A minimally invasive bronchoscopic approach for direct delivery to murine airways and application to models of pulmonary infection.

The laboratory mouse is used extensively for human disease modeling and preclinical therapeutic testing for efficacy, biodistribution, and toxicity. The variety of murine models available, and the ability to create new ones, eclipses all other species, but the size of mice and their organs create challenges for many in vivo studies. For pulmonary research, improved methods to access murine airways and lungs, and track substances administered to them, would be desirable. A nonsurgical endoscopic system with a camera, effectively a bronchoscope, coupled with a cryoimaging fluorescence microscopy technique to view the lungs in 3D, is described here that allows visualization of the procedure, including the anatomical location at which substances are instilled and fluorescence detection of those substances. We have applied it to bacterial infection studies to characterize better and optimize a chronic lung infection murine model in which we instill bacteria-laden agarose beads into the airways and lungs to extend the duration of the infection and inflammation. The use of the endoscope as guidance for placing a catheter into the airways is simple and quick, requiring only momentary sedation, and reduces post-procedural mortality compared with our previous instillation method that includes a trans-tracheal surgery. The endoscopic method improves speed and precision of delivery while reducing the stress on animals and the number of animals generated and used for experiments.

Stromal derived factor-1 plasmid as a novel injection for treatment of stress urinary incontinence in a rat model.

INTRODUCTION AND HYPOTHESIS: SDF-1 chemokine enhances tissue regeneration through stem cell chemotaxis, neovascularization and neuronal regeneration. We hypothesized that non-viral delivery of human plasmids that express SDF-1 (pSDF-1) may represent a novel regenerative therapy for stress urinary incontinence (SUI). METHODS: Seventy-six female rats underwent vaginal distention (VD). They were then divided into four groups according to treatment: pSDF-1 (n = 42), sham (n = 30), PBS (n = 1) and luciferase-tagged pSDF-1 (n = 3). Immediately after VD, the pSDF-1 group underwent immediate periurethral injection of pSDF-1, and the sham group received a vehicle injection followed by leak point pressure (LPP) measurement at the 4th, 7th and 14th days. Urogenital tissues were collected for histology. H&E and trichrome slides were analyzed for vascularity and collagen/muscle components of the sphincter. For the luciferase-tagged pSDF-1 group, bioluminescence scans (BLIs) were obtained on the 3rd, 7th and 14th days following injections. Statistical analysis was conducted using ANOVA with post hoc LSD tests. The Mann-Whitney U test was employed to make pair-wise comparisons between the treated and sham groups. We used IBM SPSS, version 22, for statistical analyses. RESULTS: BLI showed high expression of luciferase-tagged pSDF-1 in the pelvic area over time. VD resulted in a decline of LPP at the 4th day in both groups. The pSDF1-treated group demonstrated accelerated recovery that was significantly higher than that of the sham-treated group at the 7th day (22.64 cmH2O versus 13.99 cmH2O, p < 0.001). Functional improvement persisted until the 14th day (30.51 cmH2O versus 24.11 cmH2O, p = 0.067). Vascularity density in the pSDF-1-treated group was higher than in the sham group at the 7th and 14th days (p < 0.05). The muscle density/sphincter area increased significantly from the 4th to 14th day only in the pSDF-1 group. CONCLUSIONS: Periurethral injection of pSDF-1 after simulated childbirth accelerated the recovery of continence and regeneration of the urethral sphincter in a rat SUI model. This intervention can potentially be translated to the treatment of post-partum urinary incontinence.

Dynamic, Simultaneous Concentration Mapping of Multiple MRI Contrast Agents with Dual Contrast - Magnetic Resonance Fingerprinting.

