To examine bladder and urethral function in pelvic organ prolapsed lysyl oxidase like-1 (LOXL1) knockout mice.
To examine bladder and urethral function in pelvic organ prolapsed lysyl oxidase like-1 (LOXL1) knockout mice.
Female parous Loxl1 −/− mice in the stable phase of prolapse, and age-matched wild type (WT) mice (six each) had conscious cystometry, leak-point pressure (LPP) testing, and contractile responses assessed of their bladder muscle strips to KCl, electrical-field stimulation, ATP, and carbachol.
Loxl1 −/− mice voided more frequently and had lower mean (sem) bladder capacity, at 0.10 (0.01) vs 0.20 (0.01) mL, and voiding pressure, at 25.0 (1.90) vs 36.6 (4.04) cmH2O, respectively, during cystometry than had WT mice. The LPP was not significantly different between WT and Loxl1 −/− mice, at 7.05 (0.81) vs 5.22 (1.23) cmH2O, respectively. There were no significant differences between bladder strips from Loxl1 −/− mice and WT mice in their responsiveness to various stimuli.
Loxl1 −/− knockout mice had lower urinary tract dysfunction, most likely due to urethral dysfunction. Loxl1 −/− knockout mice can be used as an animal model for pelvic floor disorders. Further studies are needed to characterize the morphological and molecular alterations of the bladder and urethra.
(female) pelvic floor disorders
pelvic organ prolapse
Pelvic floor disorders (PFD) are a group of conditions that include pelvic organ prolapse (POP), urinary incontinence (UI), and other sensory and emptying abnormalities of the lower urinary tract. Prevalence data suggest that: (i) up to half of women in the USA have some level of female PFD (FPFD), i.e. UI, POP, including uterine and rectal prolapse, and associated problems, during their lifetimes ; (ii) FPFD share many common risk factors, including ageing, vaginal childbirth, obesity and diabetes mellitus, and might appear in conjunction clinically ; (iii) FPFD have a substantial aggregate impact in terms of economic costs and the quality of life . The continuing increase in the age-related incidence of obesity and diabetes, with a demographically ageing population in the USA, might be expected to provoke a dramatic increase in FPFD in the coming decades. Despite their prevalence, the pathophysiology of FPFD is not clear. There is much interest in developing animal models to characterize FPFD and to develop new treatment.
Elastic fibres are components of the extracellular matrix and confer resilience to tissues that are normally subjected to stretching and expansive forces. The female reproductive organs and pelvic floor are rich in elastic fibres . Further, these tissues undergo massive remodelling during pregnancy and childbirth . We identified an essential and specific role for lysyl oxidase like-1 (LOXL1) in elastic fibre remodelling in mice . Mice lacking LOXL1 are unable to synthesise elastin polymers in adult tissues . LOXL1 deficiency in mice results in profound FPFD-like manifestations, including POP and abnormal lower urinary tract function after pregnancy and vaginal delivery of pups [4,5]. The aim of the present study was to examine the bladder and urethral function in LOXL1 knockout mice in comparison to age- and sex-matched wild types (WT).
The generation of LOXL1-deficient mice has been previously described in detail [4,5]. All mice were shipped from Harvard Medical School to the Cleveland Clinic animal facility. At 2 weeks from the first delivery, six pairs of female Loxl1 −/− mice in the stable phase of prolapse and WT mice (without prolapse) were used in the experiment. The mice were maintained in a 12-h light/dark facility with free access to food and water. The mice were assessed using conscious cystometrography (CMG) and leak-point pressure testing (LPP). Then the urinary bladder was removed for contractility testing. Mice were euthanized with one i.p. injection of pentobarbital (200 mg/kg). The experimental protocol was approved by the Cleveland Clinic Foundation Institutional Animal Care and Use Committee (Cleveland).
A suprapubic catheter was implanted 2 days before CMG; under ketamine (100 mg/kg) and xylazine (10 mg/kg) anaesthesia, a midline longitudinal abdominal incision was made. The bladder was exposed and a circular purse-string suture of 7/0 silk was placed on the bladder wall. A small incision was made in the bladder wall, and the catheter (PE-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 closed separately.
