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PD36-05 PIOGLITAZONE ENHANCES SURVIVAL AND REGENERATION OF PELVIC GANGLIA NEURONS AFTER CAVERNOSAL NERVE INJURY
THE JOURNAL OF UROLOGYâ
e766
Vol. 193, No. 4S, Supplement, Monday, May 18, 2015
METHODS: Whole MPGs were harvested from Male SpragueDawley rats (300g) and cultured in Matrigel with or without TNF-a (20ng/ml). We measured neurite lengths from MPGs in each field at 48 and 72 hours after culture. Cultured MPGs were collected after 72 hours to evaluate gene expressions of TNF-a, inducible nitric oxide synthase (iNOS: M1 macrophage marker), arginase-1 (Arg1: M2 macrophage marker) by qPCR. RESULTS: Average neurite lengths of MPGs cultured with exogenous TNF-a at 48 and 72 hours were significantly shorter than the control (p<0.05). In the control group, histogram of neurite lengths showed a single peak at the mean length. In the TNF-a group, histogram had two peaks: one at the mean of the control and the other at much shorter length. To demonstrate this difference quantitatively, variances of the both groups were compared. The variance of the TNFa group was significantly larger than the control group’s at 72 hours (p<0.05). MPGs incubated with TNF-a had increased gene expressions of iNOS, Arg1 and TNF-a (p<0.01). CONCLUSIONS: This study demonstrates that exogenous TNF-a treatment inhibits neurite outgrowth from MPG. However, outgrowths of some neurites were not inhibited by TNF-a. Furthermore, exogenous TNF-a can induce increased expression of iNOS, Arg1 and TNF-a, suggesting that TNF-a recruits M1/M2 macrophages via positive feedback. These results imply that TNF-a inhibition may be a future therapy to prevent post-operative ED after RP. Source of Funding: NIDDK Urology Care Foundation
PD36-05 PIOGLITAZONE ENHANCES SURVIVAL AND REGENERATION OF PELVIC GANGLIA NEURONS AFTER CAVERNOSAL NERVE INJURY Eric Katz*, Daniel Heidenberg, Nora Haney, Taylor Peak, George Lasker, Margaret Knoedler, Daniel Rittenberg, Bashir Rezk, Ahmed Moustafa, Zakaria Abd Elmageed, Faysal Yafi, Suresh Sikka, Asim Abdel Mageed, Wayne Hellstrom, New Orleans, LA INTRODUCTION AND OBJECTIVES: Pioglitazone has neuroprotective effects in models of sciatic nerve ischemia, optic nerve crush injury, and traumatic brain injury. Our group previously demonstrated that it restores erectile function in a model of cavernosal nerve (CN) crush injury. In this study, we investigated the mechanism of this physiological recovery with a molecular analysis of the rat pelvic ganglia, thereby evaluating its utility for prevention of post-prostatectomy neurogenic erectile dysfunction (ED). METHODS: Sprague-Dawley rats (n¼32) aged 12 weeks were divided into four groups (n¼8 each). Groups A, B, and C underwent bilateral CN crush injury. Group D underwent sham operations. Preoperative injection of Fluorogold (FG) fluorescent tracer into the cavernosal tissue was performed. Group A received pre and post-surgical treatment with pioglitazone (6.5 mg/kg); Group B only received postsurgical treatment. Pre-surgical treatment of pioglitazone was four days and post-surgical treatment was two weeks following crush injury. Pelvic ganglia were resected from all rats after two weeks and processed for reverse transcription polymerase chain reaction (RTPCR), immunohistochemistry (IHC), and Western blot (WB) for the expression of neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), beta-III tubulin, neurturin (NTN), GDNF family receptor alpha-2 (GFRa2), and FG. RESULTS: Pre-treatment with pioglitazone (Group A) induced significantly higher expression of nNOS, NTN, GFRa2, and FG in the pelvic ganglia compared to that in Group C rats. Post-surgical treatment (Group B) induced statistically insignificant improvements in nNOS and FG compared to no treatment (Group C). The sham group (Group D) showed moderate expression of these markers, but not to the same level of rats in Group A. CONCLUSIONS: Pre-treatment with pioglitazone has neuroprotective and neuroregenerative effects on the pelvic ganglia; this may signify a novel therapeutic avenue for preventing neurogenic ED after radical prostatectomy.