Synchronous assessment of multiple MRI contrast agents in a single scanning session would provide a new "multi-color" imaging capability similar to fluorescence imaging but with high spatiotemporal resolution and unlimited imaging depth. This multi-agent MRI technology would enable a whole new class of basic science and clinical MRI experiments that simultaneously explore multiple physiologic/molecular events in vivo. Unfortunately, conventional MRI acquisition techniques are only capable of detecting and quantifying one paramagnetic MRI contrast agent at a time. Herein, the Dual Contrast - Magnetic Resonance Fingerprinting (DC-MRF) methodology was extended for in vivo application and evaluated by simultaneously and dynamically mapping the intra-tumoral concentration of two MRI contrast agents (Gd-BOPTA and Dy-DOTA-azide) in a mouse glioma model. Co-registered gadolinium and dysprosium concentration maps were generated with sub-millimeter spatial resolution and acquired dynamically with just over 2-minute temporal resolution. Mean tumor Gd and Dy concentration measurements from both single agent and dual agent DC-MRF studies demonstrated significant correlations with ex vivo mass spectrometry elemental analyses. This initial in vivo study demonstrates the potential for DC-MRF to provide a useful dual-agent MRI platform.

Smooth muscle-specific deletion of MnSOD exacerbates diabetes-induced bladder dysfunction in mice.

Bladder dysfunction in diabetes progresses gradually over time. However, the mechanisms of the development are not clear. We tested the hypothesis that oxidative stress plays a key role in the development of diabetic bladder dysfunction using an inducible smooth muscle (SM)-specific superoxide dismutase 2 (Sod2) gene knockout (SM-Sod2 KO) mouse model. Eight-week-old male Sod2lox/lox, SM-CreERT2(ki)Cre/+ mice and wild-type mice were assigned to diabetic or control groups. 4-Hydroxytamoxifen was injected into Sod2lox/lox, SM-CreERT2(ki)Cre/+ mice to activate CreERT2-mediated deletion of Sod2. Diabetes was induced by injection of streptozotocin, whereas control mice were injected with vehicle. Nine weeks later, bladder function was evaluated, and bladders were harvested for immunoblot analysis. Wild-type diabetic mice presented compensated bladder function along with increased nitrotyrosine and MnSOD in detrusor muscle. Induction of diabetes in SM-Sod2 KO mice caused deteriorated bladder function and even greater increases in nitrotyrosine compared with wild-type diabetic mice. Expression levels of apoptosis regulator Bax and cleaved caspase-3 were increased, but apoptosis regulator Bcl-2 expression was decreased in detrusor muscle of both diabetic groups, with more pronounced effects in SM-Sod2 KO diabetic mice. Our findings demonstrate that exaggerated oxidative stress can accelerate the development of bladder dysfunction in diabetic mice and the enhanced activation of apoptotic pathways in the bladder may be involved in the process.

The vaginal distention model in mice is not a reliable model of simulated birth trauma-induced stress urinary incontinence.

AIMS: Vaginal distention (VD) is a validated model of birth-related trauma in rats. Recently a mouse VD model was reported. Our study was originally conducted to evaluate the impact of age on VD in mice. This manuscript describes the study and reports on the lack of reproducibility of VD models in mice. METHODS: We utilized female C57BL/6 mice. A total of 190, 12-weeks old mice, were randomized into VD and sham groups. We inflated a modified Foley's balloon with 0.3 mL for 1 h inside the mice vagina. Afterwards, we measured the leak point pressure (LPP) at defined timepoints (0, 4, 10, 20, or 40 days). We randomized another 190, 40-week old, C57BL/6 mice into either VD or sham groups. We used an extra 20 mice as age - matched controls. RESULTS: In both 12 and 40 weeks-old mice, LPP was significantly decreased versus the negative controls at day 0. Additionally, in both 12 and 40 weeks-old mice, the decrease in LPP was significantly higher in the VD group compared to the sham group at day 0. However, the LPP results were comparable between VD and sham at any other time point thereafter. Furthermore, there was no significant change in LPP values between instrumented (VD and sham) mice and control mice at any time after day 0. CONCLUSIONS: The VD models previously described is not a reproducible model for the study of VD with large number of mice. Our results, unfortunately, do not support its use to study VD injury in mice.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis.

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

A genetic female mouse model with congenital genitourinary anomalies and adult stages of urinary incontinence.