At 2 days after implanting the bladder catheter, the mice were placed in specially modified metabolic cages for the conscious CMG. Briefly, the implanted bladder catheter was attached via a stopcock to both a pressure transducer (BP-100, CB Sciences, USA) and a flow pump (Kent Scientific Corp., Torrington, CT, USA). The bladder was filled via the catheter with room temperature 0.9% saline (1 mL/ h), while bladder pressure was recorded. Urine was collected in a beaker on a force transducer (FT-03 D, Grass Instrument Co, Quincy, MA, USA) placed beneath each cage. The pressure and force transducers were connected to an amplifier (ETH-400, CB Sciences), and multiport controller software (Polyview, Grass Instruments) was used for data recording through a computer. After the initial stabilization period, the data on at least 10 representative micturition cycles were collected for analysing the cystometric variables. The means of the collected data were reported for analysis. The bladder capacity was calculated by multiplying the time of infusion to the first void by the infusion rate. Peak voiding pressure was measured at the peak of the detrusor contraction.
To measure the LPP, the mice were anaesthetized with urethane (1.2 g/kg, i.p.) after CMG. The bladder catheter was connected via a stopcock to a P300 pressure transducer (Astro-Medical, West Warwick, Rhode Island, USA) and a Model 100 flow pump (KD Scientific, New Hope, PA, USA). The transducer was connected to an MT 9500 amplifier and polygraph (Astro-Medical), and a computer that digitized pressure data at 10 samples/s. The bladder was palpated to empty and filled with saline at 1 mL/h via the flow pump. When half the capacity was attained, gentle pressure was applied to the abdomen to simulate a mild Credé manoeuvre. Bladder pressure was continually measured via the bladder catheter. Pressure was increased until the mouse leaked saline through the urethra, when the externally applied pressure was rapidly removed . At least three measurements were obtained per mouse and the mean calculated for each.
For the contractility studies, full-thickness longitudinal strips of ≈ 3–5 mg (5 × 2 mm) were prepared from the dorsal part of the bladder body, as described previously . Bladder strips were mounted between platinum-plate electrodes and secured by small clips in a double-jacketed organ bath containing 20 mL Krebs’ solution aerated with 95% O2 and 5% CO2 to obtain a pH of 7.4 at 37 °C. The composition of the Krebs’ solution was (mM): 133 NaCl, 4.7 KCl, 2.5 CaCl2, 16.3 NaHCO3, 1.35 NaH2PO4, 0.6 MgSO4, and 7.8 dextrose. The strip length was adjusted 3–5 times to obtain maximum active tension, and all subsequent work was done at the length at which this was achieved. Isometric contraction was recorded with a computerized data acquisition program (Biobench, National Instruments Corporation, TX, USA) at a rate of 50 Hz and stored on a hard drive for later analyses.
KCl, electric-field stimulation (EFS), ATP, and carbamylcholine chloride (carbachol) were used to stimulate each strip, in that order. Testing with different stimuli was separated by at least two washes with drug-free Krebs’ solution over a 10-min period. The responses to KCl (20–120 mm) were obtained sequentially and not cumulatively by stepwise increases in concentration. Frequency-response curves (0.5, 1, 2, 4, 8, 16 and 32 Hz) were elicited by stimulating the tissues using an electric stimulator (A310 Accupulser, New Haven, CT, USA) delivering 0.1 ms rectangular shock-wave pulses over 10 s at 2 min intervals. The concentration-response curves for ATP (10 µm−30 mm) and carbachol (0.1 µm−100 µm) were obtained by adding increasing amounts of these chemicals, cumulatively, directly to the organ bath. At the end of the experiment, the length and weight of each muscle strip between the suspension clips were measured. Contraction responses were expressed as tension/cross-sectional area, the latter calculated as strip mass/(length × density). The density of all preparations was assumed to be 1.05 mg/mm .
