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MP26-10 NEUROGENIC DETRUSOR UNDERACTIVITY: SHOULD WE TARGET THE BLADDER?
THE JOURNAL OF UROLOGYâ
Vol. 197, No. 4S, Supplement, Saturday, May 13, 2017
alteration of histopathology and expression of TLR4 and NLRP3 inflammasome-related molecules in the bladder using spontaneously hypertensive rats (SHRs) as an OAB model. METHODS: Twenty-weeks-old male SHRs and Wistar Kyoto rats (control) were used. After voiding function was analyzed by using metabolic cages, the bladder was excised for analysis of histopathology and mRNA expression. Hematoxylin eosin and Masson0 s trichrome stain were performed to analyze bladder inflammatory condition and fibrosis. Immunohistostaining for NLRP3, TLR4 was also performed. Expression levels of NLRP3, IL1b, IL-18, IL6, IL8 and TGFb mRNA in the bladder were investigated by real-time PCR. Statistical analysis was performed using Mann-Whitney U test. P value less than 0.05 was considered statistically significant. RESULTS: In voiding function analyses, single urine volume was significantly decreased and voiding frequency was significantly increased in SHRs compared to control rats. In histological evaluation, suburothelial fibrosis was shown in SHRs compared to controls. Furthermore, immunohistostaining showed localized expression of NLRP3 and TLR4 in the bladder urothelium in both groups. In RT-qPCR analyses, mRNA expression levels of NLRP3, TLR4, IL1b, IL-18, IL6, IL8 and TGFb were significantly increased in SHRs in the bladder compared to controls. CONCLUSIONS: These results suggest that activation of TLR4 associated with oxidative stress is implicated in bladder chronic inflammation, which leads to frequent urination through NLRP3 inflammasome pathways. Therefore, further clarification of interactions between TLR4 and inflammasome pathways may offer new therapeutic targets for OAB associated with chronic inflammation. Source of Funding: none
MP26-10 NEUROGENIC DETRUSOR UNDERACTIVITY: SHOULD WE TARGET THE BLADDER? Karel Dewulf*, Emmanuel Weyne, Yves Deruyver, Rita Van Bree, Godelieve Verbist, Dirk De Ridder, Maarten Albersen, Wouter Everaerts, Leuven, Belgium INTRODUCTION AND OBJECTIVES: Pelvic surgery induced detrusor underactivity (DU) remains a poorly understood condition. Preganglionic pelvic nerve crush injury (PNI) in the rat has been described as a model for neurogenic DU. In this study we investigate temporal changes in detrusor contractility, detrusor fibrosis and denervation of the pelvic plexus as potential players in the pathophysiology of DU. METHODS: Male Sprague-Dawley rats (10-12w) underwent PNI 3x15s or sham surgery. One, three and nine weeks after surgery, functional and molecular assessments were planned. Detrusor contractility was examined in vitro in organ bath studies. Contractile responses tot KCl 120mM, cumulative doses of carbachol, noncumulative doses of a,b-methylene ATP (mATP) and electrical field stimulation (EFS) were recorded. Gene expression was assessed by RT-qPCR. The genes evaluated in the bladder and major pelvic ganglion (MPG) were: vesicular acetylcholine transporter (VAchT), tyrosine hydroxylase (TH), protein gene product 9.5 (PGP9.5); and in the bladder: M2 and M3 muscarinic receptors, P2X1 purinergic receptor, collagen 1 and 3 and smoothelin. Statistical analyses were performed by one-way ANOVA with Tukey-Kramer post test. RESULTS: No changes were observed in contractile responses to KCl 120mM. One week after PNI, a 82% upregulation of collagen 1 was observed with a 77% reduction at 9 weeks (p<0,0001). Smoothelin expression was reduced by 45% 1 week after PNI with recovery at 9 weeks (p<0,0001), all compared to sham. Compared to sham, maximum contractile responses to carbachol and mATP were preserved at 3 and 9 weeks following PNI. Relative expression of M3 was increased at 3 (+59%) and 9 weeks (+46%) compared to sham (p¼0,0002), but no differences were seen in M2 and P2X1 expression. Functional detrusor denervation was objectified by EFS: at 32 Hz contractile responses