AIMS: To characterize the urinary incontinence observed in adult Gli2+/- ; Gli3Δ699/+ female mice and identify the defects underlying the condition. METHODS: Gli2+/- and Gli3Δ699/+ mice were crossed to generate: wild-type, mutant Gli2 (Gli2+/- ), mutant Gli3 (Gli3Δ699/+ ), and double mutant (Gli2+/- ; Gli3Δ699/+ ) female mice, verified via Polymerase Chain Reactions. Bladder functional studies including cystometrogram (CMG), leak point pressure (LPP), and voiding testing were performed on adult female mice. Female bladders and urethras were also analyzed via ink injection and histological assays. RESULTS: CMG tracing showed no signal corresponding to the filling of the Gli2+/- ; Gli3Δ699/+ bladders. LPP were significantly reduced in Gli2+/- ; Gli3Δ699/+ mice compared to wild-type mice. CMG studies revealed a decrease in peak micturition pressure values in Gli2+/- ; Gli3Δ699/+ mice compared with all other groups. No significant differences between mutant and wild-type mice were detected in urinary output. Histological analyses revealed Gli2+/- ; Gli3Δ699/+ mice exhibited a widened urethra and a decrease in smooth muscle layer thickness in the bladder outlet and urethra, with increased mucosal folding. CONCLUSIONS: Gli2+/- ; Gli3Δ699/+ adult female mice display persistent urinary incontinence due to the malformation of the bladder outlet and urethra. This presents a consistent and reliable genetic mouse model for female urinary incontinence and alludes to the key role of genetic factors involved in the condition.

Functional and morphological alterations of the urinary bladder in type 2 diabetic FVB(db/db) mice.

AIMS: Diabetic bladder dysfunction (DBD) has been extensively studied in animal models of type 1 diabetes. We aimed to examine the functional and morphological alterations of the urinary bladder in a type 2 diabetes model, FVB(db/db) mice. METHODS: FVB(db/db) mice and age-matched FVB/NJ control mice were tested at either 12, 24 or 52weeks of age. Body weight, blood glucose and glycated hemoglobin (HbA1c) levels were measured. Bladder function was assessed by measurement of 24-h urination behavior and conscious cystometry. Bladder was harvested for Masson's Trichrome staining and morphometric analysis. RESULTS: The body weights of FVB(db/db) mice were twice as those of FVB/NJ control mice. The blood glucose and HbA1c levels were higher in FVB(db/db) mice at 12 and 24weeks, but not at 52weeks. A significant increase in the mean volume per void, but decrease in the voiding frequency, in FVB(db/db) mice was observed. Cystometry evaluation showed increased bladder capacity, voided volume, and peak micturition pressure in FVB(db/db) mice compared with FVB/NJ mice. Morphometric analysis revealed a significant increase in the areas of detrusor muscle and urothelium in FVB(db/db) mice. In addition, some FVB(db/db) mice, especially males at 12 and 24weeks, showed small-volume voiding during 24-h urination behavior measurement, and detrusor overactivity in the cystometry measurement. CONCLUSIONS: The FVB(db/db) mouse, displaying DBD characterized by not only increased bladder capacity, void volume, and micturition pressure, but also bladder overactivity, is a useful model to further investigate the mechanisms of type 2 diabetes-related bladder dysfunction.

Local release from affinity-based polymers increases urethral concentration of the stem cell chemokine CCL7 in rats.

The protein chemokine (C-C motif) ligand 7 (CCL7) is significantly over-expressed in urethral and vaginal tissues immediately following vaginal distention in a rat model of stress urinary incontinence. Further evidence, in this scenario and other clinical scenarios, indicates CCL7 stimulates stem cell homing for regenerative repair. This CCL7 gradient is likely absent or compromised in the natural repair process of women who continue to suffer from SUI into advanced age. We evaluated the feasibility of locally providing this missing CCL7 gradient by means of an affinity-based implantable polymer. To engineer these polymers we screened the affinity of different proteoglycans, to use them as CCL7-binding hosts. We found heparin to be the strongest binding host for CCL7 with a 0.323 nM dissociation constant. Our experimental approach indicates conjugation of heparin to a polymer backbone (using either bovine serum albumin or poly (ethylene glycol) as the base polymer) can be used as a delivery system capable of providing sustained concentrations of CCL7 in a therapeutically useful range up to a month in vitro. With this approach we are able to detect, after polymer implantation, significant increase in CCL7 in the urethral tissue directly surrounding the polymer implants with only trace amounts of human CCL7 present in the blood of the animals. Whole animal serial sectioning shows evidence of retention of locally injected human mesenchymal stem cells (hMSCs) only in animals with sustained CCL7 delivery, 2 weeks after affinity-polymers were implanted.

