Comparison of voiding function and nociceptive behavior in two rat models of cystitis induced by cyclophosphamide or acetone

Abstract

Aims: Nociceptive behavior and its relationship with bladder dysfunction were investigated in two cystitis models, which were induced by intraperitoneal (i.p.) injection of cyclophosphamide (CYP) or intravesical instillation of acetone, using freely moving, non-catheterized conscious rats.

Methods: Female Sprague-Dawley rats were used. Cystitis was induced by i.p. injection of CYP (100 and 200 mg/kg) or intravesical instillation of acetone (10%, 30%, and 50%) via a polyethylene catheter temporarily inserted into the bladder through the urethra. Then the incidence of nociceptive behavior (immobility with decreased breathing rates) was scored. Voided urine was collected simultaneously and continuously to measure bladder capacity. The plasma extravasation in the bladder was quantified by an Evans blue (EB) dye leakage technique.

Results: CYP (100 mg/kg, i.p.) induced nociceptive behavior without affecting bladder capacity or EB concentration in the bladder. A higher dose of CYP (200 mg/kg, i.p.) decreased bladder capacity and increased EB levels as well as nociceptive behavior. In contrast, intravesical instillation of acetone (30%) decreased bladder capacity and increased EB levels, but evoked nociceptive behavior less frequently compared with CYP-treated animals. In capsaicin-pretreated rats, nociceptive behavior induced by CYP or acetone was reduced; however, the overall effects of CYP or acetone on bladder capacity and bladder EB levels were unaffected.

Conclusions: These results suggest that there is a difference in the induction process of nociceptive behavior and small bladder capacity after two different types of bladder irritation, and that C-fiber sensitization is more directly involved in pain sensation than reduced bladder capacity.

Fig. 1

Effects of intraperitoneal administration of cyclophosphamide (CYP) (A, B and C) or intravesical instillation of acetone (D, E and F) on bladder capacity, evans blue (EB) concentration in the bladder and nociceptive behavior in normal rats. A, B, D and E, Bars represent mean ± SEM (n=5 or 6 rats/group). * p<0.05, ** p<0.01 compared with vehicle (saline)-treated rats (Dunnett's multiple comparison test). C and F, nociceptive behavior score, which represents the number of 5-second observation periods during which at least one nociceptive event was detected, during three 5-minute intervals (0-5, 15-20 or 60-65 min) following CYP treatment. The maximal nociceptive score could be 60 during each 5-minute observation period. Bars represent median and IQR (n=5 or 6 rats/group). * p<0.05, ** p<0.01 compared with vehicle (saline)-treated rats (Steel's multiple comparison test).

Fig. 2

Relationship between individual values of bladder capacity and bladder evans blue (EB) concentration (A), nociceptive behavior and bladder EB concentration (B) and nociceptive behavior and bladder capacity (C) (n=16-21/group). The nociceptive behavior score (total number) was summed for each 5-minute period (0-5, 15-20 or 60-65 min) after vehicle (saline), cyclophosphamide (CYP) treatment (100 or 200 mg/kg) or acetone treatment (10, 30 or 50%).

Fig. 3

Effects of cyclophosphamide (CYP) (200mg/kg, ip) (A, B and C) or acetone (30%) (D, E and F) on bladder capacity, bladder evans blue (EB) concentration and nociceptive behavior in rats with [capsaicin (CAP) (+)] or without C-fiber desensitization [CAP (-)]. A, B, D and E, Bars represent mean ± SEM (n=5 rats/group). * p<0.05, ** P<0.01 compared with vehicle (saline)-treated rats (Student's t-test). C and F, the nociceptive behavior score (total number), which was summed for three 5-minute periods (0-5, 15-20 or 60-65 min) after CYP or acetone treatment. Bars represent median and IQR (n=5 rats/group). * p<0.05, ** p<0.01 compared with vehicle (saline)-treated rats. # p<0.05 compared with rats without capsaicin pretreatment (Wilcoxon rank sum test).

Fig. 4

Relationship between individual values of bladder capacity and bladder evans blue (EB) concentration (n=10-20/group). A, vehicle (saline) or cyclophosphamide (CYP)-treated rats with [capsaicin (CAP) (+)] or without CAP pretreatment [CAP (-)]. B, vehicle (saline) or acetone-treated rats with [CAP (+)] or without CAP pretreatment [CAP (-)].

Fig. 5

Proposed mechanisms inducing a reduction in bladder capacity and nociceptive behavior after cyclophosphamide (CYP) or acetone treatment. The CYP metabolite, acrolein, which is excreted into urine, directly stimulates C-fiber afferents to induce pain behavior, and then elicit extravasation in the bladder and/or Aδ-fiber excitation to decrease bladder capacity. In contrast, acetone, which directly induces tissue damage, is more likely to induce extravasation in the bladder and/or Aδ-fiber stimulation to reduce bladder capacity before inducing pain behavior. Thick and thin arrows represent the high and low likelihood of the events to occur, respectively.