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were reduced at 1 week (-40%), 3 weeks (-23%) and 9 weeks (-24%) after PNI compared to sham (p<0,0001). In the bladder, VAchT expression was 4 times less at 9 weeks (p¼0,03), whereas TH expression was 76% lower 1 week following PNI with partial recovery at 9 weeks (p¼0,0067), all compared to sham. Similarly, TH expression was 57% less in the MPG 1 week after PNI compared to sham with recovery at 9 weeks (p<0,0001). In the MPG, the expression of PGP9.5 was reduced following PNI compared to sham (p¼0,002). CONCLUSIONS: Functional and molecular denervation of the bladder and MPG is observed in our rat model for neurogenic DU. However, the detrusor remains contractile to parasympathetic stimulation. Therefore, research should focus on optimizing neural regeneration of the pelvic plexus. Source of Funding: None
MP26-11 THERAPEUTIC OUTCOMES OF INSULIN-LIKE GROWTH FACTOR-1 RELEASED FROM ALGINATE-GELATIN MICROBEADS ON STRESS URINARY INCONTINENCE IN RATS WITH SIMULATED CHILDBIRTH INJURY Hao Yan*, Beijing, China, People’s Republic of; Liren Zhong, Yaodong Jiang, Jian Yang, Winston-Salem, NC; Dan Li Lin, Cleveland, OH; Xiaoyi Yuan, Wuhan, China, People’s Republic of; Mei Kuang, Anna Rietsch, Cleveland, OH; Emmanuel Opara, Winston-Salem, NC; Margot Damaser, Cleveland, OH; Yuanyuan Zhang, Winston-Salem, NC INTRODUCTION AND OBJECTIVES: Insulin-like growth factor-1 (IGF-1) treatment has been reported to accelerate recovery from stress urinary incontinence (SUI) induced by simulated childbirth injury in rats. However, a local sustained delivery method is ideal for further clinical applications to avoid side effects of IGF-1. The goal of this study was to determine the effects of controlled release of IGF1 from alginate-gelatin microbeads (IGF1-A-G-beads) on sphincter tissue repair in a rat model of stress urinary incontinence (SUI). METHODS: Forty-four female Sprague-Dawley rats were divided into 4 equal groups: sham vaginal distension (VD) + saline, VD + saline, VD + empty A-G-beads, & VD + IGF1-A-G-beads). All rats received periurethral injections of A-G-beads immediately after VD. Leak point pressure (LPP) testing and external urethral sphincter (EUS) electromyography (EMG) were performed 1 week later. Urethral tissue and anterior vagina were dissected en bloc for further analysis via histology and immunofluorescence. Quantitative data was analyzed using ANOVA on Ranks followed by a Tukey posthoc test with p < 0.05 indicating a statistically significant difference between groups. Data is presented as mean +/- standard error of the mean. RESULTS: LPP was significantly decreased 1 week after VD treated with saline only (23.9 1.3 cmH2O) compared to sham VD (44.4 3.4 cmH2O). LPP was also significantly decreased in the VD + empty A-G-beads group (21.7 0.8 cmH2O) compared to sham VD, demonstrating that the microbeads themselves do not create a bulking or obstructive effect in the urethra. In contrast, rats with VD treated with IGF1-A-G-beads (28.4 1.2 cmH2O) was significantly greater than LPP of rats with VD treated with empty AG-beads and had LPP partway between and not significantly different from either the sham VD or the VD + saline groups, demonstrating initiation of a reparative effect 1 week after VD. The increase in EUS EMG amplitude with LPP testing was significantly reduced after VD treated with saline or empty A-G-beads compared to sham VD. VD rats treated with IGF1-A-G-beads had EUS EMG amplitude response to LPP testing partway between and not significantly different from either the sham VD or VD + saline groups. Histological analysis demonstrated well-developed, wellorganized skeletal muscle fibers in the external urethral sphincter in the VD + IGF1-A-G-beads group, similar to that of sham VD rats. In