Mesenchymal stem cell therapy in a rat model of birth-trauma injury: functional improvements and biodistribution.

INTRODUCTION AND HYPOTHESIS: We evaluated the potential role of human mesenchymal stem cells (hMSCs) in improvement of urinary continence following birth-trauma injury. METHODS: Human MSCs were injected periurethrally or systemically into rats immediately after vaginal distention (VD) (n = 90). Control groups were non-VD (uninjured/untreated, n = 15), local or systemic saline (injection/control, n = 90), and dermofibroblast (cell therapy/control, n = 90). Leak-point pressure (LPP) was measured 4, 10, and 14 days later. Urethras were morphometrically evaluated. In another sets of VD and non-VD rats, the fate of periurethrally injected hMSC, biodistribution, and in vivo viability was studied using human Alu genomic repeat staining, PKH26 labeling, and luciferase-expression labeling, respectively. RESULTS: Saline- and dermofibroblast-treated control rats demonstrated lower LPP than non-VD controls at days 4 and 14 (P < 0.01). LPP after systemic hMSC and periurethral hMSC treatment were comparable with non-VD controls at 4, 10, and 14 days (P > 0.05). Local saline controls demonstrated extensive urethral tissue bleeding. The connective tissue area/urethral section area proportion and vascular density were higher in the local hMSC- versus the saline-treated group at 4 and 14 days, respectively. No positive Alu-stained nuclei were observed in urethras at 4, 10, and 14 days. PKH26-labelled cells were found in all urethras at 2 and 24 h. Bioluminescence study showed increased luciferase expression from day 0 to 1 following hMSC injection. CONCLUSIONS: Human MSCs restored the continence mechanism with an immediate and sustained effect in the VD model, while saline and dermofibroblast therapy did not. Human MSCs remained at the site of periurethral injection for <7 days. We hypothesize that periurethral hMSC treatment improves vascular, connective tissue, and hemorrhage status of urethral tissues after acute VD injury.

Bladder function in mice with inducible smooth muscle-specific deletion of the manganese superoxide dismutase gene.

Manganese superoxide dismutase (MnSOD) is considered a critical component of the antioxidant systems that protect against oxidative damage. We are interested in the role of oxidative stress in bladder detrusor smooth muscle (SM) in different disease states. In this study, we generated an inducible, SM-specific Sod2(-/-) mouse model to investigate the effects of MnSOD depletion on the function of the bladder. We crossbred floxed Sod2 (Sod2(lox/lox)) mice with mice containing heterozygous knock-in of a gene encoding a tamoxifen-activated Cre recombinase in the SM22α promoter locus [SM-CreER(T2)(ki)(Cre/+)]. We obtained Sod2(lox/lox),SM-CreER(T2)(ki)(Cre/+) mice and injected 8-wk-old males with 4-hydroxytamoxifen to induce Cre-mediated excision of the floxed Sod2 allele. Twelve weeks later, SM-specific deletion of Sod2 and depletion of MnSOD were confirmed by polymerase chain reaction, immunoblotting, and immunohistochemistry. SM-specific Sod2(-/-) mice exhibited normal growth with no gross abnormalities. A significant increase in nitrotyrosine concentration was found in bladder SM tissue of SM-specific Sod2(-/-) mice compared with both wild-type mice and Sod2(+/+), SM-CreER(T2)(ki)(Cre/+) mice treated with 4-hydroxytamoxifen. Assessment of 24-h micturition in SM-specific Sod2(-/-) mice revealed significantly higher voiding frequency compared with both wild-type and SM-specific Cre controls. Conscious cystometry revealed significantly shorter intercontraction intervals and lower functional bladder capacity in SM-specific Sod2(-/-) mice compared with wild-type mice. This novel model can be used for exploring the mechanistic role of oxidative stress in organs rich in SM in different pathological conditions.

