Cross-Talk and Sensitization of Bladder Afferent Nerves

Abstract

Chronic pelvic pain (CPP) disorders are a heterogenous group of overlapping conditions involving the bladder, bowel, and other pelvic visceral structures. CPP is a poorly understood, often debilitating condition affecting both men and women. The development of pelvic organ cross-sensitization, and often ensuing CPP, following acute or chronic pelvic visceral irritation or stimulation is facilitated by anatomic apposition, coordinated physiologic and reflexive pathways (cross-talk), and convergent sensory input of the pelvic viscera, their associated striated sphincters, muscular components of the pelvic floor, pelvic abdominal wall, and perineum and corresponding cutaneous components, which together facilitate daily coordinated conscious pelvic physiologic activity. Correspondingly, the causes of CPP are numerous and may include gynecologic, urologic, gastrointestinal, musculoskeletal, neural, or psychologic origins, as well as combinations thereof. Although efficacious therapeutic options are limited for those afflicted (the US estimate for women alone is 9.2 million [1]), further research and multi-disciplinary approaches will no doubt improve patient quality of life and decrease direct and indirect annual costs to society, which currently is estimated to exceed ≈$3 billion [1]. CPP not only encompasses pain syndromes of the pelvic cavity, such as interstitial cystitis/painful bladder syndrome (IC/PBS) and irritable bowel syndrome (IBS) but also includes those of the pelvic floor, including vulvodynia, orchialgia, urethral syndrome, and chronic prostatitis (male CPP syndrome) [2]. Since the distal colon and urinary bladder are two pelvic organs whose functions are an integral part of daily, conscious, physiologic pelvic activity, it is not surprising that IBS and IC/PBS, analogous disorders of pelvic visceral pain and urgency, are probably the most common CPP disorders in the general population [1–4].

IC/PBS is a CPP disorder affecting the urinary bladder and is characterized by unpleasant urinary symptoms, such as urinary frequency, urgency, pain, and dyspareunia [5,6], all in the absence of organic disease. IBS is the analogous disorder affecting the distal colorectum and is characterized by chronic or recurrent lower abdominal pain or discomfort associated with altered stool consistency and frequency [7]. Although the etiologies of IC/PBS, IBS, and other CPP disorders have been studied extensively (although mostly independently), few investigations have proposed multi-organ mechanisms in their development, despite their striking clinical overlap [8–9]. As such, 40% to 60% of patients with IBS also exhibit symptoms consistent with IC/PBS, while up to 52% of patients with IC/PBS also have symptoms consistent with IBS (30% with diagnosed IBS) [10–12]. Moreover, frank inflammatory conditions of the colon, such as diverticulitis and inflammatory bowel disease, are also associated with dysuria and IC/PBS [11, 13]. Likewise in one study, patients with IC/PBS had concurrent pain of the vulva or vulvodynia [14], and in another, 45% of males with chronic prostatitis (male CPP) exhibited pain with bladder filling, a classic feature of IC/PBS [3]. Similarly, 22% of men with male CPP or its symptoms had IBS [15–17]. The high concurrence rate of CPP disorders further supports a role of sensitized convergent afferent pathways, which may occur due to infectious, idiopathic, inflammatory, neurogenic, metabolic, or other neuropathic mechanisms. The consequences of afferent sensitization include pain and aberrant smooth muscle activity in response to normally non-noxious or physiologic stimuli. The concept that afferent sensitization of one pelvic organ may adversely influence and sensitize innervation of another structure via direct neuronal connections or reflexes, or via changes in central processing, has emerged as an important area of insight into the development of overlapping CPP disorders.

Neural Mechanisms of Pelvic Organ Cross-Sensitization

The development of pelvic organ cross-sensitization requires cross-afferent stimulation in the pelvis. Afferent information from the major pelvic organs, such as the bladder, colorectum, and uterus, is conducted through hypogastric, splanchnic, pelvic, and pudendal nerves to cell bodies in thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia (DRG) [18]. Typically, noxious prodromic (peripheral→central) noxious afferent stimulation from an irritated pelvic organ leads to antidromic (central→peripheral) afferent stimulation and co-sensitization of another “non-irritated” pelvic organ. These reflexive pathways may occur locally (peripherally) via axon collaterals (dichotomizing afferents), in the spinal cord (dorsal root reflexes), and/or in the central nervous system (CNS) [19]. Consequently, neurogenic sensitization or neurogenic “inflammation” via antidromic pathways may produce functional changes in the uninsulted organ with no or little evidence of an organic etiology.

