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Keywords:

  • IPD-1151T;
  • rat;
  • interstitial cystitis;
  • cystometry;
  • HCl-induced cystitis

Abstract

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

OBJECTIVE

To examine the effects of suplatast tosilate (IPD-1151T), a Th2 cytokine inhibitor recently recognized to improve the symptoms in patients with interstitial cystitis (IC), in a rat model of HCl-induced chronic cystitis, to elucidate the possible mechanisms by which the drug improves the symptoms of IC.

MATERIALS AND METHODS

Chronic cystitis was induced by intravesical instillation of 0.2 mL of 0.4 m HCl in female adult rats. After a once-daily oral administration of IPD-1151T (0.1–100 mg/kg) or prednisolone (5 mg/kg) for 7 days, cystometry was performed under urethane anaesthesia. The bladder from HCl-induced cystitis rats was also assessed histopathologically.

RESULTS

On cystometrography there was frequent voiding in cystitis rats. Administration of IPD-1151T for 7 days after intravesical HCl instillation dose-dependently increased the micturition volume and intercontraction intervals. Treatment with prednisolone had similar therapeutic effects. Histological analyses in the bladder from cystitis rats revealed oedema and infiltration of inflammatory cells such as mast cells and eosinophils in the lamina propria and the transitional epithelial thickening. These histological changes and the number of mast cells and eosinophils were reduced by administration of IPD-1151T or prednisolone.

CONCLUSION

The present results indicate that IPD-1151T improves bladder function and pathological changes in HCl-induced cystitis rats, as previously observed in patients with IC. The rat cystitis model induced by HCl could provide useful information for studying proposed therapies for IC which might involve T cell-dependent inflammatory responses as one of its potential pathophysiologies.


Abbreviation
IC

interstitial cystitis.

INTRODUCTION

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

Interstitial cystitis (IC) is a painful bladder syndrome characterized by chronic pelvic pain, urinary frequency and urgency, which are often very difficult to treat regardless of currently proposed treatments such as glycosaminoglycan, dimethylsulphoxide and amitriptyline [1]. Although the underlying cause of IC is still uncertain, several mechanisms have been proposed, including autoimmune responses, unidentified bacteria or virus, a defect in the bladder lining, toxic substances in the urine or neurogenic inflammation [1,2]. It was also reported that mast cell infiltration in the bladder wall is a prominent characteristic of IC, suggesting an association with inflammatory responses. In addition, as more than half of patients with IC reportedly have allergic diseases such as asthma, the involvement of allergic reaction has also been proposed in the pathogenesis of IC [3]. Thus, it seems important to control the inflammatory and/or allergic reactions for treating IC.

Ueda et al.[4] recently reported that oral suplatast tosilate (IPD-1151T) was effective for treating the symptoms of IC in a pilot clinical study. IPD-1151T is an immunoregulator that inhibits the production of Th2 cytokines such as interleukin-4 or -5, selectively suppresses IgE synthesis, and suppresses Th2 cell-dependent eosinophils in cellular infiltrates [5,6]. It was also reported that IPD-1151T improved the control of asthma and reduced steroid use in patients taking high doses of inhaled corticosteroid [7]. Thus the improvement of IC symptoms by IPD-1151T might be attributable to suppression of the immunogenic responses associated with chronic inflammation in IC. Therefore, we sought to investigate the mechanisms of action of IPD-1151T in an animal model of HCl-induced chronic cystitis. Although good animal models of IC are not available, except for cats with naturally occurring IC [8], previous studies reported that the HCl-induced cystitis in the rat gave histological changes similar to those seen in human IC [9]. Thus, we examined the effects of IPD-1151T, and prednisolone, which is known to suppress the production of both Th2 and Th1 cytokines [10], on bladder function and histological changes in this rat model of chronic cystitis, to elucidate the mechanisms by which IPD-1151T improves IC symptoms.

