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Keywords:

  • interstitial cystitis;
  • autoimmunity;
  • cystometrography;
  • interferon-γ;
  • mice

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 bladder function in a newly developed experimental autoimmune cystitis (EAC) model in female SWXJ strain mice, as a potential animal model for interstitial cystitis (IC).

MATERIALS AND METHODS

In all, 20 SWXJ female mice were divided into two groups: an EAC group immunized with mouse bladder homogenate in complete Freund’s adjuvant (CFA) and a control group immunized with CFA alone. At 4 months after injection, the bladder function of some mice (six) was studied with 24-h micturition habits using metabolic cages and conscious cystometrography (CMG). The bladder and lung were harvested for histological examination and to assess interferon-γ (IFN-γ) mRNA expression.

RESULTS

Histology examination showed obviously thickened lamina propria, infiltration of lymphocytes, giant cells, and increased mast cells in the detrusor muscle of the EAC mice. The lungs of EAC mice showed normal histology. The IFN-γ mRNA expression increased significantly in the bladder, but not in the lung of the EAC mice. The 24-h micturition habits measurements showed increased frequency of urination in the EAC mice compared with the controls. Similarly, CMG showed decreased intercontraction intervals and voided volumes per micturition in the EAC mice compared with the controls. However, there were no significant differences in peak voiding pressure or total voiding volume between the EAC and control mice.

CONCLUSIONS

Our murine EAC model has comparable functional and histological alterations to those seen in human IC, and may provide a useful model for the study of the pathogenesis and treatment of IC.


Abbreviations
EAC

experimental autoimmune cystitis

IC

interstitial cystitis

CFA

complete Freund’s adjuvant

CMG

cystometrography

IFN-γ

interferon-γ

DDDH2O

double-distilled deionized H2O

RT-PCR

reverse transcriptase PCR

GAPDH

glyceraldehyde-3-phosphate dehydrogenase.

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 chronic bladder disorder that mainly affects women [1]. Although the symptoms vary from case to case, the most commonly reported IC symptoms are bladder/pelvic pain, urgency, frequency and nocturia [1,2]. As such, the terminology of painful bladder syndrome has also been used to describe the complex nature of the disease. Several aetiological theories have been proposed, which have included leaky epithelium, occult infection, chronic inflammation with neurogenic origin, autoimmunity, mast cell activation, the presence of toxic substances in the urine, and genetic defects [3]. However, no one pathological process has been identified in every patient with IC. The syndrome of IC may have multiple aetiologies, all of which result in a similar clinical manifestation. Another view is that many of the pathological processes may act in concert to produce the features of IC. At least 16 animal models have been used for investigation of the pathogenesis of IC in the past 20 years [4]. No model of bladder injury in healthy animals currently reproduces all the features of IC in humans.

The autoimmunity theory of the pathophysiology of IC continues to trigger interest with reports on the association between IC and other autoimmune diseases such as lupus erythematosis, rheumatoid arthritis, ulcerative colitis and thyroiditis [5–7]. In addition, the incidence of autoantibodies in patients with IC is relatively high [7]. Therefore, autoimmunity may be one component in the pathogenesis of IC, although there is no compelling evidence that autoimmune reactivity has either a primary or a secondary role in the pathophysiology of IC. Subsequently, interest in developing animal models with autoimmune reactivity remains an active area of IC translational research [8].

Previous studies have shown that the SWXJ strain of mice are susceptible to the development of several autoimmune diseases initiated by Th1-type responses [9,10], including autoimmune encephalomyelitis, and autoimmune myocarditis, thus we aimed to create and characterize the phenotype of a recently developed experimental autoimmune cystitis (EAC) model in female SWXJ strain mice.

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

SWXJ (H-2q.s) mice were generated by mating SJL/J (H-2s) males with SWR/J (H-2q) females at the Jackson Laboratory (Bar Harbor, ME, USA). Bladders from 8–10-week-old female SWXJ mice were homogenized in double-distilled deionized H2O (DDDH2O) using a Dremel Moto-Tool (Dremel, Racine, WI, USA). The homogenate was centrifuged at 1000g for 10 min, and the supernatant was lyophilized overnight. The lyophilate was dissolved in DDDH2O, so that each bladder was reduced to a total volume of 0.1 mL. Ten of the female SWXJ mice were immunized s.c. in the abdominal flank with 0.2 mL of an emulsion of 0.1 mL DDDH2O containing the lyophilate from one SWXJ bladder and 0.1 mL complete Freund’s adjuvant (CFA) containing 400 µg Mycobacteria tuberculosis H37RA (Difco, Detroit, MI, USA). Ten age- and sex-matched control mice were immunized with an emulsion containing only CFA. Some of the immunized mice were used for functional experiments (six), the others (four) were used for histological and reverse transcriptase (RT)-PCR studies. All experimental protocols and procedures were approved by the Cleveland Clinic Institutional Animal Care and Use Committee (Cleveland, OH, USA).

