Differential effects of intravesical resiniferatoxin on excitability of bladder spinal neurons upon colon-bladder cross-sensitization

Abstract

Cross-sensitization in the pelvis may contribute to etiology of functional pelvic pain disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS). Increasing evidence suggests the involvement of transient receptor potential vanilloid 1 (TRPV1) receptors in the development of neurogenic inflammation in the pelvis and pelvic organ cross-sensitization. The objective of this study was to test the hypothesis that desensitization of TRPV1 receptors in the urinary bladder can minimize the effects of cross-sensitization induced by experimental colitis on excitability of bladder spinal neurons. Extracellular activity of bladder neurons was recorded in response to graded urinary bladder distension (UBD) in rats pretreated with intravesical resiniferatoxin (RTX, 10(-7)M). Colonic inflammation was induced by intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). The duration of excitatory responses to noxious UBD during acute colonic inflammation (3 days post-TNBS) was significantly shortened in the group with RTX pretreatment (25.3±1.5s, n=49) when compared to the control group (35.1±4.2s, n=43, p<0.05). The duration of long-lasting excitatory responses, but not short-lasting responses of bladder spinal neurons during acute colitis was significantly reduced by RTX from 52.9±6.6s (n=21, vehicle group) to 34.4±2.1s (RTX group, n=21, p<0.05). However, activation of TRPV1 receptors in the urinary bladder prior to acute colitis increased the number of bladder neurons receiving input from large somatic fields from 22.7% to 58.2% (p<0.01). The results of our study provide evidence that intravesical RTX reduces the effects of viscerovisceral cross-talk induced by colonic inflammation on bladder spinal neurons. However, RTX enhances the responses of bladder neurons to somatic stimulation, thereby limiting its therapeutic potential.

Fig. 1

Histological evaluation of inflammation in the distal colon and expression of TRPV1 protein. (A) Hematoxylin and eosin (H&E) staining of the distal colon from the following groups of animals: control (top panel), acute colonic inflammation (middle panel, TNBS – 3 days) and resolved inflammation (bottom pane, TNBS – 30 days, 4×). Please note that only group with acute colitis (3 days post-TNBS) showed significant tissue damage including sites of infiltration, thickening of the muscle layer, and disruption of the colonic crypts. (B) Western blotting with anti-TRPV1 antibody of the proteins isolated from the urothelium (Uro), detrusor smooth muscle (DSM), and distal colon (Colon). The amount of protein loaded per lane was 40 μg for all tissues. Alpha tubulin was used as a loading control. (C) Normalized TRPV1/α-tubulin values in different groups of animals (N=4 in each group). Ctrl-vehicle in the bladder, RTX (B) – RTX in the bladder (3 days post-RTX), RTX(B)+TNBS(C) – RTX in the bladder followed by intracolonic TNBS (3 days post-TNBS treatment). * – p≤0.05 between the groups. (D) The levels of MPO in the distal colon in the control groups and 3 days after the induction of TNBS colitis (acute phase).

Fig. 2

Comparison of the duration of excitatory and inhibitory responses induced by UBD in bladder neurons. (A) Duration of excitatory (E) and inhibitory (I) responses in the groups of animals with intravesical vehicle vs. intravesical RTX followed by acute experimental colitis. (B) Excitability of bladder spinal neurons recorded from rats with resolved TNBS-induced colitis. * – p≤0.05 between the groups.

Fig. 3

Short- and long-lasting excitatory and inhibitory responses of bladder spinal neurons in rats with and without intravesical RTX followed by acute colonic inflammation. (A) Examples of bladder neurons with short-lasting excitatory (SL-E) and long-lasting excitatory (LL-E) responses. In these panels top trace is the rate histogram and bottom trace shows raw recordings of cell acitivity. (B) Latency of short-lasting excitatory (SL-E) and inhibitory (SL-I) neurons in rats with intravesical RTX during acute experimental colitis. (C) Comparison of latency of long-lasting excitatory (LL-E) and inhibitory (LL-E) neurons in animals with acute colonic inflammation. (D) Duration of short-lasting excitatory and inhibitory bladder neurons in rats with colitis. (E) Duration of long-lasting excitatory and inhibitory bladder neurons in animals with acutely inflamed colon. Imp/s – number of action potentials per second.

Fig. 4

Responses of low thereshold (LT) and high threshold (HT) bladder neurons to increasing volumes of UBD in rats with experimental colitis. (A) A recording from a low threshold excitatory (LT-E) neuron during innocuous (0.5 ml), subnoxious (1.0 ml) and noxious (1.5 ml) UBD (top left panel). Top right panel shows the activity of high threshold excitatory (HT-E) neuron in response to graded UBD. Lower panel includes recordings from low threshold inhibitory (LT-I, left) and high threshold excitatory-inhibitory (HT-EI, right) spinal neurons. (B) Number of total LT and HT neurons in rats with acute colitis pretreated with intravesical RTX. Please note that pretreatment with RTX increased the number of HT neurons in this group (* – p≤0.05 to vehicle control). (C) Comparison of total population of LT and HT neurons in rats with resolved colitis.

Fig. 5

Intravesical RTX increases the number of urinary bladder spinal neurons with large somatic field input in rats with acute colonic inflammation. (A) Representative traces of low (top panel), wide dynamic (middle panel) and high (lower panel) threshold neurons responded to UBD and either innocuous (brush, Br) or noxious (pinch, Pi) stimulation of the somatic field. (B) Schematic presentation of the somatic field input from small (top) and large (bottom) area on the rat lower abdomen or hindquarter. (C) Number of bladder neurons responded to somatic stimulation in animals with intravesical instillation of a TRPV1 agonist during acute colitis. (D) Percentage of neurons responded to brush and pinch after resolved colonic inflammation.