MATERIALS AND METHODS

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

In experiment 1 (cystometric evaluation) cystitis was induced with HCl using the method reported by Rivas et al.[9] with a slight modification. Female Sprague-Dawley rats (250–300 g, Charles River Laboratories, Japan) were divided into seven groups of 8–10 each and anaesthetized with pentobarbital (50 mg/kg, i.p.). The periurethral area of the rats was cleaned with iodophor solution, and the urethra was cannulated with a sterilized polyethylene catheter (PE-50, Becton Dickinson, Japan). In six of the seven groups the bladder was emptied by aspiration of urine, and then 0.2 mL of 0.4 m HCl was instilled into the bladder using a sterile syringe. Thereafter, in each group, once-daily oral administration of either IPD-1151T (0.1, 1, 10 or 100 mg/kg), prednisolone (5 mg/kg) or distilled water (control) was started on the day after HCl instillation and continued for 7 days. The remaining group of rats (10; sham group) had identical urethral catheterization and urine aspiration, except that 0.2 mL of sterile saline was instilled intravesically instead of HCl, and the rats were treated with oral distilled water for 7 days. Prophylactic antimicrobial treatment was provided with cephazolin (7 mg/kg) given s.c. after intravesical instillation of HCl or saline.

At 8 days after an intravesical application of saline or HCl, the rats were anaesthetized with urethane (1.0 g/kg, i.p.) A polyethylene catheter (PE-50) was inserted into the bladder from the dome and held in place with a purse-string suture. The rats were then placed in a Ballman cages (Yamashita Giken, Japan), and the bladder catheter was connected via a T-tube to a pressure transducer and to a microinjection pump (Teru Fusion Syringe Pump STC-521, Terumo, Japan). Warmed saline (37 °C) was infused into the bladder at 3.0 mL/h. A pressure amplifier, via a pressure transducer (Disposable Blood Pressure Monitoring Life Kit DX-360, Nihon Kohden, Japan) was used to measure the intravesical pressure, which was recorded with a Mac Laboratory system (Chart Program V3.3 data recording and analysing software, AD Instruments Pty, Ltd, Australia). Voided urine volume was recorded simultaneously using a scale. Cystometry was started 20 min after the surgical procedures. All micturition cycles were then recorded for 20 min. The following urodynamic variables were measured: the intercontraction interval (the mean interval between contractions), urinary frequency (number of contractions), basal pressure, threshold pressure, micturition pressure (the maximum bladder pressure during micturition), bladder capacity (the volume of infused saline), voided volume (the volume of expelled urine), and compliance (bladder capacity/(threshold pressure – basal pressure)).

In experiment 2 (histological evaluation), using the same techniques as described above, 0.4 m HCl (0.2 mL; five groups of 10 rats each) or saline (one group of 10 rats, sham) was infused into the bladder to induce chronic cystitis. Thereafter, the rats were treated with IPD-1151T (1, 10 or 100 mg/kg) orally for 7 consecutive days, starting on the day after inducing cystitis. On the day after the final oral administration, the urinary bladder was removed for histological evaluation.

For the histopathological analysis, bladder tissues were fixed with 20% phosphate-buffered formalin (pH 7.4) for 96 h. Transverse sections were then prepared from the middle part of the bladders, embedded in paraffin wax, cut into 4 µm sections, and then stained with haematoxylin-eosin (H-E) and Luna staining for histopathological analyses of mast cell and eosinophil counts, respectively. Mast cells and eosinophils were counted on the entire cross-sections obtained from the bladder in each rat, with separate cell counts in the lamina propria, muscle layer and adventitia/serosal layer. The severity of tissue damage was scored using a four-grade scale, i.e. −, no change; +, scattered foci; ++, multiple foci; and +++, diffuse foci. All histopathological analyses and cell counts in different groups were done by one pathologist unaware of the treatment, except for the sham group, which was disclosed before the start of the examination.

All variables were expressed as the mean (sem); Student’s t-test, Wilcoxon’s test, Williams’ multiple comparison and Shirley-Williams’ multiple comparison (one-sided) were used for statistical analyses. When Student’s t-test and/or Wilcoxon’s test was used, P < 0.05 was considered to indicate statistical significance. When Williams’ and/or Shirley-Williams’ multiple comparison (one-sided) was used, P < 0.025 was considered to indicate significance.

