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Keywords:

  • bladder;
  • muscle;
  • cross-sensitization;
  • interstitial cystitis;
  • fibromyalgia

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

What’s known on the subject? and What does the study add?

Bladder pain syndrome/interstitial cystitis (BPS/IC) and fibromyalgia syndrome (FMS) often occur concomitantly. In this study, we found that somatic (gluteus)-to-visceral (bladder) cross sensitization might underlie bladder hypersensitivity in patients with FMS.

OBJECTIVE

• To examine the correlation between muscular pain and bladder hypersensitivity in order to clarify the pathogenesis of comorbidity of bladder pain syndrome/interstitial cystitis with other chronic pain conditions such as fibromyalgia syndrome (FMS).

MATERIALS AND METHODS

• Under isoflurane anaesthesia, 0.2 mL of hydrochloric acid (HCl) solution (pH 4.0) was injected into the bilateral gluteus muscles of female Sprague–Dawley rats to produce an FMS model, as the gluteus is one of the specific tender points in patients with FMS. Control rats received saline injection (0.2 mL).

• The mechanical sensitivity of the plantar was evaluated using the mean number of bilateral hindlimb withdrawals in response to tactile stimulation with a 2.0-g von Frey filament at 1, 2 and 3 weeks after the HCl injection.

• In a separate rat group, cystometry was performed with the rats awake during saline infusion (0.06 mL/min) into the bladder before and after 1% lidocaine injection (0.2 mL) into the bilateral gluteus 1, 2 and 3 weeks after the HCl injection.

RESULTS

• The mean number of hindlimb withdrawals was significantly higher in FMS rats than in controls at 1 and 2 weeks.

• Using cystometry, we found that the intercontraction interval (ICI) and voided volume (VV) were significantly lower in FMS rats than in controls at 1 and 2 weeks. In addition, the voiding threshold pressure, ICI and VV were significantly higher after lidocaine injection in FMS rats, but not in controls, at 1 and 2 weeks.

CONCLUSIONS

• HCl injection (pH 4.0) into the gluteus can induce plantar hypersensitivity and urinary frequency for up to 2 weeks after the injection, suggesting that somatic (gluteus)-to-visceral (bladder) cross-sensitization might underlie bladder hypersensitivity in patients with FMS.

• Moreover, intervention at specific tender points outside the bladder could be effective in treating urinary frequency because lidocaine injection into the gluteus normalized bladder function in FMS rats for up to 2 weeks.


Abbreviations
BPS/IC

bladder pain syndrome/interstitial cystitis

FMS

fibromyalgia syndrome

HCl

hydrochloric acid

ICI

intercontraction interval

VV

voided volume

BP

baseline pressure

TP

threshold pressure

MVP

maximal voiding pressure

PVR

postvoid residual urine volume

VE

voiding efficiency

DRG

dorsal root ganglion

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Bladder pain syndrome/interstitial cystitis (BPS/IC) is defined as chronic (for 6 months or more) pelvic pain, pressure or discomfort perceived to be related to the bladder, accompanied by at least one other urinary symptom, e.g. a persistent urge to void or frequency [1]. It is well known that BPS/IC is significantly associated with other chronic pelvic pain conditions, e.g. irritable bowel syndrome and endometriosis, as well as with more systemic pain conditions such as fibromyalgia syndrome (FMS) [2–4]. It has been reported that 12–18% of patients diagnosed with BPS/IC also exhibit symptoms and fulfill diagnostic criteria for FMS [4,5] and that 23–27% of patients with FMS have BPS/IC-like symptoms such as bladder fullness, urgency and pelvic pain [5]. However, while the pathogenesis of BPS/IC, irritable bowel syndrome, FMS and other chronic pelvic pain disorders have been studied mostly independently, few investigators have examined multi-organ mechanisms in the development of chronic pain disorders, despite their striking clinical overlap [6].