Short-term diabetes- and diuresis-induced alterations of the bladder are mostly reversible in rats.

OBJECTIVES: To determine whether diabetes mellitus- and diuresis-induced alterations in the bladder can be reversed in rats. METHODS: Male Sprague-Dawley rats were randomly distributed into eight groups (n = 16 per group): 3 weeks and 11 weeks age-matched controls, 3 weeks and 11 weeks after streptozotocin-induced diabetes mellitus, 3 weeks after diabetes mellitus induction then treated with insulin for 8 weeks, 3 weeks and 11 weeks after 5% sucrose-induced diuresis, and 3 weeks after 5% sucrose-induced diuresis followed by removal of 5% sucrose for 8 weeks. Bodyweight, blood glucose and glycated hemoglobin A1c were monitored. At the designated time-points, 24-h urinary habits were examined, and cystometry was carried out in half of the animals. The bladders from the remaining animals were harvested for histological examination, and quantification of smooth muscle, urothelium and collagen components. RESULTS: Insulin treatment reversed hyperglycemia and polyuria in diabetic animals successfully, which was shown by normalization of blood glucose, glycated hemoglobin A1c and 24-h urinary habits. Subsequently, bodyweight, bladder weight and percentage change of bladder components (smooth muscle, collagen, urothelium) in total bladder cross-sectional area were reversed to almost normal levels, and the bladder dysfunction was mostly reversed by 8 weeks of glycemic control, seen in the cystometry study. Similar alterations and reversed effects were seen in diuretic rats without and with 5% sucrose removal, respectively. CONCLUSIONS: Short-term (3-week induction) diabetes- and polyuria-induced functional and morphological alterations of the bladder can mostly be reversed in rats.

Chronic pelvic allodynia is mediated by CCL2 through mast cells in an experimental autoimmune cystitis model.

The cause of chronic pelvic pain in interstitial cystitis/painful bladder syndrome (IC/PBS) remains unclear; autoimmunity is a possible etiology. We have recently shown that injection of a single immunogenic peptide of uroplakin 3A (UPK3A 65-84) induces experimental autoimmune cystitis (EAC) in female BALB/cJ mice that is unique among experimental models in accurately reflecting both the urinary symptoms and pelvic pain of IC/PBS. The aim of this project was to identify the roles of mast cells and mast cell chemoattractant/activator monocyte chemoattractant protein-1 [chemokine (C-C motif) ligand 2 (CCL2)] in the allodynia in this model. We immunized 6- to 8-wk-old female BALB/cJ mice with UPK3A 65-84 peptide and, 5-40 days later, observed increased responses to stimulation of the suprapubic abdominal and hindpaw surfaces with von Frey monofilaments compared with mice injected with adjuvant alone. Suprapubic and hindpaw tactile allodynia responses by EAC mice were blocked by instillation of lidocaine into the bladder but not by lidocaine in the uterus, confirming the bladder as the source of the hypersensitivity. Markedly increased numbers of activated mast cells and expression of CCL2 were found in the bladder after immunization with UPK3A 65-84. Hypersensitive responses were inhibited by mast cell stabilizer cromolyn sodium and antagonists of histamine receptors 1 and 2. Furthermore, BALB/cJ mice with deletion of the Ccl2 or chemokine (C-C motif) receptor 2 gene exhibited markedly reduced allodynia and accumulation of mast cells after UPK3A 65-84 immunization. These results show that UPK3A 65-84 immunization causes chronic visceral allodynia and suggest that it is mediated by CCL2-driven mast cell accumulation in the bladder.

Stem cell homing factor, CCL7, expression in mouse models of stress urinary incontinence.