RESULTS

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

Intravesical instillation of HCl resulted in significantly greater decreases in both the intercontraction interval and voided volume in the control group, than in the sham group (intravesical saline and oral distilled water treatment). The decrease in these variables induced by intravesical HCl was significantly suppressed by IPD-1151T treatment, dose-dependently at doses of ≥ 1 mg/kg (Figs 1 and 2). Treatment with prednisolone significantly prevented the HCl-induced decrease in micturition volume but had no significant effect on the HCl-induced change in intercontraction interval (Fig. 2).

image

Figure 1. Cystometry in a rat model of chronic cystitis after HCl treatment. The intravesical pressure and voided volume were recorded for 20 min under urethane anaesthesia. Upper traces: intravesical pressure. Lower traces: voided volume. A, Sham: a rat treated with intravesical saline in place of the HCl. B, Control: a rat treated with intravesical HCl that had more bladder contractions and decreased voided volume per micturition. C, IPD-1151T: a rat treated with intravesical HCl followed by IPD-1151T (100 mg/kg, orally for 7 days). After treatment with IPD-1151T, frequent voiding and decreased voided volume per micturition were prevented.

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image

Figure 2. Effects of IPD-1151T and prednisolone (orally for 7 days) on the intercontraction interval and voided volume/micturition in rats with HCl-induced cystitis. Values are expressed as the mean (sem); *P < 0.025, **P < 0.005, significantly different from the value obtained from control rats (Shirley-Williams’ multiple comparison, one-sided). ♯P < 0.05, ♯♯P < 0.01, significantly different from the value obtained from control rats (Student’s t-test). N: Number of rats. S: sham, Cont: control, PDN: prednisolone.

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Frequency of urination and basal pressure were significantly greater in the control group (intravesical HCl and oral distilled water treatment) than in the sham group. The HCl-induced increase in frequency of urination was suppressed by IPD-1151T treatment at doses of ≥10 mg/kg (Table 1). The increase in basal pressure was prevented by IPD-1151T treatment only at 100 mg/kg. Treatment with prednisolone significantly reduced the HCl-induced increase in frequency of urination but did not affect basal pressure (Table 1).

Table 1.  Effects of IPD-1151T (0.1–100 mg/kg, orally for 7 days) and prednisolone (5 mg/kg, orally for 7 days) on cystometric variables and the histology of bladder tissues in rats with HCl-induced cystitis
VariableShamControlIPD, mg/kg
0.1110100Prednisolone
  • *

    P < 0.025,

  • P < 0.005, significantly different from control rats by Williams’ multiple comparison (one-sided; urinary frequency was tested by Shirley-Williams’ multiple comparison);

  • **

    P < 0.05,

  • P < 0.01, significantly different from sham rats by Student’s t-test (urinary frequency was evaluated by the Wilcoxon’s test);

  • §

    P < 0.05, significantly different from control rats by Student’s t-test (urinary frequency was evaluated by the Wilcoxon’s test).

  • P < 0.05;

  • °

    P < 0.01 vs control (Wilcoxon).

Mean (sem)
No. of rats10 9 9 9 910 8
Urinary frequency (N/20 min) 2.8 (0.5) 11.2 (2.0) 6.9 (1.4) 6.6 (1.6) 4.1 (0.6) 2.8 (0.3) 4.8 (0.9)§
Basal pressure, mmHg 4.1 (0.3) 8.2 (1.8)** 6.7 (1.4) 5.1 (0.6) 4.8 (1.0) 4.6 (0.7)* 4.9 (0.7)
Micturition pressure, mmHg21.9 (1.0)21.2 (2.6)20.0 (1.6)18.9 (1.2)23.4 (2.7)18.7 (1.4)21.4 (1.9)
Threshold pressure, mmHg 11.1 (1.2) 11.5 (1.6) 11.8 (1.4) 11.1 (1.2)10.1 (1.5) 9.9 (1.0) 8.4 (1.3)
Bladder capacity, mL 0.45 (0.10) 0.13 (0.03) 0.22 (0.05) 0.30 (0.08)* 0.28 (0.03)* 0.34 (0.04) 0.27 (0.05)§
Compliance, mL/mmHg 0.084 (0.025) 0.029 (0.004) 0.045 (0.010) 0.040 (0.007) 0.058 (0.010) 0.091 (0.027) 0.091 (0.038)
Site/finding, scored as −/+/++/+++ on 10 rats
Urinary bladder
Transitional epithelium
Thickening (hypertrophy and/or  proliferation)10/0/0/0 0/1/2/7 0/0/8/2 1/1/5/3 4/2/3/1° 2/2/5/1
Lamina propria
Inflammatory cell infiltration10/0/0/0 0/5/5/0 0/9/1/0 1/7/2/0 2/7/1/0° 3/6/1/0°
Oedema10/0/0/0 0/1/4/5 0/1/9/0° 1/1/7/1 3/3/4/0 2/2/5/1°
Swelling of fibroblast10/0/0/0 1/2/4/3 0/9/1/0° 1/7/2/0° 3/7/0/0 2/7/1/0°
Muscle
Interstitial oedema10/0/0/0 1/3/5/1 1/2/7/0 2/3/4/1 5/3/2/0° 5/3/1/1
Inflammatory cell infiltration10/0/0/0 3/7/0/0 5/5/0/0 8/2/0/0° 9/1/0/010/0/0/0
Adventitial membrane/serosa
Oedema10/0/0/0 2/8/0/0 5/5/0/0 7/3/0/0° 8/2/0/0 8/2/0/0
Inflammatory cell infiltration10/0/0/0 3/7/0/0 8/2/0/0° 8/2/0/0° 8/2/0/0° 9/1/0/0