General characteristics, CMG variables and the LPP between groups were compared using Student’s t-test. Contraction responses to stimuli are expressed as the mean (sem) tension/cross-sectional area. Contraction responses and components were compared using a repeated-measures anova; in all tests, P < 0.05 was considered to indicate statistical significance.
The pelvic organ defects in the LOXL1 knockout mice were striking (Fig. 1); the prolapsed tissues retracted after 1–2 weeks and remained in an occult type of prolapse indefinitely (stable stage). Gross examination of the Loxl1 −/− mice showed a stable level of prolapse of the genitalia that was readily distinguishable from the WT. A large bulge appeared in the urogenital region, indicating internal pelvic organ descent. Loxl1 −/− mice also developed mild rectal prolapse. Consistent with our previous study , WT mice showed well defined uterine, cervical and vaginal structures. The urethra was tightly adherent to the suburethral vaginal wall along its length, and the urinary bladder was firmly attached at a position near the uterine cervix. By contrast, the upper portion of the urethra in Loxl1 −/− mice was typically detached from the vaginal wall. In the present study there were no significant differences between the body weights or bladder weights among the two groups of mice (Fig. 2).
On conscious CMG, both Loxl1 −/− and WT mice showed a regular and periodic emptying of the bladder; Figure 3 is a representative tracing of CMG of WT (upper panel) and Loxl1 −/− mice (lower panel). Loxl1 −/− mice voided more often during CMG than did WT mice, and had a lower bladder capacity, at 0.10 (0.01) vs 0.20 (0.01) mL and micturition pressure, at 25.03 (1.90) vs 36.60 (4.04) cmH2O, respectively, during CMG than WT mice (Fig. 4). The LPP in Loxl1 −/− mice was lower than that in WT mice, at 5.22 (1.23) vs 7.05 (0.81) cmH2O, respectively, but with no statistical difference between the groups. The infused volume for LPP measurement, however, was significantly lower in Loxl1 −/− mice than WT mice at 0.37 (0.06) versus 0.54 (0.03) mL, respectively. The bladder contractility in response to high K+, EFS or direct cholinergic and purinergic stimuli showed similar trends in dose-dependent responses, but there were no significant differences between the bladder strips from Loxl1 −/− and WT mice in their responsiveness to various stimuli (Fig. 5).
The present study was designed to examine bladder and urethral function in Loxl1 −/− mice by using in vivo and in vitro methods. The LOXLs are a group of a copper-dependent monoamine oxidases that are required for the covalent cross-linking of elastin and collagen polymers . In addition to the prototype LOX, genes coding for four LOX-like proteins are present in the mammalian genome, and termed LOXL-1 to 4. LOXL1 is essential in maintaining the elastic fibre network [4,5]. Elastic fibres confer elasticity and resilience to tissues that are normally subjected to stretching and expansive forces . The major component of these fibres is an amorphous polymer made of the protein elastin. The covalent cross-linking of tropoelastin requires an initial step of oxidative deamination of lysine residues catalysed by a LOX . Our group has shown that LOXL1 is invariably found to co-localize with elastic fibres in all tissues examined [5,11]. During pregnancy and parturition, tissues in the reproductive tract undergo profound changes that include breakdown and re-synthesis of elastic fibres. An inability to rebuild the elastic fibre network might be anticipated to produce some structural and functional deficits in these tissues. Mice lacking LOXL1 develop severe POP after giving birth, and have a higher voiding frequency accompanied by a corresponding decrease in volume [5,11]. The cause of the increased frequency lies with the lower urinary tract and could be associated with the bladder, the urethra, or both. The possible mechanisms might involve an overactive bladder, atonic bladder and/or inefficient urethral closure.
