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023 Harnessing the Role of Microtubules in Neural Regeneration to Improve Erectile Function Outcomes Following Cavernous Nerve Injury in a Rat Model of Radical Prostatectomy
Proceedings of the 21st Annual Fall Scientific Meeting of SMSNA, Las Vegas, Nevada, USA, November 19-22, 2015
S11
neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NTF3). Additional MPGs were cultured as above for 72 hours, fixed and prepared for immunoflourescent imaging of neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), and CXCR4. Result(s): SDF-1 enhanced neurite outgrowth from the MPG. Significant (p<0.05) increases in neurite length with SDF-1 treatment compared to control were seen at 48 (746±45 uM vs 575±34 uM) and 72 hours (1045±46 uM vs 739±34 uM). Compared to controls, MPGs cultured with SDF-1 demonstrated a 3-fold increase in SDF-1 gene expression (p¼0.048) but no difference in CXCR4 or CXCR7 expression. SDF-1 treatment was associated with 53% increase in BDNF (p¼0.02) and a 73% increase in NGF (p¼0.03), but no difference in NT3 expression. Immunofluorescence microscopy demonstrated nNOS, TH, and CXCR4 expression along regenerating neurites. Conclusion(s): This study demonstrates that SDF-1 treatment facilitates neurite outgrowth from the MPG by utilizing positive feedback autocrine SDF-1 signaling and increasing neurotrophin expression. This study suggests that SDF-1 plays an important role in stem cell independent peripheral nerve regeneration and may provide a novel therapeutic target. Disclosure: Work supported by industry: no.
TGF b1, 2 and 3 were measured in the cell culture media using multiplexed immunoassay kits. Results: Nine PD plaque cultures and six TA control cultures were analyzed. We found significantly higher concentrations of MMP2, MMP12 and MMP13 in PD cells compared to the controls. There were also significantly higher amounts of TIMP1, TIMP2, and TIMP3 in the PD plaque cells. No significant measureable differences were detected in TGF-B and IL1 concentrations from the two groups. Conclusions: MMPs are key regulators of extracellular matrix (ECM) turnover during normal and pathological processes. A fine balance between the levels of MMPs and TIMPs control the extent of local ECM degradation. Our data show higher concentrations of TIMPs and lower concentrations of MMPs in Peyronie’s disease, implying an imbalance in the healing equilibrium, such that mediators of fibrosis predominate. This information will be helpful in the development of specific immunologic based therapies for the treatment and prevention of Peyronie’s disease. Disclosure: Work supported by industry: no. The presenter or any of the authors act as a consultant, employee (part time or full time) or shareholder of an industry.
022 MATRIX METALLOPROTEINASES AND TISSUE INHIBITORS OF MATRIX METALLOPROTEINASES IN THE PATHOGENESIS OF PEYRONIE’S DISEASE Campbell, J.1; DeYoung, L.2,*; Chung, E.2; Brock, G.1 1 Western University, Department of Surgery, Division of Urology, London, ON, Canada; 2Lawson Health Research Institute, London, ON, Canada
HARNESSING THE ROLE OF MICROTUBULES IN NEURAL REGENERATION TO IMPROVE ERECTILE FUNCTION OUTCOMES FOLLOWING CAVERNOUS NERVE INJURY IN A RAT MODEL OF RADICAL PROSTATECTOMY Tar, M.T.1; Baker, L.2; Nacharaju, P.1; Friedman, J.M.1; Sharp, D.J.1; Davies, K.P.1,* 1 Albert Einstein College of Medicine, USA; 2Albert Einstein College of Medicine