In addition, bladder capacity was significantly smaller in the control than in the sham group. The HCl-induced decrease in bladder capacity was suppressed by IPD-1151T treatment dose-dependently at doses of ≥1 mg/kg (Table 1). Treatment with prednisolone also significantly blocked the HCl-induced decrease in bladder capacity. There were no significant differences between the control and sham group in other variables, e.g. micturition pressure, threshold pressure, and compliance (Table 1).

Table 1 also shows the results of the histopathological analysis; 8 days after intravesical delivery of HCl, the major sites of the lesions were in the epithelium and the lamina propria. There was a significant thickening of the transitional epithelium (hypertrophy and/or proliferation of the transitional cell epithelium). The lamina propria was oedematous and infiltrated with chronic inflammatory cells (eosinophils, mast cells, etc.). There was also fibroblast swelling in the lamina propria. In addition, there were oedema and inflammatory cell infiltration in the muscle layer and the adventitia/serosa. Histopathological analyses revealed that IPD-1151T reduced transitional epithelial thickening, and inflammatory cell infiltration and oedema, in the lamina propria induced by intravesical HCl (Fig. 3).

image

Figure 3. Photomicrographs showing the effects of IPD-1151T (100 mg/kg, orally for 7 days) on bladder histology (H-E staining) in rats with HCl-induced cystitis. Sham: a rat treated with intravesical saline in place of the HCl. B, Control: a rat treated with intravesical HCl that showed thickening of the transitional epithelium (hypertrophy and/or proliferation of the transitional cell epithelium) and inflammatory cell infiltration and oedema in the lamina propria. C, IPD-1151T: a rat treated with intravesical HCl followed by IPD-1151T (100 mg/kg, orally for 7 days). Thickening of the transitional epithelium and inflammatory cell infiltration and oedema in the lamina propria was reduced after the treatment with IPD-1151T. ×25.

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Figure 4 shows the data on the eosinophil and mast cell counts. Intravesical HCl resulted in significantly more bladder eosinophil and mast cells in the control group than in the sham group. These increases were suppressed in a dose-dependent manner by treatment with IPD-1151T, with the differences at doses of ≥10 mg/kg being statistically significant. Treatment with prednisolone also significantly suppressed the increases bladder eosinophil and mast cell counts.

image

Figure 4. Effects of IPD-1151T and prednisolone (orally for 7 days) on the number of eosinophils and mast cells in rats with HCl-induced cystitis. Values are expressed as the mean (sem); *P < 0.025, **P < 0.005, significantly different from the value obtained from control rats (Shirley-Williams’ multiple comparison, one-sided). ♯P < 0.05, ♯♯P < 0.01, significantly different from the value from control rats (Wilcoxon’s test). N: Number of rats. S: sham, Cont: control, PDN: prednisolone.

