Urinary bladder function and somatic sensitivity in vasoactive intestinal polypeptide (VIP)-/- mice

Abstract

Vasoactive intestinal polypeptide (VIP) is an immunomodulatory neuropeptide widely distributed in neural pathways that regulate micturition. VIP is also an endogenous anti-inflammatory agent that has been suggested for the development of therapies for inflammatory disorders. In the present study, we examined urinary bladder function and hindpaw and pelvic sensitivity in VIP(-/-) and littermate wildtype (WT) controls. We demonstrated increased bladder mass and fewer but larger urine spots on filter paper in VIP(-/-) mice. Using cystometry in conscious, unrestrained mice, VIP(-/-) mice exhibited increased void volumes and shorter intercontraction intervals with continuous intravesical infusion of saline. No differences in transepithelial resistance or water permeability were demonstrated between VIP(-/-) and WT mice; however, an increase in urea permeability was demonstrated in VIP(-/-) mice. With the induction of bladder inflammation by acute administration of cyclophosphamide, an exaggerated or prolonged bladder hyperreflexia and hindpaw and pelvic sensitivity were demonstrated in VIP(-/-) mice. The changes in bladder hyperreflexia and somatic sensitivity in VIP(-/-) mice may reflect increased expression of neurotrophins and/or proinflammatory cytokines in the urinary bladder. Thus, these changes may further regulate the neural control of micturition.

Figure 1

Nociceptive behavioral scoring and somatic sensitivity testing in WT and VIP^-/- mice after cyclophosphamide (CYP) treatment. A. Cumulative nociceptive scores for wildtype (WT) and VIP^-/- mice. Mice were observed every 30 min for 5 min after CYP treatment for a period of 4 hours (h). Cumulative scores were assigned based upon the following behaviors: (1) normal (score 0); (2) piloerection (score 2); (3) strong piloerection (score 3); (4) labored breathing (score 4); licking of the abdomen (score 5) and (6) stretching or contraction of the abdomen (score 6). Data represent the mean ± S.E.M. of n = 10 mice per group. B. Pelvic region testing with calibrated von Frey hairs in WT and VIP^-/- mice after CYP treatment (4 h). The von Frey hairs were applied in an up-down method for 1-3 sec with an interstimulus interval of 15 sec. For pelvic region stimulation, stimulation was confined to the lower abdominal area overlying the urinary bladder. The following behaviors were considered positive responses to pelvic region stimulation: sharp retraction of the abdomen, jumping, or immediate licking or scratching of the pelvic area (Rudick et al., 2007). VIP^-/- mice exhibited significantly (*, p ≤ 0.001) increased pelvic response frequency with all von Frey hairs (0.1 - 4 g) tested. Data represent the mean ± S.E.M. of n = 14 mice per group. C. Hindpaw sensitivity testing with calibrated von Frey hairs in WT and VIP^-/- mice after CYP treatment (4 h). VIP^-/- mice exhibited a significant (*, p ≤ 0.01) decrease in the paw pressure threshold required to elicit a positive response at 4 h and 24 h after CYP treatment; sharp withdrawal of the paw or licking of the tested hindpaw (Rudick et al., 2007). Data represent the mean ± S.E.M. of n = 10 mice per group. All somatic testing and behavioral scoring was performed in a blinded manner with respect to treatment and mouse strain.

Figure 2

Representative cystometrogram traces from conscious, unrestrained VIP^-/- (A) and wildtype (WT; B) mice with continuous intravesical infusion (25 μl/min) of room temperature saline under control (no inflammation; Aa, b; Ba, b) conditions or after acute treatment with cyclophosphamide (CYP; Ac, d; Bc, d). Bladder pressure (cm H₂0) and voided volume (ml) are shown. Data presented are from the same WT mouse or VIP^-/- mouse in the absence and presence of CYP-induced cystitis. Arrows indicate non-voiding contractions.

Figure 3

Summary bar graphs of the voided volume (μl; A) and intercontraction interval (seconds, s; B) using conscious cystometry in conscious, unrestrained WT and VIP^-/- mice with continuous infusion of saline in non-inflamed mice or mice treated with CYP (4 h). A. Voided volumes in VIP^-/- mice were significantly (p ≤ 0.001) greater in control mice without bladder inflammation (white bars). Both WT and VIP^-/- mice exhibited a significant (p ≤ 0.001) decrease in voided volume after bladder inflammation with CYP (black bars). The magnitude of reduction in the voided volume was significantly greater in VIP^-/- mice. Data represent the mean ± S.E.M. of n = 10 mice per group. B. Intercontraction intervals were significantly longer in VIP^-/- mice compared to WT mice in control mice without bladder inflammation (white bars). Both WT and VIP^-/- mice exhibited a significant decrease in intercontraction interval duration after CYP treatment (black bars). The magnitude of reduction in the intercontraction interval was significantly greater in VIP^-/- mice. Data represent the mean ± S.E.M. of n = 10 mice per group.

Figure 4

Bladder pressures in WT and VIP^-/- mice under control (no CYP treatment; A) and inflamed urinary bladder (CYP; 4 h; B) conditions as determined with conscious cystometry and continuous intravesical infusion of saline. No differences in maximum voiding pressure, threshold pressure or baseline resting pressure were determined in WT and VIP^-/- mice without CYP treatment (A) or after CYP treatment (B). Bladder pressures were not different between mouse strains or after CYP treatment. Data represent the mean ± S.E.M. of n = 10 mice per group.

Figure 5

Transepithelial resistance (TER) and permeability measurements in urinary bladder from WT and VIP^-/- mice. A. No difference in TER was demonstrated in WT or VIP^-/- mice. B. No change in permeability to ³H-water permeability was demonstrated in WT or VIP^-/- mice. C. A significant (p ≤ 0.01) increase in ¹⁴C-urea permeability was demonstrated in VIP^-/- mice. Bar graphs in A-C represent the mean ± S.E.M. of n = 16 VIP^-/- mice and n = 18 WT mice.

Figure 6

Capacitance of urinary bladder from WT (A) and VIP^-/- mice (B) in response to bladder stretch or no stretch. WT and VIP^-/- mouse bladder tissue was equilibrated in Krebs solution. No differences in capacitance were observed between urinary bladder from WT and VIP^-/- mice with or without stretch. Line graphs in A-B represent the mean ± S.E.M. of n = 8 VIP^-/- mice with stretch; n = 4 VIP^-/- mice without stretch; n = 3 WT mice with stretch; n = 4 WT mice without stretch.