0.001 comparing day 8 or day 14 with day 0 of PHE treated animals, in both test, Kruskal-Wallis test followed by Dunn’s test). (C) Graph bar showing lower abdominal mechanical pain threshold using Von Frey filaments. After chronic adrenergic stimulation with 2.5 mg of PHE/kg body weight, animals presented a decrease in the mechanical pain threshold (*** P < 0.001 comparing day 0 with day 8 and day 14, Kruskal-Wallis test followed by Dunn’s test). Pre-treatment with capsaicin abolished abdominal mechanical pain threshold.

Fig 2

(A) Bar graph showing a dose dependent increase in the mean number of bladder reflex contractions induced by saline (vehicle), 0.5 mg PHE/Kg of body weight (PHE0.5), 2.5 mg PHE/Kg of body weight (PHE2.5), 2.5 mg PHE/Kg of body weight following capsaicin pre-treatment (CAP+PHE2.5). (B) Bar graph shows that PHE dose-dependently increased the mean number of Fos immunoreactive cells/section at L6 spinal cord segment after 2h of cystometry. Pre-treatment with capsaicin abolished PHE-induced Fos immunoreactivity (P>0.05, comparing vehicle with CAP 75 mg/kg + PHE 2.5 mg/kg, *** P < 0.001 comparing 0.5 mg of PHE /kg body weight or 2.5 mg of PHE /kg body weight with vehicle, Kruskal-Wallis test followed by Dunn’s test).

Fig. 3

(A) Micrograph of a typical control bladder and of a typical area of urothelial thinning following administration of 2.5 mg PHE/Kg body weight, subcutaneously, daily for 14 days. (B) Micrograph showing intense cytokeratin 20 immunolabelling in the umbrella cells of the urothelium of control animals. (C) Micrograph showing areas of decrease or absent cytokeratin 20 immunolabelling (white arrows head) following administration of 2.5 mg PHE/Kg body weight, indicating a loss of umbrella cells. (D) Micrograph showing immunolabelling of the active form of caspase 3 in the urothelium after administration of 2.5 mg PHE/Kg body weight. (E) Micrograph showing bax immunolabelling in the urothelium after administration of 2.5 mg PHE/Kg body weight. (F) Micrograph showing co-localization of alpha 1 adrenoreceptor and TRPV1 imunnolabelling in suburothelial nerve fibres of control animals.

Fig. 4

Cystitis induces sympathetic fiber sprouting. Bar graph showing the mean number of VMAT2-immunopositive fiber length/mm², in (A) suburothelium of the dome, the body and trigone and in the (B) muscular layers of the dome, body and trigone, of both vehicle and 2 mg/kg of LPS treated animals. (*P<0.05, comparing sham suburothelium dome with LPS suburothelium dome, **P<0.01 comparing sham suburothelium body with LPS suburothelium body, and sham muscular dome with LPS muscular dome, ***P<0.001 comparing sham muscular body with LPS muscular body, Kruskal-Wallis test followed by Dunn’s test). Micrographs representative of sections from the dome immunoreactive for VMAT2 are shown in C-F. (C) Bladder suburothelium of vehicle treated animals. (D) Bladder suburothelium of LPS treated animals. (E) Bladder muscular layer of vehicle treated animals. (F) Bladder suburothelium layer of LPS-treated animals.