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DISCUSSION

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

The present results show that oral treatment with an immunoregulator, IPD-1151T, is effective in improving bladder function and histopathological changes in the bladder, including more bladder eosinophils and mast cells, in a rat model of chronic cystitis induced by HCI.

In the present study, rats with HCl-induced cystitis had histological characteristics such as infiltration of mast cells and eosinophils in the bladder wall. Previous clinical studies showed increased number and activation of mast cells in human IC bladders [11]. In addition, an increase in the level of urinary eosinophil electropositive protein in patients with IC was reported [12]. Thus the present data confirm the previous findings that intravesical instillation of HCl in rats results in histological appearances similar to those seen in humans with IC [9]. It was reported that cats with naturally occurring IC are the only appropriate animal model of IC [8], but this cat model is not easily available or accessible, and therefore animal models that could be conveniently and consistently produced are needed for basic research into IC. Thus, rats with HCl-induced chronic cystitis, which share some similar characteristics with human IC, could be a good model for studying the pathogenesis of IC and the development of new treatments for IC.

IPD-1151T suppresses T cell-dependent inflammatory responses [7]; it inhibits the synthesis of interleukin-4 and -5 in both human and murine T cells [5,6], IgE synthesis in both mice and humans, and murine mast cell differentiation [13]. Studies in animal models of allergic inflammation also show that local eosinophilia induced by Th2-type cytokines in mice [5], and infiltration of eosinophils and T cells into the airways after allergen challenge in guinea pigs, were inhibited by IPD-1151T [14]. Ueda et al.[4] reported that the therapeutic effects of IPD-1151T in patients with IC were accompanied by a reduction in serum IgE levels and in the numbers of blood eosinophils, CD20-positive cells and urine T cell marker. In a recent study by Sugaya et al.[15], interleukin-4 gene expression was significantly higher in patients with IC than in control subjects. Thus it seems reasonable to assume that T cell-dependent inflammatory responses are involved at least in part in the pathophysiology of IC, and that T cell-dependent cytokine production could be an important target for treating IC.

In the present study, oral IPD-1151T treatment improved urinary frequency and histopathological changes by suppressing infiltration of mast cells and eosinophils in rats with HCl-induced cystitis. Furthermore, we showed that treatment with prednisolone, which is known to suppress the production of both Th2 and Th1 cytokines [10], also produced a significant improvement in frequent voiding and histological changes in the same rat model. Thus we suggest that rats with chronic cystitis induced by intravesical HCl instillation might be a suitable model for studying new treatments for IC that can modulate T cell-dependent inflammatory responses.

It was proposed that sensitization of bladder afferent pathways induced by chronic bladder inflammation might be involved in the mechanisms inducing bladder pain and urinary frequency in IC [16]. Previous studies showed that there was proliferation of nerve fibres [17], and a specific increase in nerve fibres containing substance P in bladders with IC [18]. Lecci et al.[19] also reported that i.p. administration of cyclophosphamide induced subacute to long-lasting bladder inflammation and bladder overactivity elicited by neuropeptides such as substance P released from primary afferent nerves. Cytokines and inflammatory mediators are known to be involved in sensitization of bladder afferent pathways under inflammatory conditions [16]. Boucher et al.[20] reported the effect of intravesical IPD-1151T on the release of histamine, and TNF-α in urine and in the media from bladder explants of female C57BL mice in which bladder inflammation was induced by intravesical administration of carbachol or lipopolysaccharide. Thus, it is likely that the therapeutic effects of IPD-1151T in patients with IC, and on HCl-induced cystitis in rats, are at least in part attributable to suppression of inflammatory responses and subsequent activation of bladder afferent pathways induced by cytokines or pro-inflammatory mediators released from infiltrated inflammatory cells.

In conclusion, the present study indicated that rats with HCl-induced cystitis had urinary frequency and pathological changes in the bladder, e.g. oedema in the lamina propria and infiltration of inflammatory cells, which are often seen in human IC bladders. As IPD-1151T improved these symptoms in this rat cystitis model, as previously reported in patients with IC [4], we suggest that targeting T cell-dependent inflammatory responses could be a reasonable approach for treating IC, and that the HCl-induced rat cystitis model could provide useful information for future research on newly proposed therapies for IC.

REFERENCES

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