Vol. 197, No. 4S, Supplement, Saturday, May 13, 2017
alteration of histopathology and expression of TLR4 and NLRP3 inflammasome-related molecules in the bladder using spontaneously hypertensive rats (SHRs) as an OAB model. METHODS: Twenty-weeks-old male SHRs and Wistar Kyoto rats (control) were used. After voiding function was analyzed by using metabolic cages, the bladder was excised for analysis of histopathology and mRNA expression. Hematoxylin eosin and Masson0 s trichrome stain were performed to analyze bladder inflammatory condition and fibrosis. Immunohistostaining for NLRP3, TLR4 was also performed. Expression levels of NLRP3, IL1b, IL-18, IL6, IL8 and TGFb mRNA in the bladder were investigated by real-time PCR. Statistical analysis was performed using Mann-Whitney U test. P value less than 0.05 was considered statistically significant. RESULTS: In voiding function analyses, single urine volume was significantly decreased and voiding frequency was significantly increased in SHRs compared to control rats. In histological evaluation, suburothelial fibrosis was shown in SHRs compared to controls. Furthermore, immunohistostaining showed localized expression of NLRP3 and TLR4 in the bladder urothelium in both groups. In RT-qPCR analyses, mRNA expression levels of NLRP3, TLR4, IL1b, IL-18, IL6, IL8 and TGFb were significantly increased in SHRs in the bladder compared to controls. CONCLUSIONS: These results suggest that activation of TLR4 associated with oxidative stress is implicated in bladder chronic inflammation, which leads to frequent urination through NLRP3 inflammasome pathways. Therefore, further clarification of interactions between TLR4 and inflammasome pathways may offer new therapeutic targets for OAB associated with chronic inflammation. Source of Funding: none
MP26-10 NEUROGENIC DETRUSOR UNDERACTIVITY: SHOULD WE TARGET THE BLADDER? Karel Dewulf*, Emmanuel Weyne, Yves Deruyver, Rita Van Bree, Godelieve Verbist, Dirk De Ridder, Maarten Albersen, Wouter Everaerts, Leuven, Belgium INTRODUCTION AND OBJECTIVES: Pelvic surgery induced detrusor underactivity (DU) remains a poorly understood condition. Preganglionic pelvic nerve crush injury (PNI) in the rat has been described as a model for neurogenic DU. In this study we investigate temporal changes in detrusor contractility, detrusor fibrosis and denervation of the pelvic plexus as potential players in the pathophysiology of DU. METHODS: Male Sprague-Dawley rats (10-12w) underwent PNI 3x15s or sham surgery. One, three and nine weeks after surgery, functional and molecular assessments were planned. Detrusor contractility was examined in vitro in organ bath studies. Contractile responses tot KCl 120mM, cumulative doses of carbachol, noncumulative doses of a,b-methylene ATP (mATP) and electrical field stimulation (EFS) were recorded. Gene expression was assessed by RT-qPCR. The genes evaluated in the bladder and major pelvic ganglion (MPG) were: vesicular acetylcholine transporter (VAchT), tyrosine hydroxylase (TH), protein gene product 9.5 (PGP9.5); and in the bladder: M2 and M3 muscarinic receptors, P2X1 purinergic receptor, collagen 1 and 3 and smoothelin. Statistical analyses were performed by one-way ANOVA with Tukey-Kramer post test. RESULTS: No changes were observed in contractile responses to KCl 120mM. One week after PNI, a 82% upregulation of collagen 1 was observed with a 77% reduction at 9 weeks (p<0,0001). Smoothelin expression was reduced by 45% 1 week after PNI with recovery at 9 weeks (p<0,0001), all compared to sham. Compared to sham, maximum contractile responses to carbachol and mATP were preserved at 3 and 9 weeks following PNI. Relative expression of M3 was increased at 3 (+59%) and 9 weeks (+46%) compared to sham (p¼0,0002), but no differences were seen in M2 and P2X1 expression. Functional detrusor denervation was objectified by EFS: at 32 Hz contractile responses