Objectives: Peyronie’s disease (PD) is a fibrotic condition of penile tunica albuginea (TA). The expanding role of medical management in PD mandates that we more fully elucidate the underlying causative pathophysiologic processes. The dense plaques identified in Peyronie’s disease result from abnormal wound healing caused by an imbalance of fibrosis and fibrinolysis. Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play a central role in the regulation of this dynamic process. To investigate the role of these proteins in Peyronie’s disease, we examined the concentrations of MMPs, TIMPs and inflammatory cytokines expressed in cell culture media of human PD cells compared to control TA cells. Methods: Human tissue samples from PD plaques and normal TA where collected intraoperatively. The third passage of the primary cell cultures of PD and controls were sub-cultured on Bio Flex-Pro Nectin plates. Media obtained from these final cell cultures were collected and analyzed. We measured Interleukin-1 (IL-1) levels with aliquots of cell culture media using an enzymelinked immunosorbent assay. The protein concentrations of MMP1, 2, 3, 7, 8, 9, 10, 11 and 12; TIMPS 1, 2, 3, and 4; and
Objective: Growing evidence demonstrates that pharmacological stabilization of microtubules has the potential to promote neural regeneration. Recently we reported that local depletion of a newly discovered endogenous microtubule regulator called Fidgetin-like 2 (FL2) promotes the closure and regeneration of cutaneous wounds. In the studies reported here we investigated if depletion of FL2 at the site of cavernous nerve (CN) injury in a rat (a model of radical prostatectomy (RP) might improve erectile function outcomes. Material and Methods: Two rat models of CN injury were used; mild (a smooth clamp was applied for 2 minutes to the CN) and moderate (a serrated clamp was applied for 4 minutes to the CN). At the time of injury two formulations (nanoparticle or liposomal) for delivery of FL2-siRNA (experimental) or control-siRNA were applied. At several time points following injury (up to 4 weeks) erectile function was determined by measuring the intracorporal pressure/blood pressure ratio following electro-simulation of the CN. Results: In both models of CN injury there was an improved erectile response in the FL2-siRNA treated animals compared to controls (indicated by a greater ICP/BP response at a particular
J Sex Med 2016;13:S1eS71
023
S12
Proceedings of the 21st Annual Fall Scientific Meeting of SMSNA, Las Vegas, Nevada, USA, November 19-22, 2015
level of electro-simulation of the CN. This improvement was significant two-three weeks following treatment. Conclusion: This is the first ever demonstration that modulating microtubule processes can repair peripheral nerve damage, specifically CN injury. This study suggests the potential for depletion of FL2 in the recovery of erectile function in a rat model of RP and identifies novel molecular mechanisms involving the microtubule cytoskeleton that are responsible for this recovery. Disclosure: Work supported by industry: no. The presenter or any of the authors act as a consultant, employee (part time or full time) or shareholder of an industry. 024 TNF-ALPHA INHIBITS NEURITE OUTGROWTH FROM THE MAJOR PELVIC GANGLION BY INDUCING APOPTOSIS OF NITRERGIC NEURONS EX VIVO Matsui, H.1,*; Sopko, N.A.1; Weyne, E.2; Reinhardt, A.A.3; Kates, M.1; Lough, D.M.1; Liu, X.1; Albersen, M.2; Hannan, J.L.4; Bivalacqua, T.J.1 1 The Johns Hopkins School of Medicine, USA; 2KU Leuven, Belgium; 3Ripon College, USA; 4East Carolina University, USA Objective(s): Cavernous nerve (CN) injury leads to erectile dysfunction (ED) following radical prostatectomy (RP). Previously, we demonstrated that TNF-a was increased in the major pelvic ganglion (MPG) following bilateral CN injury. Increase in TNF-a was shown to induce neuronal apoptosis following sciatic nerve injury. We hypothesized that TNF-a also induces neuronal apoptosis in the major pelvic ganglion (MPG). To test this hypothesis, we examined the effect of exogenous TNF-a (eTNF-a) on neurite outgrowth form the MPG ex vivo. Material and Method(s): Whole MPGs were harvested from male Sprague-Dawley rats (300-350g) and cultured in Matrigel with eTNF-a in the