Hypothesis / aims of study
A relationship between chronic stress and bladder pain syndrome/ interstitial cystitis (BPS/IC) is well established. Patients with BPS/IC have symptom exacerbation during stress events while at experimental level the Water Avoidance Stress (WAS) was shown to increased bladder pain through a mechanism dependent of intense stimulation of alfa1A adrenoceptors. One other hand, clinical studies linked BPS/IC and urinary nerve growth factor (NGF). Recent meta-analysis showed that BPS/IC courses with increased levels of NGF in the urine and that treatment with antibodies anti-NGF have a beneficial symptomatic effect on in women with that condition.
In this study, we hypothesized that in animals subjected to chronic stress there is an increase in NGF expression and that the high affinity TrkA blockade will prevent alterations in the bladder function.
Study design, materials and methods
Adult female Wistar rats (200–250 g) were submitted to WAS test only or to WAS while receiving orally with the TrkA antagonist GW441756, 58µg/kg/day (Biotechne). Sham animals were used as controls. Visceral pain behavioral tests and mechanical pain threshold estimation by von Frey filaments in the lower abdomen were performed at day 0 and day 10. At day 11, blood and urine were collected to analyze NGF levels by ELISA (RayBio Human beta-NGF Kit). After, rats were urethane anaesthetized and bladder reflex activity was determined by cystometry (saline infusion at 6 ml/h).
Two visceral pain test were used. Test A included animal breathing rate, eyes aperture, and body posture were scored on a 0–10 scale for each parameter. Visceral pain test B scored normal 0, piloerection 1, strong piloerection 2, labored breathing 3, licking the abdomen 4, and stretching and contractions of the abdomen 5. Mechanical hyperalgesia was evaluated using a Von Frey test in the lower abdominal region.
The bladders of intact rats were collected, and bladder strips were left in a culture medium or in culture medium with 1.25 g/l of phenylephrine. After 24 h, the medium was collected and NGF was measured as indicated above
Statistical analysis was performed using Graph Pad Instat 3.0. When comparing two groups, significance was estimated using T test. When comparing more than two groups, significance was estimated using Kruskal-Wallis followed from Dunn’s multiple comparisons test.
NGF concentration in the plasma and urine in the sham group were 32.17 ± 18.32 and 17.17 ± 1.73 pg/ml, respectively. The WAS group had a marked increased both in the plasmatic and the urinary NGF levels 2088.47 ± 1027.16 pg/ml and 50.20 ± 25.37 pg/ml, respectively (p< 0.001). NGF in the culture medium of the bladder strips contained the same amount of NGF, whether adrenergic stimulated or not (4.97 ± 1.06 and 4.75 ± 0.94 pg/ml.mg bladder strip, respectively).
In the WAS group, at the beginning of the study, the pain score of test A and test B were 0. Ten days later, an increase in score was shown in both tests. Test A increased to 6 ± 5 and test B to 8 ± 4 (p < 0.05 for both tests). Von Frey test performed at day 0 and day 10 in WAS group showed a decrease in mechanical pain threshold from 40 ± 24 g to 6 ± 2 g (p<0.05). Cystometry showed an increase in the number of reflex voiding contractions between the sham and the WAS group (0.45 ± 0.05 vs 0.83 ± 0.11; p<0.001).
In the WAS + GW441756 group, the pain score of test A and test B were 0. At day 10 no changes in the score was observed compared to basal values. The treatment prevented the decrease in mechanical pain threshold during (day 0: 37 ± 19 g; day 10: 25 ± 18 g; p=0.38). TrkA antagonist reduced the number of reflex bladder contractions to 0.6 ± 0.07, p<0.05 against WAS group).
Interpretation of results
The stress conditions induce a marked increase in the systemic NGF levels, as seen by the massive increase in the plasmatic levels. The levels in the urine were more modest. Although also increased during stress, we favor that such increase reflects the systemic increase rather than the local production. In fact, the bladder strips in culture, NGF release was not enhanced by adrenergic stimulation.
The blockade of TrkA receptors caused a marked improvement in visceral pain behavior and in mechanical pain threshold in the lower abdomen. In addition it normalized bladder function.
Altogether this data suggest that chronic stress induce painful behavior and bladder changes by an NGF dependent sensitization of sensory fibers, by binding TrkA, the high affinity receptor for the neurotrophin.