The high concurrence rate of different chronic pelvic pain disorders further supports a role of sensitized convergent afferent pathways, which may occur as a result of infectious, idiopathic, inflammatory, neurogenic, metabolic or other neuropathic mechanisms [7]. A rat model of neural cross-talk and sensitization in the pelvis developed by Pezzone et al.[8] revealed that acute cystitis, induced by intravesical infusion of protamine sulphate combined with potassium chloride to produce a BPS/IC-like model, lowered distal colonic sensory thresholds to colorectal distention, and that acute intracolonic irritation with trinitrobenzene sulfonic acid to produce an irritable bowel syndrome-like model led to the development of bladder hypersensitivity. In addition, a rat model with surgically induced endometriosis had reduced micturition thresholds and increased Evans Blue dye extravasation in the bladder [9].

An FMS-like rat model induced by hydrochloric acid (HCl) injection (pH 4.0) into the unilateral gastrocnemius muscle that could produce mechanical hyperalgesia of the bilateral plantar without evidence of tissue damage has been developed by Sluka et al.[10]. Although colonic hyperalgesia has been shown in a FMS-like rat model induced by HCl injection to the hindlimb muscle [11], it is not known whether muscular pain can induce bladder hypersensitivity, so we examined time-dependent changes in bladder function and plantar sensitivity in a rat model with FMS induced by HCl injection (pH 4.0) into the bilateral gluteus. In addition, we determined whether lidocaine injection into the same area of the gluteus could recover bladder function because it has been reported that pretreatment with intravesical instillation of lidocaine attenuated mechanical hyperalgesia of the plantar induced by intravesically instilled acrolein [12].

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Under isoflurane (Mylan, Canonsburg, PA, USA) anaesthesia, 0.2 mL of HCl solution (pH 4.0; Sigma, St. Louis, MO, USA) was injected into the bilateral gluteus muscles of female Sprague–Dawley rats weighing 220–280 g (n= 24; FMS rats) because the afferent nerves innervating to the bladder and this area of the gluteus arise from the same L6 level of the spinal cord (Fig. 1) [13], and the gluteus muscle is one of the specific tender points in patients with FMS [14]. Control rats (n= 24) received 0.2 mL of saline injection. All experiments were conducted in accordance with institutional guidelines and approved by the Jikei University Institutional Animal Care and Use Committee.

image

Figure 1. A chart showing the site of HC1 injection (pH4.0) into the bilateral gluteus muscles

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We placed FMS (n= 6) and control (n= 6) rats individually in a small acrylic cage with a wire mesh floor 1, 2 and 3 weeks after the HCl or saline injection. After all the rats had adapted to the environment for about 1 h, a 2.0-g von Frey filament (Stoelting, Wood Dale, IL, USA) was pressed vertically against the mid-plantar surface of the right and left hindlimbs through the mesh floor for 3 s until the filament slightly buckled. The mean number of bilateral hindlimb withdrawals per 10 applications of the filament at 5-s intervals was used as a measure of mechanical sensitivity in the plantar 1, 2 and 3 weeks after the injection. In our preliminary experiments, a hindlimb withdrawal response occurred in about 10% of rats when a 2.0-g von Frey filament was applied 10 times to the right or left plantar in control rats, so this tactile stimulation was thought to be suitable for the assessment of mechanical hypersensitivity in the plantar.