concentrations of 0, 10, 20, 30 ng/mL (n¼5/group). Media and eTNF-a were changed every 24 hours. Neurite lengths were measured at 72 hours after culture. MPGs were processed for qPCR to evaluate gene expression of neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), and Caspase (CASP)-1, CASP3, and CASP9 (n¼5/group). Additional MPGs were cultured in Matrigel with or without eTNF-a 20 ng/mL and fixed at 72 hours after culture to perform immunofluorescence for TH and nNOS. Result(s): eTNF-a inhibited neurite outgrowth from the MPG in a dose-dependent fashion (neurite lengths: 586±20.5, 455±11.5, 408±28.7, 361±2.65 mm; eTNF-a 0, 10, 20, 30 ng/ mL, respectively: p<0.01). eTNF-a groups demonstrated bimodal distribution in the histograms of neurite lengths, while control group demonstrated normal distribution. MPGs cultured with eTNF-a displayed increased gene expression of CASP1 (p¼0.03) and decreased gene expression of nNOS (p<0.05). Gene expression of CASP3, CASP9, and TH remained unchanged (p>0.05). Immunofluorescence demonstrated that nNOS-positive neurites were shorter than TH-positive neurites in the MPGs cultured with
eTNF-a 20 ng/mL, while nNOS-positive neurites were as long as TH-positive neurites in the control group. Conclusion(s): This study demonstrated that eTNF-a impaired neurite outgrowth from the MPG in a dose-dependent fashion by upregulating gene expression of apoptosis marker. Furthermore, eTNF-a downregluated the gene expression of nNOS and inhibited neurite outgrowth of nNOS-positive neurites, indicating that eTNF-a selectively inhibited neurite outgrowth of nitrergic neurons. These results suggest that TNF-a antagonists may prevent post-RP ED by inhibiting apoptosis of nitrergic neurons. Disclosure: Work supported by industry: no. 025 NEW METHODOLOGY FOR INVESTIGATING EJACULATION DYSFUNCTION: MEASURING INTRALUMINAL SEMINAL VESICLE PRESSURE IN RATS WITH TELEMETRIC DEVICE Lee, S.W.1,*; Sung, H.H.1; Moon, K.H.2; Hyun, J.S.3; Chai, M.R.1 1 Sungkyunkwan University School fo Medicine, Samsung Medical Center, South Korea; 2Yeungnam University School of Medicine, South Korea; 3Gyeongsang national University Hospital, South Korea Objective: Ejaculation dysfunction is the most common male sexual disorder. Despite its prevalence and adverse impact on patients, little attention has been given to investigating the ejaculation dysfunction. We introduce a new in vivo method for the evaluating ejaculation dysfunction in rats with telemetric device. Materials and Methods: A pressure sensor (C40; Data Sciences) was surgically implanted in the seminal vesicles of 7-week-old male SpragueeDawley rats. One week later, the rats were subcutaneously administered to tamsulosin (3 and 10 ug/kg), silodosin 1mg/ kg or normal saline (control), respectively. And 30 minutes later, intraluminal seminal vesicle pressure (ISVP) was recorded in freely moving rats after apomorphine (80ug/kg). Sexual events were visually identified and recorded. Ejaculation was confirmed by inspection of copulatory plug in the tip of penis. We compared the maximal ISVP and the area under the curve (AUC). Results: The mean numbers of ejaculation during inspection time was 1.5±0.1. The maximal ISVP values in rats receiving 3ug/kg (30.0±5.2mmHg) tamsulosin and 10ug/kg (15.1±1.6 mmHg) tamsulosin, and silodosin 1mg/kg (12.9±2.2 mmHg) were significantly lower than that of control (61.4±13.4 mmHg P<0.05). The AUC of tamsulosin 3ug/kg (72.7±18.9mmHg*s) and 10ug/kg (23.5±6.1mmHg*s), and silodosin 1mg/kg (23.9±8.0mmHg*s) were also lower than that of control (162.6±34.3 mmHg*s, p<0.05). Conclusions: The present report is the first to describe the use of telemetric device for measuring ISVP in rats. Telemetric ISVP is reliable and feasible for investigating ejaculation physiology in seminal vesicle. Disclosure: Work supported by industry: no.