Evidence for Dichotomizing Bowel and Bladder Afferents

Overlapping CPP disorders could originate peripherally via axonal collaterals (the simplest reflexive afferent pathway) through which a single primary afferent supplies two structures (dichotomizing afferent). Dichotomizing neurons within the DRG were first proposed by Sinclair [20] and have seen been shown in several species to range from 0.5% to 15% of all afferent nerve fibers [21]. Supporting their role in referred sacral pain via dichotomizing sacral afferents, there are 2.3 times as many peripheral nerve fibers as there are cell bodies in the corresponding sacral DRG [22]. Thus, irritation of one pelvic organ may lead to referred pain and/or neurogenic inflammation in another by means of antidromic activation of afferent terminals emanating from the same DRG neuron.

Using fluorescent cholera toxin subunit B (CTB) conjugates to identify potential cross-organ sensitizing that dichotomizes DRG neurons in the pelvis, we performed concomitant retrograde labeling of both urinary bladder and distal colon afferents in Sprague-Dawley rats and C57Bl/6 mice [23]. In preparation for immunohistochemical localization of labeled afferents, animals were perfused 4–5 days after subserosal organ injections, and T10-S2 DRG were removed, sectioned, and analyzed using confocal microscopy. CTB-positive bladder afferent fibers in the rat were nearly 3 times more numerous than those from the distal colon, while in the mouse, each organ was equally represented. In both species, the majority of colon and bladder afferent fibers (both single- and dual-organ projections, the majority of which were single-organ projections) projected from LS ganglia and to a lesser extent, from TL ganglia. In the rat, 17% of the total CTB-positive neurons were retrogradely labeled from both organs, with 11% localized in the TL, 6% in the LS, and 0.8% in the thoracic (TH) ganglia (dual-organ projections were more prominent in the TL ganglia). In the mouse, 21% of the total CTB-positive neurons were dually-labeled, with 12% localized in the LS, 4% in the TH, and 4% in the TL ganglia. These findings support the existence of dichotomizing pelvic afferents that provide a pre-existing neuronal network for potential immediate and sustained pelvic organ cross-sensitization, which may play a role in CPP disorders.

A Role of Pre-existing Neural Pathways, Substantiated in an in vivo Model of Acute, Bi-Directional, Pelvic Organ Cross-Sensitization

Neural “cross-talk” in the pelvis is necessary for the normal regulation of sexual, bladder, and bowel function; convergent sensory pathways in the spinal cord are involved [24–29]. For example, overlapping central projections of pelvic and pudendal afferents allow integration of somatic and parasympathetic motor activity in the pelvis and facilitate the orchestration of sacral reflexes [30], perfect illustrations of neural “cross-talk” at work. Likewise, convergence of afferent nerve fibers from the bladder and bowel is a common feature of visceral interneurons that are thought to mediate vesico- and colono-sphincteric reflexes and colono-vesical cross-inhibitory interactions [31]. Because a neural substrate for pelvic organ cross-talk exists under normal conditions, alterations in these neural pathways by disease or injury may play a role in the development of overlapping CPP disorders and pelvic organ cross-sensitization [32,33].

Prior to our original studies, there had been little experimental evidence of pelvic organ cross-sensitization. We demonstrated that acute cystitis can lower colorectal sensory thresholds to balloon distension and that acute colitis can produce an acute irritative micturition pattern [32]. Precisely, prior to bladder irritation, graded colorectal distensions (CRDs) to 40 cm H₂0 produced no notable changes in abdominal wall electromyographic (EMG) activity, a visceromotor measure of visceral pain. Dramatic increases in abdominal wall EMG activity in response to CRD were observed at much lower distension pressures after acute bladder irritation, suggesting colonic afferent sensitization. Similarly, acute colonic irritation increased bladder contraction frequency by 66%, suggesting sensitization of lower urinary tract afferent nerve fibers. The acute development of pelvic organ cross-sensitization further confirms a role for, and subsequent modulation of, pre-existing afferent pathways in the pelvis, perhaps via antidromic release of neuropeptides with subsequent modulation of these same afferent fibers [32].