OBJECTIVES: Animal models of vaginal distention (VD) have demonstrated increased expression of chemokine (C-C motif) ligand 7 (CCL7) In this study, we investigated the expression of CCL7 in mice models of simulated birth trauma-induced urinary incontinence using VD and pudendal nerve transection (PNT). METHODS: Forty-nine mice were divided into 6 groups: VD, sham VD, PNT, sham PNT, anesthesia, and age-matched controls. The urethra, vagina, and rectum were harvested for the expression of CCL7 immediately or 24 hours after assigned procedure. Venous sampling for quantification of serum CCL7 was also performed. An analysis of variance model was used to compare the relative expression of CCL7 in each group. RESULTS: Urethral CCL7 expression in the VD group was significantly higher than control group after 24 hours (P < 0.01). There was no difference in the urethral CCL7 expression in PNT, sham PNT, sham VD, or anesthesia groups compared with the controls. No statistically significant difference was noted in the vaginal and rectal expression of CCL7 between any of the groups except for sham PNT. Statistically significant differences were noted in the serum CCL7 expression in the VD, PNT, and sham PNT (P < 0.01 in all) groups after 24 hours compared with the control group. CONCLUSIONS: This study demonstrates overexpression of urethral CCL7 after VD but not PNT. This suggests that nerve injury does not contribute to the CCL7 overexpression. The overexpression of CCL7 in the serum of mice after VD suggests a translational potential where CCL7 measurement could be used as a surrogate for injury after delivery.

Setting a new standard: updating the vaginal distention translational model for stress urinary incontinence.

AIMS: The vaginal distention (VD) translational model for postpartum stress urinary incontinence (SUI) is potentially biased for use in evaluating animals with increasing phenotypic size (obesity) due to a fixed VD volume. Our study had three principle and two secondary aims. First, to examine both ex vivo and in vivo catheter pressure changes during volume distention. Secondly, to determine mean pressure at current volume standard for use as target pressure (TP) for VD under isobaric (IB) conditions. Thirdly, to demonstrate feasibility and equivalence of VD at TP versus isovolumetric (IV) standard. Secondary aims were to demonstrate decreased variability (IB vs. IV) and to review the effect of weight. METHODS: Ten French modified Foley catheters were inflated sequentially to 3.0 ml while connected (both in vivo and ex vivo) to a pressure transducer. Mean result generated TP. Thirty Sprague-Dawley rats (280-300 g) were then randomized to one of three groups: IV, IB at TP, or sham VD. Student's t-test was used to compare groups' leak point pressures (LPP) and simple linear regression was used to evaluate the effect of weight as a continuous variable. RESULTS: Catheter pressure/volume responses were demonstrated. Calculated TP was 531 mm Hg. LPP under conditions of IB and IV were statistically equivalent and were statistically lower than Sham. Variability was not statistically different between IB and IV groups. When treated as a continuous variable, weight had no effect on LPP. CONCLUSIONS: VD injury based on TP is feasible and reproducible. Understanding catheter pressure dynamics is valuable for investigating alternative rat phenotypes.

Autoimmunity to uroplakin II causes cystitis in mice: a novel model of interstitial cystitis.