In a separate group of FMS (n= 18) and control (n= 18) rats, under isoflurane anaesthesia, the bladder was exposed through a lower midline abdominal incision and a polyethylene (PE-50) catheter (Clay Adams, Parsippany, NJ, USA) was implanted into the bladder through the bladder dome. The intravesical catheter was passed through the abdominal wall when the wound was closed with sutures, and a local anaesthetic (EMLA cream; AstraZeneca, Wilmington, DE, USA) was applied to the abdominal wound. The animals were then placed into a sling-suit harness (Lomir Biomedical, Malone, NY, USA) and allowed to recover from the anaesthesia for about 1 h, so that cystometry was performed in an awake condition. The intravesical catheter was connected via a three-way stopcock to a pressure transducer (ADInstruments, Castle Hill, NSW, Australia) and a syringe pump (KD Scientific, Holliston, MA, USA). Saline solution was infused at 0.06 mL/min for about 1 h, until rhythmic bladder contractions became stable. Cystometric values were then recorded for 1 h before and after 1% lidocaine (Sigma) injection (0.2 mL) into the bilateral gluteus at 1, 2 and 3 weeks in FMS or control rats (n= 6, respectively). Intercontraction interval (ICI), baseline pressure (BP), voiding threshold pressure (TP) and maximal voiding pressure (MVP) were recorded using data-acquisition software (sampling at 10 Hz, Chart; ADInstruments) on a computer system equipped with an analogue-to-digital converter (PowerLab; ADInstruments). Saline voided from the urethral orifice was also collected and measured to determine voided volume (V V). After constant VVs were collected the infusion was stopped and postvoid residual urine volume (PVR) was measured by dropping the catheter and withdrawing intravesical fluid through the catheter using gravity. Voiding efficiency (VE) was calculated using the formula V V/(V V + PVR) × 100. All animals were killed after the experiments.

All data were represented as mean (sem) values. Statistical analysis software (Prism; GraphPad Software, San Diego, CA, USA) was used to compare the results in each group. Changes in cystometric values before and after 1% lidocaine injection at 1, 2 and 3 weeks in FMS or control rats were analysed using a paired Student’s t-test. The other measurements used to compare FMS with control rats at 1, 2 and 3 weeks were analysed using an unpaired Student’s t-test. A P value <0.05 was considered to indicate statistical significance.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

The mean number of bilateral hindlimb withdrawals was significantly higher in FMS rats than in controls at 1 week (FMS: 3.0 ± 0.5/10 trials vs. control: 1.2 ± 0.1/10 trials) and 2 weeks (FMS: 2.1 ± 0.2/10 trials vs. control: 1.0 ± 0.1/10 trials). However, there was no significant difference in the mean number of hindlimb withdrawals between FMS and control rats at 3 weeks (FMS: 1.3 ± 0.1/10 trials vs. control: 1.1 ± 0.1/10 trials; Fig. 2).

image

Figure 2. Time-dependent changes in the number of bilateral hindlimb withdrawals in response to tactile stimulation with 2.0 g von Frey filament.

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The cystometry results for FMS and control rats, with or without 1% lidocaine injection, at 1, 2 and 3 weeks are shown in Table 1 in addition to the representative cystometrograms (Fig. 3). The ICI was significantly lower in FMS rats than in control rats at 1 week (FMS: 517.4 ± 45.5 s vs. control: 785.3 ± 90.4 s) and 2 weeks (FMS: 524.6 ± 50.3 s vs. control: 755.9 ± 86.7 s). V V was also significantly lower in FMS rats than in control rats at 1 week (FMS: 0.47 ± 0.05 mL vs. control: 0.75 ± 0.09 mL) and at 2 weeks (FMS: 0.48 ± 0.06 mL vs. control: 0.71 ± 0.08 mL). However, the significant difference in ICI and V V between FMS and control rats was not seen at 3 weeks, nor was there any significant difference in the other cystometric measures such as BP, voiding TP, MVP, PVR or VE between FMS and control rats without lidocaine injection at 1, 2 and 3 weeks.

Table 1.  Cystometry results for FMS and control rats, with or without 1% lidocaine injection, at 1, 2 and 3 weeks
Rat groupBP, cmH2OTP, cmH2OMVP, cmH2OICI, sV V, mLPVR, mLVE, %
  1. Data are mean (sem) values.