J Sex Med 2016;13:S1eS71
S11
neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NTF3). Additional MPGs were cultured as above for 72 hours, fixed and prepared for immunoflourescent imaging of neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), and CXCR4. Result(s): SDF-1 enhanced neurite outgrowth from the MPG. Significant (p<0.05) increases in neurite length with SDF-1 treatment compared to control were seen at 48 (746±45 uM vs 575±34 uM) and 72 hours (1045±46 uM vs 739±34 uM). Compared to controls, MPGs cultured with SDF-1 demonstrated a 3-fold increase in SDF-1 gene expression (p¼0.048) but no difference in CXCR4 or CXCR7 expression. SDF-1 treatment was associated with 53% increase in BDNF (p¼0.02) and a 73% increase in NGF (p¼0.03), but no difference in NT3 expression. Immunofluorescence microscopy demonstrated nNOS, TH, and CXCR4 expression along regenerating neurites. Conclusion(s): This study demonstrates that SDF-1 treatment facilitates neurite outgrowth from the MPG by utilizing positive feedback autocrine SDF-1 signaling and increasing neurotrophin expression. This study suggests that SDF-1 plays an important role in stem cell independent peripheral nerve regeneration and may provide a novel therapeutic target. Disclosure: Work supported by industry: no.
TGF b1, 2 and 3 were measured in the cell culture media using multiplexed immunoassay kits. Results: Nine PD plaque cultures and six TA control cultures were analyzed. We found significantly higher concentrations of MMP2, MMP12 and MMP13 in PD cells compared to the controls. There were also significantly higher amounts of TIMP1, TIMP2, and TIMP3 in the PD plaque cells. No significant measureable differences were detected in TGF-B and IL1 concentrations from the two groups. Conclusions: MMPs are key regulators of extracellular matrix (ECM) turnover during normal and pathological processes. A fine balance between the levels of MMPs and TIMPs control the extent of local ECM degradation. Our data show higher concentrations of TIMPs and lower concentrations of MMPs in Peyronie’s disease, implying an imbalance in the healing equilibrium, such that mediators of fibrosis predominate. This information will be helpful in the development of specific immunologic based therapies for the treatment and prevention of Peyronie’s disease. Disclosure: Work supported by industry: no. The presenter or any of the authors act as a consultant, employee (part time or full time) or shareholder of an industry.
022 MATRIX METALLOPROTEINASES AND TISSUE INHIBITORS OF MATRIX METALLOPROTEINASES IN THE PATHOGENESIS OF PEYRONIE’S DISEASE Campbell, J.1; DeYoung, L.2,*; Chung, E.2; Brock, G.1 1 Western University, Department of Surgery, Division of Urology, London, ON, Canada; 2Lawson Health Research Institute, London, ON, Canada
HARNESSING THE ROLE OF MICROTUBULES IN NEURAL REGENERATION TO IMPROVE ERECTILE FUNCTION OUTCOMES FOLLOWING CAVERNOUS NERVE INJURY IN A RAT MODEL OF RADICAL PROSTATECTOMY Tar, M.T.1; Baker, L.2; Nacharaju, P.1; Friedman, J.M.1; Sharp, D.J.1; Davies, K.P.1,* 1 Albert Einstein College of Medicine, USA; 2Albert Einstein College of Medicine
Objectives: Peyronie’s disease (PD) is a fibrotic condition of penile tunica albuginea (TA). The expanding role of medical management in PD mandates that we more fully elucidate the underlying causative pathophysiologic processes. The dense plaques identified in Peyronie’s disease result from abnormal wound healing caused by an imbalance of fibrosis and fibrinolysis. Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play a central role in the regulation of this dynamic process. To investigate the role of these proteins in Peyronie’s disease, we examined the concentrations of MMPs, TIMPs and inflammatory cytokines expressed in cell culture media of human PD cells compared to control TA cells. Methods: Human tissue samples from PD plaques and normal TA where collected intraoperatively. The third passage of the primary cell cultures of PD and controls were sub-cultured on Bio Flex-Pro Nectin plates. Media obtained from these final cell cultures were collected and analyzed. We measured Interleukin-1 (IL-1) levels with aliquots of cell culture media using an enzymelinked immunosorbent assay. The protein concentrations of MMP1, 2, 3, 7, 8, 9, 10, 11 and 12; TIMPS 1, 2, 3, and 4; and
Objective: Growing evidence demonstrates that pharmacological stabilization of microtubules has the potential to promote neural regeneration. Recently we reported that local depletion of a newly discovered endogenous microtubule regulator called Fidgetin-like 2 (FL2) promotes the closure and regeneration of cutaneous wounds. In the studies reported here we investigated if depletion of FL2 at the site of cavernous nerve (CN) injury in a rat (a model of radical prostatectomy (RP) might improve erectile function outcomes. Material and Methods: Two rat models of CN injury were used; mild (a smooth clamp was applied for 2 minutes to the CN) and moderate (a serrated clamp was applied for 4 minutes to the CN). At the time of injury two formulations (nanoparticle or liposomal) for delivery of FL2-siRNA (experimental) or control-siRNA were applied. At several time points following injury (up to 4 weeks) erectile function was determined by measuring the intracorporal pressure/blood pressure ratio following electro-simulation of the CN. Results: In both models of CN injury there was an improved erectile response in the FL2-siRNA treated animals compared to controls (indicated by a greater ICP/BP response at a particular
J Sex Med 2016;13:S1eS71
023
S12
Proceedings of the 21st Annual Fall Scientific Meeting of SMSNA, Las Vegas, Nevada, USA, November 19-22, 2015
level of electro-simulation of the CN. This improvement was significant two-three weeks following treatment. Conclusion: This is the first ever demonstration that modulating microtubule processes can repair peripheral nerve damage, specifically CN injury. This study suggests the potential for depletion of FL2 in the recovery of erectile function in a rat model of RP and identifies novel molecular mechanisms involving the microtubule cytoskeleton that are responsible for this recovery. Disclosure: Work supported by industry: no. The presenter or any of the authors act as a consultant, employee (part time or full time) or shareholder of an industry. 024 TNF-ALPHA INHIBITS NEURITE OUTGROWTH FROM THE MAJOR PELVIC GANGLION BY INDUCING APOPTOSIS OF NITRERGIC NEURONS EX VIVO Matsui, H.1,*; Sopko, N.A.1; Weyne, E.2; Reinhardt, A.A.3; Kates, M.1; Lough, D.M.1; Liu, X.1; Albersen, M.2; Hannan, J.L.4; Bivalacqua, T.J.1 1 The Johns Hopkins School of Medicine, USA; 2KU Leuven, Belgium; 3Ripon College, USA; 4East Carolina University, USA Objective(s): Cavernous nerve (CN) injury leads to erectile dysfunction (ED) following radical prostatectomy (RP). Previously, we demonstrated that TNF-a was increased in the major pelvic ganglion (MPG) following bilateral CN injury. Increase in TNF-a was shown to induce neuronal apoptosis following sciatic nerve injury. We hypothesized that TNF-a also induces neuronal apoptosis in the major pelvic ganglion (MPG). To test this hypothesis, we examined the effect of exogenous TNF-a (eTNF-a) on neurite outgrowth form the MPG ex vivo. Material and Method(s): Whole MPGs were harvested from male Sprague-Dawley rats (300-350g) and cultured in Matrigel with eTNF-a in the concentrations of 0, 10, 20, 30 ng/mL (n¼5/group). Media and eTNF-a were changed every 24 hours. Neurite lengths were