Single-Unit Afferent Recordings: Direct Evidence of Cross-Organ Afferent Sensitization

Acute colitis can increase the excitability of bladder nociceptive neurons, as measured by enhanced responses to capsaicin (CP) and accentuated sodium currents in dispersed bladder DRG cells [34]. To directly test the hypothesis that colonic irritation can alter the mechano-and chemo-sensitive properties of urinary bladder afferents, we recorded single-unit bladder C-fiber activity from fine filaments of the pelvic nerve in urethane-anesthetized Sprague-Dawley female rats, and assessed their responsiveness to bladder distension and intravesical CP, bradykinin, or substance P stimulation before and 1 hour after (acute) intracolonic administration of trinitrobenzenesulfonic acid (TNBS) [33]. Colonic irritation increased the resting firing rate of bladder afferents 2-fold (1.0 ± 0.2 vs. 0.49 ± 0.2 imp/sec, respectively, p<0.05). Moreover, a greater percentage of bladder afferents responded to low pressure urinary bladder distensions (UBDs, 10–20 mm Hg) 1 hour after vs before TNBS administration (73% vs. 27%, respectively, p<0.05), and this observation was consistent with lowered afferent sensory thresholds or activation of silent nociceptors as proposed previously [35]. Although the magnitude of the afferent response to UBD was unchanged at low pressures, the response was greatly enhanced at pressures of 30 mm Hg and above (2.36 ± 0.56 vs. 8.55 ± 0.73 imp/sec, respectively, p<0.05). Responses to CP, bradykinin, and Substance P were also significantly increased after TNBS, and all responses were blocked by bladder denervation [33].

These studies confirm that acute colonic irritation with TNBS directly sensitizes the mechano- and chemo-receptive properties of urinary bladder C-fibers traveling within the pelvic nerve, and shed further light on our initial observations regarding cross-organ sensitization [32]. We propose that such cross-organ afferent pathways may originate centrally via spinal or supra-spinal circuits (including spinal antidromic dorsal root reflexes), and/or peripherally directly from the colon via antidromic axon reflexes from a single dichotomizing primary afferent supplying two structures (prespinal convergence).

Depletion of Neuropeptides From C-fibers, Confirming Their Role

Given the findings in our acute afferent studies, we proposed that continued pelvic cross-afferent stimulation would result in continued antidromic release of neuropeptides from afferent endings and lead to receptor sensitization and neurogenic “inflammation.” To test this hypothesis, we recorded single-unit bladder C-fiber activity from fine filaments of the pelvic nerve in urethane-anesthetized Sprague-Dawley female rats and assessed their responsiveness to mechanical (UBD) and chemical (intravesical CP, bradykinin, or substance P) stimulation 10 days after intracolonic administration of TNBS (subacute phase) or vehicle [36]. To eliminate the contribution of C-fibers and their associated neuropeptides, some animals were pretreated systemically with CP 3 days prior to colonic irritation [37].

UBD increased bladder afferent firing in proportion to intravesical pressure in control animals. At intravesical pressures of 30 mm Hg and above, the percent increase in afferent firing was significantly increased after TNBS compared with animals that did not receive TNBS (1222 ± 176% vs. 624 ± 54%, respectively, p<0.01). The response to intravesical CP was also increased after vs. before intracolonic TNBS (4126 ± 775% vs. 1979 ± 438%, respectively, p<0.01). Systemic depletion of neuropeptides from sensory nerves with CP prior to TNBS abolished these effects. Histologic examination of the bladders revealed a dramatic increase in mast-cell density in TNBS-treated animals compared to controls (18.02 ± 1.25 vs. 3.11 ± 0.27 mast cells per 100× field, respectively, p<0.01). Bladder mast cell (MC) numbers in TNBS-treated animals were significantly decreased by systemic CP pre-treatment (10.25 ± 0.95/100x field, p<0.05 vs. TNBS-treated control animals). These findings confirm that subacute colonic irritation (10 days post-TNBS) in the rat sensitizes urinary bladder afferents to noxious stimuli and causes mast-cell infiltration in the bladder. Inhibition of C-fiber afferent function diminished these effects, supporting their role in the effect of TNBS-induced colitis and neurogenic cystitis.