At 1 week       
 Control       
  Without lindocaine, n= 64.9 (0.7)7.8 (0.5)20.8 (1.3)785.3 (90.4)0.75 (0.09)0.04 (0.01)94.5 (1.3)
  With lindocaine, n= 64.9 (0.4)7.8 (0.3)20.9 (1.0)834.1 (92.6)0.80 (0.09)0.04 (0.01)95.3 (0.9)
 FMS       
  Without lindocaine, n= 64.7 (0.4)7.1 (0.3)20.5 (1.0)517.4 (45.5)0.47 (0.05)0.05 (0.01)89.9 (2.1)
  With lindocaine, n= 64.6 (0.3)8.4 (0.3)22.9 (1.1)867.2 (64.3)0.82 (0.06)0.05 (0.01)94.5 (0.6)
At 2 weeks       
 Control       
  Without lindocaine, n= 65.0 (0.3)8.1 (0.4)21.1 (1.1)755.9 (86.7)0.71 (0.08)0.04 (0.01)94.4 (0.7)
  With lindocaine, n= 64.9 (0.3)8.0 (0.2)21.7 (1.2)793.3 (88.4)0.75 (0.09)0.04 (0.01)94.9 (1.0)
 FMS       
  Without lindocaine, n= 64.6 (0.7)6.5 (0.7)21.4 (0.9)524.6 (50.3)0.48 (0.06)0.05 (0.01)90.2 (3.0)
  With lindocaine, n= 64.5 (0.6)7.6 (0.8)20.9 (0.8)761.9 (94.0)0.72 (0.09)0.04 (0.01)94.4 (1.4)
At 3 weeks       
 Control       
  Without lindocaine, n= 64.5 (0.7)7.1 (0.6)20.6 (1.6)786.4 (68.0)0.76 (0.07)0.03 (0.01)95.5 (0.9)
  With lindocaine, n= 64.3 (0.6)7.3 (0.5)22.2 (1.6)850.4 (84.4)0.82 (0.09)0.04 (0.01)95.7 (0.8)
 FMS       
  Without lindocaine, n= 64.1 (0.4)6.5 (0.4)21.6 (1.5)681.6 (103.2)0.65 (0.10)0.04 (0.01)94.1 (1.6)
  With lindocaine, n= 64.4. (0.7)7.1 (0.5)20.8 (0.8)737.0 (86.6)0.70 (0.09)0.04 (0.01)94.8 (1.4)
image

Figure 3. Representative cystometrograms in FMS and control rats with or without 1% lidocaine injection into the bilateral gluteus muscles.

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In FMS rats after lidocaine injection, mean voiding TP increased significantly from 7.1 (0.3) cmH2O to 8.4 (0.3) cmH2O at 1 week and from 6.5 (0.7) cmH2O to 7.6 (0.8) cmH2O at 2 weeks. In addition, mean ICI increased significantly from 517.4 (45.5) s to 867.2 (64.3) s at 1 week and from 524.6 (50.3) s to 761.9 (94.0) s at 2 weeks, with VV increasing significantly from 0.47 (0.05) mL to 0.82 (0.06) mL at 1 week and from 0.48 (0.06) mL to 0.72 (0.09) mL at 2 weeks. However, cystometric values before and after lidocaine injection were not different in FMS rats at 3 weeks or in control rats at 1, 2 and 3 weeks.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Although there are no perfect animal models of BPS/IC, except for a naturally occurring model in cats [15], rodents with i.p. injection of cyclophosphamide [16,17], or with intravesical instillation of mustard oil [18], HCl [19], acetone [20] and protamine sulphate [8,21] have often been used to study the disease process of BPS/IC. Recently, in a rat model of irritable bowel syndrome induced by intracolonic injection of trinitrobenzene sulfonic acid, colonic irritation can induce BPS/IC-like changes in the bladder [8,22]; this has been proposed as a new model of BPS/IC because of colon-to-bladder cross-sensitization. Although there is one FMS-like animal model that is characterized by muscular pain induced by HCl injection (pH 4.0) into the gastrocnemius muscle [10], it is not known whether muscular pain can induce bladder hypersensitivity. The present study has shown that HCl injection (pH 4.0) into the bilateral gluteus muscles can induce both plantar hypersensitivity and urinary frequency (i.e. a reduction in the VV and ICI) for up to 2 weeks, suggesting that somatic (gluteus)-to-visceral (bladder) cross-sensitization might underlie bladder hypersensitivity in patients with FMS. We observed bladder hypersensitivity in FMS rats for 2 weeks and that lower voiding TP in the FMS rats, which indicates the activation of afferent inputs from the bladder [23], became significantly higher (the same as that in the control rats) after lidocaine injection.