BACKGROUND: The pathophysiology of interstitial cystitis (IC) is unknown. Deficits in urothelial cell layers and autoimmune mechanisms may play a role. OBJECTIVE: To examine whether immunization of mice with recombinant mouse uroplakin II (rmUPK2), a bladder-specific protein, would provoke an autoimmune response sufficient to create an IC phenotype. DESIGN, SETTING, AND PARTICIPANTS: RmUPK2 complementary DNA was generated, transferred into a bacterial expression vector, and the generated protein was purified. Eight-week-old SWXJ female mice were immunized with rmUPK2 protein via subcutaneous injection of 200µg of rmUPK2 protein in 200µl of an emulsion. MEASUREMENTS: Mice were euthanized 5 wk after immunization. Axillary and inguinal lymph node cells were tested for antigen-specific responsiveness and cytokine production, serum isotype antibody titers against rmUPK2 were determined, and gene expression of inflammatory mediators was measured in the bladder and other organs. For functional analysis, mice were placed in urodynamic chambers for 24-h micturition frequency and total voided urine measurements. RESULTS AND LIMITATIONS: Immunization with rmUPK2 resulted in T-cell infiltration of the bladder urothelium and increased rmUPK2-specific serum antibody responses in the experimental autoimmune cystitis (EAC) mice models compared with controls. The ratio of bladder to body weight was increased in EAC mice. Quantitative reverse transcriptase polymerase chain reaction analysis showed elevated gene expression of tumor necrosis factor α, interferon γ, interleukin (IL)-17A, and IL-1ß in bladder urothelium but not in other organs. Evaluation of 24-h micturition habits of EAC mice showed significantly increased urinary frequency (p<0.02) and significantly decreased urine output per void (p<0.021) when compared with control mice. CONCLUSIONS: Our study showed that a bladder-specific autoimmune response sufficient to induce inflammation and EAC occurs in mice following immunization with rmUPK2. EAC mice displayed significant evidence of urinary frequency and decreased urine output per void. Further phenotype characterization of EAC mice should include evidence for pain and/or afferent hypersensitivity, and evidence of urothelial cell layer damage.

Expression of monocyte chemotactic protein 3 following simulated birth trauma in a murine model of obesity.

OBJECTIVE: To determine the effect of obesity on simulated birth trauma in leptin-deficient obese mice as measured by relative monocyte chemotactic protein 3 (MCP-3) expression. MATERIALS AND METHODS: A total of 25 wild-type and 25 obese C57BL/6 virgin female mice underwent 1 hour of vaginal distension (VD), sham VD, or anesthesia without VD. Pelvic organ tissues were then harvested either immediately or 24-hours post VD and subsequent real-time polymerase chain reaction analysis was performed. RESULTS: Urethral MCP-3 levels in wild-type mice were elevated from baseline at 0 hours with a return to baseline at 24 hours in both VD and sham VD groups. In obese mice, there was a 6-fold elevation in MCP-3 levels at 0 hours after sham VD vs control (P <.05), which then returned to baseline levels at 24 hours. After undergoing VD, MCP-3 levels increased to 6-fold baseline values (P = .002) at 0 hours, with continued elevation in MCP-3 levels to 15 times control levels (P = .0003) at 24 hours. CONCLUSIONS: MCP-3 is significantly over-expressed in the urethral tissues of both wild-type and obese mice immediately after any urethral manipulation. At 24 hours, the MCP-3 expression patterns become divergent between VD and sham VD in obese mice. With a greater degree of trauma, MCP-3 continued to rise at 24 hours, suggesting that the underlying obesity resulted in alterations in response to tissue injury, paralleling the degree of injury. Such associations warrant further investigation into the role of MCP-3 as a chemokine for stem cell migration, with implications for subsequent tissue repair mechanisms after birth trauma.

Bladder dysfunction in mice with experimental autoimmune encephalomyelitis.

The vast majority of patients with multiple sclerosis (MS) develop bladder control problems including urgency to urinate, urinary incontinence, frequency of urination, and retention of urine. Over 60% of MS patients show detrusor-sphincter dyssynergia, an abnormality characterized by obstruction of urinary outflow as a result of discoordinated contraction of the urethral sphincter muscle and the bladder detrusor muscle. In the current study we examined bladder function in female SWXJ mice with different defined levels of neurological impairment following induction of experimental autoimmune encephalomyelitis (EAE), an animal model of central nervous system inflammation widely used in MS research. We found that EAE mice develop profound bladder dysfunction characterized by significantly increased micturition frequencies and significantly decreased urine output per micturition. Moreover, we found that the severity of bladder abnormalities in EAE mice was directly related to the severity of clinical EAE and neurologic disability. Our study is the first to show and characterize micturition abnormalities in EAE mice thereby providing a most useful model system for understanding and treating neurogenic bladder.

Lower urinary tract phenotype of experimental autoimmune cystitis in mouse: a potential animal model for interstitial cystitis.

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-gamma (IFN-gamma) 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-gamma 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.