e323
were reduced at 1 week (-40%), 3 weeks (-23%) and 9 weeks (-24%) after PNI compared to sham (p<0,0001). In the bladder, VAchT expression was 4 times less at 9 weeks (p¼0,03), whereas TH expression was 76% lower 1 week following PNI with partial recovery at 9 weeks (p¼0,0067), all compared to sham. Similarly, TH expression was 57% less in the MPG 1 week after PNI compared to sham with recovery at 9 weeks (p<0,0001). In the MPG, the expression of PGP9.5 was reduced following PNI compared to sham (p¼0,002). CONCLUSIONS: Functional and molecular denervation of the bladder and MPG is observed in our rat model for neurogenic DU. However, the detrusor remains contractile to parasympathetic stimulation. Therefore, research should focus on optimizing neural regeneration of the pelvic plexus. Source of Funding: None
MP26-11 THERAPEUTIC OUTCOMES OF INSULIN-LIKE GROWTH FACTOR-1 RELEASED FROM ALGINATE-GELATIN MICROBEADS ON STRESS URINARY INCONTINENCE IN RATS WITH SIMULATED CHILDBIRTH INJURY Hao Yan*, Beijing, China, People’s Republic of; Liren Zhong, Yaodong Jiang, Jian Yang, Winston-Salem, NC; Dan Li Lin, Cleveland, OH; Xiaoyi Yuan, Wuhan, China, People’s Republic of; Mei Kuang, Anna Rietsch, Cleveland, OH; Emmanuel Opara, Winston-Salem, NC; Margot Damaser, Cleveland, OH; Yuanyuan Zhang, Winston-Salem, NC INTRODUCTION AND OBJECTIVES: Insulin-like growth factor-1 (IGF-1) treatment has been reported to accelerate recovery from stress urinary incontinence (SUI) induced by simulated childbirth injury in rats. However, a local sustained delivery method is ideal for further clinical applications to avoid side effects of IGF-1. The goal of this study was to determine the effects of controlled release of IGF1 from alginate-gelatin microbeads (IGF1-A-G-beads) on sphincter tissue repair in a rat model of stress urinary incontinence (SUI). METHODS: Forty-four female Sprague-Dawley rats were divided into 4 equal groups: sham vaginal distension (VD) + saline, VD + saline, VD + empty A-G-beads, & VD + IGF1-A-G-beads). All rats received periurethral injections of A-G-beads immediately after VD. Leak point pressure (LPP) testing and external urethral sphincter (EUS) electromyography (EMG) were performed 1 week later. Urethral tissue and anterior vagina were dissected en bloc for further analysis via histology and immunofluorescence. Quantitative data was analyzed using ANOVA on Ranks followed by a Tukey posthoc test with p < 0.05 indicating a statistically significant difference between groups. Data is presented as mean +/- standard error of the mean. RESULTS: LPP was significantly decreased 1 week after VD treated with saline only (23.9 1.3 cmH2O) compared to sham VD (44.4 3.4 cmH2O). LPP was also significantly decreased in the VD + empty A-G-beads group (21.7 0.8 cmH2O) compared to sham VD, demonstrating that the microbeads themselves do not create a bulking or obstructive effect in the urethra. In contrast, rats with VD treated with IGF1-A-G-beads (28.4 1.2 cmH2O) was significantly greater than LPP of rats with VD treated with empty AG-beads and had LPP partway between and not significantly different from either the sham VD or the VD + saline groups, demonstrating initiation of a reparative effect 1 week after VD. The increase in EUS EMG amplitude with LPP testing was significantly reduced after VD treated with saline or empty A-G-beads compared to sham VD. VD rats treated with IGF1-A-G-beads had EUS EMG amplitude response to LPP testing partway between and not significantly different from either the sham VD or VD + saline groups. Histological analysis demonstrated well-developed, wellorganized skeletal muscle fibers in the external urethral sphincter in the VD + IGF1-A-G-beads group, similar to that of sham VD rats. In