A Role of Sensitized Mast Cells in Chronic Neurogenic Cystitis

Pre-existing afferent pathways and associated neuropeptide release from C-fibers appear to play important roles in an animal model of acute (1 hr) and sub-acute (10 days) pelvic organ cross-sensitization. The chronic effects of cross-afferent stimulation (colon→bladder) on physiologic function of the urinary bladder and vice versa have not been previously studied. Chronic morphologic changes in sensory and motor neurons innervating the bladder and alterations in the number and distribution of bladder mast cells (MCs) were noted following chemical, immune, and mechanical irritation of the bladder [38]. Neuroplasticity observed in these models of direct bladder irritation could explain long-term symptoms, including pain, seen even after the resolution of bladder inflammation [38], and one may likewise hypothesize that this neuroplasticity may not necessarily be limited to the bladder itself and may involve other pelvic organs, as suggested in pelvic cross-sensitization studies [8,9,32,33]. With continued stimulation of this cross-organ neural circuitry, physiologic changes could develop not only in the affected pelvic organ, but also in other pelvic organs with convergent afferent input that could lead to development and overlap of CPP disorders [32].

A putative role of MCs in IC [39], IBS [40–42], and other disorders characterized by hyperalgesia and neurogenic inflammation [43] has been implicated. Not only do MCs contain a formidable armamentarium of nociceptive molecules, including adenosine phosphates, bradykinin, histamine, leukotrienes, potassium, lymphokines, tumor necrosis factor (TNF), and prostaglandins [44], their close apposition to nerve fibers in the human gastrointestinal tract [45,46] and urinary bladder [47] also provides anatomical evidence supporting MC-mediated modulation of afferent nerve function. Lastly, and also supporting our hypotheses, CNS-induced neurogenic cystitis following pseudorabies virus infection was also associated with bladder mast cell degranulation in the rat [48].

To further explore this hypothesis, we evaluated the physiologic effects of colonic irritation on urinary bladder function 60–90 days after TNBS-induced colonic irritation (ie, during the resolution phase of this model) [49]. Bladder MCs were enumerated, and those activated (containing <20% of cellular staining) were quantified to begin to identify histologic and biochemical changes in afferent and end-organ pathways that may be responsible for neurogenic inflammation and afferent cross-sensitization. TNBS was instilled into the colons of another group of rats, and RT-PCR was used to quantify abundance of mRNA for stem-cell factor (SCF) or MC growth factor and nerve growth factor (NGF), a neurotrophic factor and also an MC-stimulatory factor that acts synergistically with SCF [50–42] in urinary bladders and L6-S1 DRG (corresponding to LS-afferent input from the bladder and distal colorectum of the rat). Urine voiding volume was reduced (p<0.005), and voiding frequency was increased (p<0.05), both by ≈50%, after intra-rectal TNBS and was consistent with chronic irritative micturition.

Although total MC counts were not statistically increased as in the sub-acute phase of this model (10 days), both the percentage and density of activated bladder MCs were significantly increased (p<0.05). SCF and NGF mRNA expression were both increased 2-fold in the bladder (p<0.005 and p<0.01, compared to controls), but there were no significant changes in the L6-S1 DRG. Thus, chronic cystitis in the rat, as evidenced by changes in micturition and recruitment of activated MCs to the bladder wall, was observed during the resolution of TNBS colitis. These changes, which appear to be maintained with time, are clearly a consequence of antidromically stimulated convergent pelvic afferent pathways and the consequential expression of neurotrophic factors in the target (uninsulted) organ (bladder).

Summary

Colonic irritation and cross-afferent pelvic stimulation lead to bladder cross-sensitization, as evidenced by changes in physiology, mast cell histology, and neurotrophin expression. Bladder afferent sensitization is a key component in this process, and there is substantial evidence that the mast cell is an important intermediary. Mast-cell migration, activation, and sensitization, expression of multiple neurotrophic, mast-cell stimulatory, and afferent-sensitizing factors collectively influence the physiology of the indirectly affected pelvic organ (ie, even in the absence of direct irritation). Future studies are needed to further identify additional putative pathways involved in induction, maintenance, reactivation, and perhaps overlap of CPP disorders.