measured at 72 hours after culture. MPGs were processed for qPCR to evaluate gene expression of neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), and Caspase (CASP)-1, CASP3, and CASP9 (n¼5/group). Additional MPGs were cultured in Matrigel with or without eTNF-a 20 ng/mL and fixed at 72 hours after culture to perform immunofluorescence for TH and nNOS. Result(s): eTNF-a inhibited neurite outgrowth from the MPG in a dose-dependent fashion (neurite lengths: 586±20.5, 455±11.5, 408±28.7, 361±2.65 mm; eTNF-a 0, 10, 20, 30 ng/ mL, respectively: p<0.01). eTNF-a groups demonstrated bimodal distribution in the histograms of neurite lengths, while control group demonstrated normal distribution. MPGs cultured with eTNF-a displayed increased gene expression of CASP1 (p¼0.03) and decreased gene expression of nNOS (p<0.05). Gene expression of CASP3, CASP9, and TH remained unchanged (p>0.05). Immunofluorescence demonstrated that nNOS-positive neurites were shorter than TH-positive neurites in the MPGs cultured with
eTNF-a 20 ng/mL, while nNOS-positive neurites were as long as TH-positive neurites in the control group. Conclusion(s): This study demonstrated that eTNF-a impaired neurite outgrowth from the MPG in a dose-dependent fashion by upregulating gene expression of apoptosis marker. Furthermore, eTNF-a downregluated the gene expression of nNOS and inhibited neurite outgrowth of nNOS-positive neurites, indicating that eTNF-a selectively inhibited neurite outgrowth of nitrergic neurons. These results suggest that TNF-a antagonists may prevent post-RP ED by inhibiting apoptosis of nitrergic neurons. Disclosure: Work supported by industry: no. 025 NEW METHODOLOGY FOR INVESTIGATING EJACULATION DYSFUNCTION: MEASURING INTRALUMINAL SEMINAL VESICLE PRESSURE IN RATS WITH TELEMETRIC DEVICE Lee, S.W.1,*; Sung, H.H.1; Moon, K.H.2; Hyun, J.S.3; Chai, M.R.1 1 Sungkyunkwan University School fo Medicine, Samsung Medical Center, South Korea; 2Yeungnam University School of Medicine, South Korea; 3Gyeongsang national University Hospital, South Korea Objective: Ejaculation dysfunction is the most common male sexual disorder. Despite its prevalence and adverse impact on patients, little attention has been given to investigating the ejaculation dysfunction. We introduce a new in vivo method for the evaluating ejaculation dysfunction in rats with telemetric device. Materials and Methods: A pressure sensor (C40; Data Sciences) was surgically implanted in the seminal vesicles of 7-week-old male SpragueeDawley rats. One week later, the rats were subcutaneously administered to tamsulosin (3 and 10 ug/kg), silodosin 1mg/ kg or normal saline (control), respectively. And 30 minutes later, intraluminal seminal vesicle pressure (ISVP) was recorded in freely moving rats after apomorphine (80ug/kg). Sexual events were visually identified and recorded. Ejaculation was confirmed by inspection of copulatory plug in the tip of penis. We compared the maximal ISVP and the area under the curve (AUC). Results: The mean numbers of ejaculation during inspection time was 1.5±0.1. The maximal ISVP values in rats receiving 3ug/kg (30.0±5.2mmHg) tamsulosin and 10ug/kg (15.1±1.6 mmHg) tamsulosin, and silodosin 1mg/kg (12.9±2.2 mmHg) were significantly lower than that of control (61.4±13.4 mmHg P<0.05). The AUC of tamsulosin 3ug/kg (72.7±18.9mmHg*s) and 10ug/kg (23.5±6.1mmHg*s), and silodosin 1mg/kg (23.9±8.0mmHg*s) were also lower than that of control (162.6±34.3 mmHg*s, p<0.05). Conclusions: The present report is the first to describe the use of telemetric device for measuring ISVP in rats. Telemetric ISVP is reliable and feasible for investigating ejaculation physiology in seminal vesicle. Disclosure: Work supported by industry: no.
J Sex Med 2016;13:S1eS71