Source of Funding: This project was funded by the SMSNA 2014 Research Award. Additional support was provided by the Urology department at Tulane University School of Medicine.
PD36-06 EFFICACY OF ROLIPRAM LOADED NANOSPHERES IN LOCALIZED DELIVERY TO SITES OF NERVE INJURY FOR PREVENTION OF POST-PROSTATECTOMY ERECTILE DYSFUNCTION USING A RAT MODEL OF CAVERNOUS NERVE INJURY Neal Patel*, Amirali H. Salmasi, Michael Dinizo, Izak Faiena, New Brunswick, NJ; Ritu Goyal, Piscataway, NJ; Geun Taek Lee, New Brunswick, NJ; Johanna L. Hannan, Trinity J. Bivalacqua, Baltimore, MD; Joachim Kohn, Piscataway, NJ; Isaac Y. Kim, New Brunswick, NJ INTRODUCTION AND OBJECTIVES: Currently, there is no effective therapy to re-establish penile innervation and regenerate damaged cavernous nerves in post- radical prostatectomy (RP) patients. We previously reported potential neuroprotective mechanism of Phosphodiesterase type 4 inhibitors using cell culture data. We now explore the efficacy of Rolipram, a selective PDE-4 inhibitor, on postnerve injury erectile dysfunction using our nanosphere (NSP) based delivery system to local sites of nerve injury. METHODS: Rolipram loaded NSP which are tyrosine-derived copolymers that spontaneously self-assemble in aqueous media were produced. Twenty-four male Sprague Dawley rats were randomized into 4 groups. Three groups of rats received bilateral crush nerve injury (BCNI) and one group received no injury (sham, n¼6). Of the rats that underwent BCNI, one group received no intervention (BNCI, n¼6), while one group received rolipram loaded NSP over site of induced crush injury bilaterally (BCNIþRolipramþNSP, n¼6), and the last group was treated with empty nanospheres after BCNI (NSP, n¼6). After 14 days, each rat underwent erectile function testing and immunohistochemistry of phalloidin, neurofilaments, and neuronal nitric oxide
e766
Vol. 193, No. 4S, Supplement, Monday, May 18, 2015
METHODS: Whole MPGs were harvested from Male SpragueDawley rats (300g) and cultured in Matrigel with or without TNF-a (20ng/ml). We measured neurite lengths from MPGs in each field at 48 and 72 hours after culture. Cultured MPGs were collected after 72 hours to evaluate gene expressions of TNF-a, inducible nitric oxide synthase (iNOS: M1 macrophage marker), arginase-1 (Arg1: M2 macrophage marker) by qPCR. RESULTS: Average neurite lengths of MPGs cultured with exogenous TNF-a at 48 and 72 hours were significantly shorter than the control (p<0.05). In the control group, histogram of neurite lengths showed a single peak at the mean length. In the TNF-a group, histogram had two peaks: one at the mean of the control and the other at much shorter length. To demonstrate this difference quantitatively, variances of the both groups were compared. The variance of the TNFa group was significantly larger than the control group’s at 72 hours (p<0.05). MPGs incubated with TNF-a had increased gene expressions of iNOS, Arg1 and TNF-a (p<0.01). CONCLUSIONS: This study demonstrates that exogenous TNF-a treatment inhibits neurite outgrowth from MPG. However, outgrowths of some neurites were not inhibited by TNF-a. Furthermore, exogenous TNF-a can induce increased expression of iNOS, Arg1 and TNF-a, suggesting that TNF-a recruits M1/M2 macrophages via positive feedback. These results imply that TNF-a inhibition may be a future therapy to prevent post-operative ED after RP. Source of Funding: NIDDK Urology Care Foundation
PD36-05 PIOGLITAZONE ENHANCES SURVIVAL AND REGENERATION OF PELVIC GANGLIA NEURONS AFTER CAVERNOSAL NERVE INJURY Eric Katz*, Daniel Heidenberg, Nora Haney, Taylor Peak, George Lasker, Margaret Knoedler, Daniel Rittenberg, Bashir Rezk, Ahmed Moustafa, Zakaria Abd Elmageed, Faysal Yafi, Suresh Sikka, Asim Abdel Mageed, Wayne Hellstrom, New Orleans, LA INTRODUCTION AND OBJECTIVES: Pioglitazone has neuroprotective effects in models of sciatic nerve ischemia, optic nerve crush injury, and traumatic brain injury. Our group previously demonstrated that it restores erectile function in a model of cavernosal nerve (CN) crush injury. In this study, we investigated the mechanism of this physiological recovery with a molecular analysis of the rat pelvic ganglia, thereby evaluating its utility for prevention of post-prostatectomy neurogenic erectile dysfunction (ED). METHODS: Sprague-Dawley rats (n¼32) aged 12 weeks were divided into four groups (n¼8 each). Groups A, B, and C underwent bilateral CN crush injury. Group D underwent sham operations. Preoperative injection of Fluorogold (FG) fluorescent tracer into the cavernosal tissue was performed. Group A received pre and post-surgical treatment with pioglitazone (6.5 mg/kg); Group B only received postsurgical treatment. Pre-surgical treatment of pioglitazone was four days and post-surgical treatment was two weeks following crush injury. Pelvic ganglia were resected from all rats after two weeks and processed for reverse transcription polymerase chain reaction (RTPCR), immunohistochemistry (IHC), and Western blot (WB) for the expression of neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), beta-III tubulin, neurturin (NTN), GDNF family receptor alpha-2 (GFRa2), and FG. RESULTS: Pre-treatment with pioglitazone (Group A) induced significantly higher expression of nNOS, NTN, GFRa2, and FG in the pelvic ganglia compared to that in Group C rats. Post-surgical treatment (Group B) induced statistically insignificant improvements in nNOS and FG compared to no treatment (Group C). The sham group (Group D) showed moderate expression of these markers, but not to the same level of rats in Group A. CONCLUSIONS: Pre-treatment with pioglitazone has neuroprotective and neuroregenerative effects on the pelvic ganglia; this may signify a novel therapeutic avenue for preventing neurogenic ED after radical prostatectomy.
Source of Funding: This project was funded by the SMSNA 2014 Research Award. Additional support was provided by the Urology department at Tulane University School of Medicine.
PD36-06 EFFICACY OF ROLIPRAM LOADED NANOSPHERES IN LOCALIZED DELIVERY TO SITES OF NERVE INJURY FOR PREVENTION OF POST-PROSTATECTOMY ERECTILE DYSFUNCTION USING A RAT MODEL OF CAVERNOUS NERVE INJURY Neal Patel*, Amirali H. Salmasi, Michael Dinizo, Izak Faiena, New Brunswick, NJ; Ritu Goyal, Piscataway, NJ; Geun Taek Lee, New Brunswick, NJ; Johanna L. Hannan, Trinity J. Bivalacqua, Baltimore, MD; Joachim Kohn, Piscataway, NJ; Isaac Y. Kim, New Brunswick, NJ INTRODUCTION AND OBJECTIVES: Currently, there is no effective therapy to re-establish penile innervation and regenerate damaged cavernous nerves in post- radical prostatectomy (RP) patients. We previously reported potential neuroprotective mechanism of Phosphodiesterase type 4 inhibitors using cell culture data. We now explore the efficacy of Rolipram, a selective PDE-4 inhibitor, on postnerve injury erectile dysfunction using our nanosphere (NSP) based delivery system to local sites of nerve injury. METHODS: Rolipram loaded NSP which are tyrosine-derived copolymers that spontaneously self-assemble in aqueous media were produced. Twenty-four male Sprague Dawley rats were randomized into 4 groups. Three groups of rats received bilateral crush nerve injury (BCNI) and one group received no injury (sham, n¼6). Of the rats that underwent BCNI, one group received no intervention (BNCI, n¼6), while one group received rolipram loaded NSP over site of induced crush injury bilaterally (BCNIþRolipramþNSP, n¼6), and the last group was treated with empty nanospheres after BCNI (NSP, n¼6). After 14 days, each rat underwent erectile function testing and immunohistochemistry of phalloidin, neurofilaments, and neuronal nitric oxide