At 4 months after injection, the bladder function of the mice was studied with 24-h micturition habits with 12 h of light and 12 h of dark cycles, using metabolic cages. Voided volumes and urinary frequency during the light and dark periods were recorded consistently. Before the experiment, mice were given a residue-free diet (Lactaid brand whole milk, lactose-free) for 24 h. The liquid diet was given to prevent faeces from interfering with measurement of urine output [11]. During the entire test period, mice continued to have free access to this liquid diet and light/dark cycle times were recorded. A mouse micturition chamber, designed to measure urinary output in real time, was custom built by Medical Associates Inc. (St. Albans, VT, USA). Key features of the micturition chamber included a wire mesh bottom. The bottom of the chamber was designed for unobstructed collection of urine droplets, but it would not sequester solid droppings. Directly below the bottom opening was a balance. The data port of the balance was connected to a data acquisition system. Changes in the weight of the collection were recorded at a sampling speed of four times/s (Origin 7.5, OriginLab Corporation, Northampton, MA, USA). The whole system (chamber and balance) was encased in a Plexiglas outer casing, which served to reduce evaporation and the effect of air draft.

Catheter implantation was performed 2 days before cystometrography (CMG) as previously described [12]. Under ketamine (100 mg/kg) and xylazine (10 mg/kg) anaesthesia, a midline longitudinal abdominal incision was made, 0.5 cm above the urethral meatus. The bladder was exposed and a circular purse-string suture of 5/0 silk was placed on the bladder wall. A small incision was made in the bladder wall, and the catheter (polyethylene-10 tubing with a flared tip) was implanted. The purse-string suture was tightened around the catheter. The catheter was tunnelled s.c. and externalized at the back of the neck, out of reach of the mouse. The distal end of the tubing was sealed, and the skin and abdominal incisions were closed separately.

For conscious CMG the mice were placed in the special metabolic cages (Medical Associates Inc., St. Albans, VT, USA) at 2 days after implantation of the suprapubic catheter for CMG for 70–80 min. Briefly, the bladder catheter was connected to both the syringe pump and the pressure transducer. All bladder pressures were referenced to air pressure at the level of the bladder. Pressure and force transducer signals were amplified, recorded on a chart recorder and digitized for computer data collection at four samples/s (Origin 7.5, OriginLab Corp.). The bladder was then filled with room temperature 0.9% saline at 1 mL/h through the bladder catheter, while bladder pressure was recorded. Urine was collected in a beaker on a balance placed beneath each cage. Changes in the weight of the collection were recorded at four times/s (Origin 7.5, OriginLab Corp.). Saline infusion was continued until rhythmic bladder micturition contractions became stable, typically 15–30 min. After the initial stabilization period, the data on five representative micturition cycles were collected for analysing all of the CMG variables. The means of the collected data were reported for analysing. Voided volume is the volume expelled at micturition and the peak voiding pressure was measured at the peak of the detrusor contraction. The intercontraction interval between two successive contractions was calculated in each micturition cycle.

For histological assessment the mice were killed and their bladders and lungs were harvested. Half of the tissues were fixed in 10% buffered formalin, which were used for haematoxylin and eosin staining. The specimens were examined and photographed using light microscopy. The other half of the tissues were preserved in RNAlater RNA Stabilization Reagent (Qiagen Inc, Valencia, CA, USA) and used for total RNA extraction.

For RNA isolation and RT-PCR analysis of interferon-γ (IFN-γ), the total RNA was isolated from the bladder and lung tissues using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer’s directions. First the tissue samples were placed in 2 mL microcentrifuge tubes containing 600 µL Buffer RLT Working Solution. The samples were centrifuged at 15 804g for 3 min. Afterwards, the supernatants were transferred to clean 2 mL microcentrifuge tubes and the pellets were discarded. The solutions then went through a series of washes with 70% ethanol, Buffer RW1 and Buffer RPE using special spin columns provided by the manufacturer. RNA samples were eventually washed off of the columns using RNase-free water by centrifugation for 1 min at 13 467g. The RNA was quantified by spectrophotometric analysis at 260 nm. IFN-γ mRNA expressions in the bladder and lung were determined by RT-PCR. The total RNA were reverse-transcribed into cDNA using the RETROScript Kit (Ambion, Austin, TX, USA); 10 µL of each RNA sample was added to 2 µL oligo(dt) primer in separate 0.5 mL microcentrifuge tubes. Samples were incubated for 3 min at 75 °C. To each sample was added 2 µL 10 × RT buffer, 4 µL dNTP Mix, 1 µL RNase inhibitor, and 1 µL MMLV-RT. Samples were then incubated twice; first for 60 min at 55 °C, then for 10 min at 92 °C. The resulting cDNA was amplified using specific primers for murine IFN-γ. Primers for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were included in all assays to ensure equal loading of nucleic acid. The sequences of forward and reverse primers used in the study are shown in Table 1. Briefly, PCR amplification was performed by mixing 3 µL of cDNA, 2.5 µL dNTP, 1.25 µL forward primer, 1.25 µL reverse primer, 5 µL 10 × PCR buffer, 35.5 µL nuclease-free water, and 1.5 µL Taq DNA polymerase. The mixtures were incubated at 94 °C for 4 min and then subjected to the following amplification profile: 30 s at 94 °C, 30 s at 58 °C, and 40 s at 72 °C for duration of 40 cycles. This was followed by a final extension for 10 min at 72 °C. The PCR products were separated on agarose gels (2% in 1 × TBE buffer) and visualized under ultraviolet light after staining with ethidium bromide.

Table 1.  Primers used in RT-PCR
Target mRNADirectionsPrimers
IFN-γForward5′-GCTCTGAGACAATGAACGCTACAC-3′
Reverse5′-CATCCTTTTGCCAGTTCCTCCAGA-3′
GAPDHForward5′-TTCACCACCATGGAGAAGGC-3′
Reverse5′-GGCATGGACTGTGGTCATGA-3′

Quantitative data are presented as the mean (sem) for each group. Student’s t-test was used for statistical analysis, with P < 0.05 considered to indicate statistical significance.

RESULTS

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

After histological evaluation of the bladder tissues, there were marked differences in the urothelium, lamina propia and muscularis propia of EAC mice when compared to those of the control mice (Fig. 1). There was submucosal oedema circumferentially, with areas of urothelial detachment from the lamina propria. The lamina propria in the EAC mice showed obvious thickening and infiltration of lymphocytes and giant cells (Fig. 1b,d). There were increased mast cells in the detrusor muscle layer in the EAC mice. The inflammatory reaction in EAC mice was predominantly localized to the submucosa. There was no significant difference in histology of the lung between the EAC and control mice (Fig. 2), indicating the bladder organ-specific nature of the induced autoimmunity.

image

Figure 1. Representative haematoxylin and eosin-stained images of bladder in control (a, ×40; c, ×200) and EAC mice (b, ×40; d, ×200). Histological examination showed thickened lamina propria, infiltration of mast cells, lymphocytes and giant cells in the bladder of EAC mice (b, d) compared with that of the control mice (a, c).

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image

Figure 2. Representative haematoxylin and eosin-stained images of lung in control (a, ×40; c, ×100) and EAC mice (b, ×40; d, ×100). Image shows normal histology in the lung of EAC mice (b, d).

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High levels of IFN-γ mRNA were detected in the bladders of EAC mice, with no detection of IFN-γ mRNA in the bladders of the control mice. IFN-γ mRNA was not detected in the lungs in either the control or EAC mice (Fig. 3).

image

Figure 3. IFN-γ was expressed strongly in the bladder of EAC mice (lane 2). Lane 1, 100 base-pair DNA ladder; lane 2, IFN-γ in the bladder of EAC mice; lane 3, IFN-γ in the bladder of control mice; lane 4, IFN-γ in the lung of EAC mice; lane 5, IFN-γ in the lung of control mice; lane 6, GAPDH in the bladder of EAC mice; lane 7, GAPDH in the bladder of control mice; lane 8, GAPDH in the lung of EAC mice; lane 9, GAPDH in the lung of control mice.

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The 24-h micturition habits data are summarized in Table 2. The typical patterns in a control and an ECA mouse are also shown in Fig. 4. The voiding interval is significantly shorter in the EAC mice than in the controls during both the dark or light period, at 12.75 (0.48) vs 19.67 (0.67) min in the dark period and 22.02 (1.56) vs 38.16 (2.57) min in the light period. The voided volume per micturition in the dark and light period was significantly lower in EAC mice compared with the controls, at 0.299 (0.016) vs 0.429 (0.007) g in the dark period and 0.299 (0.014) vs 0.523 (0.005) g in the light period (P < 0.001). However, the total voided volume in 24 h was similar between the EAC and control mice, at 24.570 (1.112) vs 24.071 (1.005) g (P = 0.744).

Table 2.  The voiding interval and voided volume in the light and dark periods of the 24-h micturition habit measurements in control and EAC mice
Variable, mean (sem)PeriodControl miceEAC mice
Voiding interval, minLight38.16 (2.57)22.02 (1.56)
Dark19.67 ( 0.67)12.75 (0.48)
Voided volume per micturition, gLight0.523 (0.005)0.299 (0.014)
Dark0.429 (0.007)0.299 (0.016)
image

Figure 4. 24-h micturition habits showed that the EAC mice (B) had a higher frequency of micturition and a lower voided volume per micturition than the control mice (A).

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The pertinent data on the CMG variables in the different groups are summarized in Table 3.Figure 5 also shows representative CMG tracings of a control and a EAC mouse. The intercontraction interval was significantly shorter in the EAC mice than in the controls, at 3.64 (0.15) vs 7.29 (0.33) min, respectively. Compatible with the 24-h micturition data, the voided volume per micturition was significantly lower in the EAC mice than in the controls, at 0.041 (0.002) vs 0.083 (0.003) g. However, there were no significant differences in peak voiding pressures between the EAC and control mice, at 16.31 (2.24) vs 17.24 (1.08) cmH2O (P = 0.448).

Table 3.  CMG variables in the control and EAC mice
Variable, mean (sem)Control miceEAC mice
Intercontraction interval, min7.29 (0.33)3.64 (0.15)
Voided volume per micturition, g0.083 (0.003)0.041 (0.002)
Peak voiding pressure, cmH2O17.24 (1.08)16.31 (2.24)
image

Figure 5. Representive tracings of CMG in control (A) and EAC mice (B). Upper panel shows voiding events. Middle panel shows intravesical pressure. Lower panel shows voided volume.

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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

IC is a chronic pelvic pain syndrome (painful bladder syndrome) with an illusive nature. The regional and global prevalence of IC is also a subject of debate, depending on the definition criteria. Recent results obtained in an office setting in the USA reported that the rate of probable cases of painful bladder syndrome/IC determined using the O’Leary-Sant Problem and Symptom index was 575 per 100 000 individuals, but increased to 12 600 per 100 000 when using the Pelvic Pain and Urgency/Frequency patient symptom scale [13]. In reported case series [1,14–16], 84–98% of painful bladder syndrome/IC reported urgency, 80–92% had frequency, 63–92% had pain, and 61–89% patients had nocturia. Other types of urinary and pain symptoms have also been reported to a lesser extent. Held et al. [17] reported that 47% of patients with IC said that they had difficulty starting urine flow and 51% reported difficulty emptying the bladder.

The aetiology and pathogenesis of IC remain unknown. Suggestions that have been put forward include fastidious infection [18], damage to the proteoglycan surface of the epithelium [19] and immune mechanisms [20]. It is plausible that painful bladder syndrome/IC has a multifactorial or sequential pathogenesis, which could involve autoimmunity at some point during the natural history of the disease [6,7]. Autoimmunity may be the pathogenesis for a subpopulation of patients with IC. To date, there is no consistent evidence that autoimmune reactivity has either a primary or a secondary role in the pathophysiology of IC. However, it is unlikely that there is a direct autoimmune attack on the bladder in IC as there is no compelling evidence for bladder-specific autoantibodies and only some patients with IC have autoantibodies. A presumed theory is there could be primary bladder wall damage at first. Such damage could be due to genetic defect, physical damage to the bladder surface or a defect in the glycosaminoglycan-containing mucosal outer layer that provides a protective barrier to entry of toxic materials in the urine and infectious pathogens. Once the damage occurs, a self-perpetuating chronic inflammatory process could be initiated with autoantibodies being produced to ‘altered’ bladder antigens or produced because of cell and tissue destruction [7]. Although the role of autoimmunity may be of an indirect nature, it is obvious that patients with serum autoantibodies are manifesting components of an autoimmune response that might be contributing directly or indirectly to disease progression and symptomatology. Those patients with autoantibodies may represent a separate subpopulation of the IC patient group, with a different set of symptoms and perhaps a need for different treatment methods [7]. Therefore, it is helpful to create an animal model with autoimmune reactivity for the study of the pathogenesis and treatment of IC.

The EAC murine model was previously developed in Balb/cAN, C57BL/6 and C3H/HEN mice by Bullock et al. [8]. These mice have many of the characteristics of ulcerative IC: glomerulations upon hydrodistention, a significant decrease in the bladder capacity, urothelial detachment, increased bladder permeability with epithelial leakage, and histopathology showing lymphocytic infiltration, fibrosis, and oedema. However, the investigators did not provide the functional consequences.

To develop an EAC model we used female SWXJ mice known to be susceptible to the induction of several autoimmune diseases including autoimmune encephalomyelitis [9] and autoimmune myocarditis [10]. Our primary goal was to characterize the functional phenotype of EAC mice. To this end we waited until 4 months after immunization with bladder homogenate, so that the mice would have a substantial time to develop changes.

The present EAC mouse model shares some features with clinical IC/painful bladder syndrome. Histologically, EAC mice had an increased thickness of lamina propria and infiltration of lymphocytes and giant cells in the mucosa and submucosa of the bladder. Further, there were marked increases in mast cells in the detrusor muscle layer. Functionally, the mice had clear evidence of increased urinary frequency from both 24-micturition and CMG data. In addition, the voided volume per micturition and not the 24-h urine volume was significantly lower in the EAC mice than in the controls.

The pathogenesis of the present EAC model may involve two mechanisms: direct cellular immune response and/or autoantibodies. The present study showed that responses to homogenized bladder were characterized by proinflammatory Th1-like cytokine profiles with high levels of IFN-γ production. IFN-γ is an important proinflammatory cytokine that Th1 cells produce in autoimmune response [21]. These data are consistent with clinical studies, which have shown that some patients with IC have evidence of autoimmune activity such as bladder reactive antibodies in the serum and urine, IgG antibodies and unusual expression of class II major histocompatibility complex antigens in the bladder wall [7]. In another animal study, Mitra et al. [22] isolated a serum autoantibody in rats with EAC that specifically bound to a bladder protein with a molecular weight of 12 kDa.

To address the possibility of the autoimmune response of other organs, we also examined the histology and the expression of IFN-γ mRNA in the lung of the EAC mouse. The results showed that there was no inflammatory response and no IFN-γ mRNA was expressed in the lung in EAC mouse.

The significant changes in bladder function in the present EAC mice are similar to those seen in other animal models. This suggests that the mouse model of EAC is a valuable one because: (i) it may be useful to investigate the specific events involved in immune mechanisms of IC; (ii) it may be useful in the evaluation of therapeutic agents that target the immune mechanism in some patients with IC.

The limitations of the present study included that we used a tissue homogenate and not a tissue-specific protein or peptide for immunization. Although the present data indicates the bladder organ-specific nature of the induced autoimmunity, we will continue our efforts to isolate a bladder-specific target antigen for our future immunization protocol. Also, the missing element from our phenotyping studies is the demonstration of pain in addition to urinary frequency and urgency symptoms. Obviously, demonstrating pain from a specific organ is a challenging task when using animal models. In future characterization of this EAC model, we intend to use a newly developed method to assess bladder afferent sensation [23].

In conclusion, the present study showed that EAC mice immunized with bladder homogenate develop obvious autoimmune cystitis, which is accompanied by altered histology and bladder function, parallel to those previously described in human IC and in other animal models of IC. Further studies are warranted to characterize and investigate the time-dependent changes of the immune response and bladder function, as well as demonstration of pain phenotype in this EAC model.

CONFLICT OF INTEREST

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

None declared. Source of funding: NIH-NIDDK-DK070004-01 (FD) and NIH R01-DC006422 (VKT).

REFERENCES

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