The present results show that there were no significant differences between bladder weights in Loxl1 −/− and WT mice, suggesting that long term BOO is less likely in prolapsed Loxl1 −/− mice, as reported previously . Both Loxl1 −/− and WT mice showed a regular and periodic bladder contraction during conscious CMG. There were no spontaneous non-voiding bladder contractions between micturitions in Loxl1 −/− mice. The Loxl1 −/− mice voided more frequently and had a lower micturition pressure during CMG than WT mice. UI is distinguished from normal voiding by the intent or lack thereof, which cannot be determined in rodents. However, the increased voiding frequency accompanied by a corresponding decrease in volume in this mouse model is reminiscent of human UI. These cystometry data suggest that the urinary frequency is not a result of BOO or overactive bladder, but might be the result of an atonic bladder or decreased urethral resistance.
To investigate whether the contractile response of the detrusor muscle was changed in Loxl1 −/− mice, we used four stimulators that induce detrusor smooth muscle contraction via different signalling pathways. High extracellular K+ (KCl) induces the sustained depolarization of smooth muscle, and EFS elicits nerve-mediated detrusor contraction, which acts through the release of neurotransmitters and the subsequent activation of postsynaptic receptors. ATP activates purinergic receptors and carbachol activates muscarinic receptors in detrusor muscle . The present data showed that there were no significant differences between bladder strips from Loxl1 −/− mice and WT mice in their responsiveness to various contractile stimuli, supporting the idea that decreased micturition pressure possibly results from decreased urethral resistance rather than a change in contractile forces of the bladder. Normally, the urethra was tightly adherent to the suburethral vaginal wall along its length, and the urinary bladder was firmly attached at a position near the uterine cervix. However, the upper portion of the urethra in Loxl1 −/− mice was detached from the vaginal wall, potentially allowing for a much greater degree of movement of the urethra and bladder. An effective closure of the female urethra in stress situations depends on an integrated action of various anatomical structures connected to the organ. The most important of these structures (functionally) are the suburethral vaginal wall, the pubourethral ligaments, the pubococcygeus muscles and the paraurethral connective tissues. Given the damage to the pelvic floor and paraurethral tissues, it would be reasonable to assume that there is ineffectual urethral closure.
Surprisingly, the LPP in Loxl1 −/− mice tended to be lower than that in WT mice, but not significantly. However, the volume at which LPP was measured was significantly lower in Loxl1 −/− mice than WT mice, due to their frequency of voiding. Our previous work showed that the LPP is highly consistent at all bladder volumes except low volumes, where it is greater . Therefore, the lack of a significant difference in LPP between WT and Loxl1 −/− mice might be because the LPP in the Loxl1 −/− mice was artificially high at the low volumes.
Another possible mechanism is that the bladder activity was limited in a narrowed space. All Loxl1 −/− mice showed descent of the uterine, bladder and upper vaginal tissues into the lower vaginal cavity, creating a ring-like fold in the upper middle position of the vaginal wall. The severity of the pelvic organ descent was such that the urinary bladder was trapped into this vaginal fold. Considering that the urinary bladder is trapped inside the vaginal cavity during prolapse, it will inevitably limit the extension of the bladder and lead to reduced bladder capacity.
To our knowledge, the Loxl1−/− female mouse represents the first genetic animal model that simulates major aspects of human PFD. In one study, the familial incidence of genital prolapse was 30%. It has been suggested that genetic differences might predispose individuals to UI or POP, but no genetic trait or disorder has yet been conclusively linked to human FPFD. Future investigations will assess whether a failure of elastic-fibre homeostasis also underlies the aetiology of common PFD among older women.
In conclusion, the Loxl1 −/− mice had obvious POP and lower urinary tract complications; they voided more frequently, like UI in humans. Bladder contractility was normal in the Loxl1 −/− mice, but inefficient urethral closure might contribute to the increased frequency. Further studies are needed to characterize the morphological and molecular alterations of the bladder and urethra. Loxl1 −/− mice can be used as an animal model for PFD.
We thank Dr Hui Q. Pan and Paul Zaszczurynski for their assistance with preparation of the experiment. The study was supported by NIH grant -KO8 DK02631 and Animal Models of Diabetic Complications Consortium (http://www.amdcc.org) grant DK61018–02S1. Juvenile Diabetes Research Foundation Fellowship (to G. Liu).
None declared. Source of funding: see Acknowledgements.