Recently, it has been proposed that three different, but interconnected, neural mechanisms underlie organ cross-sensitization [24]. The first mechanism involves the presence of dorsal root ganglion (DRG) neurons, with branching or multiple axons, which could serve as direct neuronal connections among different organ domains. Previous studies using dual retrograde labelling in DRG sections have identified convergent DRG neurons receiving afferent inputs from the distal colon and bladder in 14–17% of the total labelled cells [22,25], although there is no evidence of the existence of axons that branch to supply the bladder and hindlimb muscle. The second mechanism involves the converging of afferent inputs from an inflamed structure and from other uninflamed structures on the same interneuron located in the dorsal horn of the spinal cord. Previous studies in female rats have shown that the bladder, cervix and distal colon are innervated by the lumbosacral parasympathetic nerve (the pelvic nerve) derived from L6–S2 and the thoracolumbar sympathetic nerve (the hypogastric nerve) derived from T13–L1 [7,26,27]. The dorsal horns of the T13–L1 and L6–S2 segments were searched for neurons responsive to gentle mechanical stimulation of the cervix and skin areas at the perineum and hindlimbs, and to distention of the colon [28]. The cervix-responsive neurons in T13–L1 and L6–S2 responded to skin stimulation (34% and 38%, respectively), as well as to colonic distention (59% and 51%, respectively), suggesting that afferent inputs from the pelvic organ (i.e. the cervix and distal colon) and from the skin at the perineum and hindlimb regions converge in the dorsal horn of the spinal cord [28]. The third mechanism underlying organ cross-sensitization appears to involve higher centres of the brain. It is well known that Barrington’s nucleus is the primary component of the pontine micturition centre. The divergent projections of Barrington’s nucleus neurons to the parasympathetic preganglionic neurons innervating the bladder and colon have shown that individual neurons within Barrington’s nucleus are poised to integrate signals from distal pelvic viscera [29]. In addition, the study of medullary reticular formation in the brain stem has shown a significant degree of convergence from the distal colon, rectum and skin at the hindlimbs [30]. In the present study, we found that HCl stimulation at the gluteus muscle area, which is innervated by the L6 level of the spinal cord, can induce not only frequent micturition, but also referral pain (i.e. mechanical hyperalgesia) in the plantar area, which is innervated by the L3–5 level of the spinal cord [13]. Taken together, these results led us to the hypothesis that plantar hypersensitivity and urinary frequency induced by HCl injection (pH 4) into the gluteus might be attributable to somatic (gluteus)-to-visceral (bladder) and somatic (gluteus)-to-somatic (plantar) cross-sensitization in the DRG, spinal cord and/or brain levels. Further studies are needed to clarify which neural pathways are important for these cross-sensitization mechanisms.

In clinical practice, BPS/IC is significantly associated with other chronic pain syndromes including FMS [2–5]. The results of the present study suggest that the somatic (gluteus)-to-visceral (bladder) cross-sensitization mechanism might be involved in the BPS/IC-like bladder symptoms of patients with FMS. Because lidocaine injection into the gluteus normalized bladder function in FMS rats in the present study, intervention at specific tender points (e.g. the gluteus) outside the bladder might be effective for treating urinary frequency in patients with FMS. Furthermore, when patients with BPS/IC exhibit other chronic pelvic pain conditions such as FMS, irritable bowel syndrome and/or endometriosis, treatment outside the bladder to control their bladder-related symptoms is important, in addition to focal therapies such as intravesical instillation of DMSO or hydrodistention of the bladder [31].

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

This study was supported by Akaeda Medical Research Fund and Jikei University Research Fund.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES