Sandra M. Gomez-Amaya

Neural reconstruction methods of restoring bladder function

During the past century, diverse studies have focused on the development of surgical strategies to restore function of a decentralized bladder after spinal cord or spinal root injury via repair of the original roots or by transferring new axonal sources. The techniques included end-to-end sacral root repairs, transfer of roots from other spinal segments to sacral roots, transfer of intercostal nerves to sacral roots, transfer of various somatic nerves to the pelvic or pudendal nerve, direct reinnervation of the detrusor muscle, or creation of an artificial reflex pathway between the skin and the bladder via the central nervous system. All of these surgical techniques have demonstrated specific strengths and limitations. The findings made to date already indicate appropriate patient populations for each procedure, but a comprehensive assessment of the effectiveness of each technique to restore urinary function after bladder decentralization is required to guide future research and potential clinical application.

Bladder Reinnervation Using a Primarily Motor Donor Nerve (Femoral Nerve Branches) is Functionally Superior to Using a Primarily Sensory Donor Nerve (Genitofemoral Nerve)

PURPOSE We determined whether transfer of a primarily motor nerve (femoral) to the anterior vesicle branch of the pelvic nerve would allow for more effective bladder reinnervation than transfer of a primarily sensory nerve (genitofemoral). MATERIALS AND METHODS A total of 41 female mongrel dogs underwent bladder decentralization and then bilateral nerve transfer, or served as sham operated or unoperated controls. Decentralization was achieved by bilateral transection of all sacral roots that induced bladder contraction upon electrical stimulation. Retrograde neuronal labeling dye was injected in the bladder 3 weeks before sacrifice. RESULTS Increased detrusor pressure after direct stimulation of the transferred nerve, lumbar spinal cord or spinal root was observed in 12 of 17 dogs with genitofemoral nerve transfer and in 9 of 10 with femoral nerve transfer (mean ± SEM 7.6 ± 1.4 and 11.7 ± 3.1 cm H2O, respectively). Mean detrusor pressure after direct electrical stimulation of transferred femoral nerves was statistically significantly greater than after stimulation of transferred genitofemoral nerves. Retrograde labeled neurons from the bladder observed in upper lumbar cord segments after genitofemoral and femoral nerve transfer confirmed bladder reinnervation, as did labeled axons at the nerve transfer site. CONCLUSIONS While transfer of a mixed sensory and motor nerve (genitofemoral) or a primarily motor nerve (femoral) can reinnervate the bladder, using the primarily motor nerve provided greater return of nerve evoked detrusor contraction. This surgical approach may be useful to achieve bladder emptying in patients with lower motor spinal cord injury.

Neuromuscular Nicotinic Receptors Mediate Bladder Contractions Following Bladder Reinnervation with Somatic to Autonomic Nerve Transfer after Decentralization by Spinal Root Transection

PURPOSE We investigated whether the reinnervated neuronal pathway mediates contraction via the same neurotransmitter and receptor mechanisms as the original pathway. MATERIALS AND METHODS After decentralizing the bladder by transecting the sacral roots in dogs we performed peripheral nerve transfer, including bilateral genitofemoral to pelvic nerve transfer and unilateral left femoral nerve to bilateral pelvic nerve transfer. Reinnervation was assessed 7.5 months postoperatively by monitoring bladder pressure during electrical stimulation of the transferred nerves, spinal ventral roots and spinal cord. RESULTS Of the 17 dogs with genitofemoral to pelvic nerve transfer 14 (82%) demonstrated functional bladder reinnervation as evidenced by increased bladder pressure during stimulation of the transferred genitofemoral nerve, or L3 or L4 spinal ventral roots. Lumbar spinal cord stimulation caused increased bladder pressure in 9 of 10 dogs (90%) with unilateral left femoral nerve to bilateral pelvic nerve transfer. Succinylcholine virtually eliminated the bladder pressure increases induced by electrical stimulation of the transferred somatic nerves or of the lumbar spinal segments that contribute axons to these donor nerves. In unoperated or sham operated controls succinylcholine had no effect on nerve evoked bladder pressure increases but it substantially decreased the urethral and anal sphincter pressure induced by stimulating the lumbosacral spinal cord or the S2-S3 spinal ventral roots. The reinnervated detrusor muscles of dogs with genitofemoral to pelvic nerve transfer and unilateral left femoral nerve to bilateral pelvic nerve transfer also showed increased α1 nicotinic receptor subunit immunoreactivity in punctate dots on detrusor muscle fascicles and in neuronal cell bodies. This staining was not observed in controls. CONCLUSIONS Succinylcholine sensitive nicotinic receptors, which normally mediate only skeletal muscle neuromuscular junction neurotransmission, appeared in the new neuronal pathway after genitofemoral to pelvic and unilateral femoral nerve to bilateral pelvic nerve transfer. This suggests end organ neuroplasticity after reinnervation by somatic motor axons.

Clarification of the Innervation of the Bladder, External Urethral Sphincter and Clitoris: A Neuronal Tracing Study in Female Mongrel Hound Dogs: GENITOURINARY STRUCTURE INNERVATION IN DOGS

Many studies examining the innervation of genitourinary structures focus on either afferent or efferent inputs, or on only one structure of the system. We aimed to clarify innervation of the bladder, external urethral sphincter (EUS) and clitoris. Retrograde dyes were injected into each end organ in female dogs. Spinal cord, mid‐bladder, and spinal, caudal mesenteric, sympathetic trunk and pelvic plexus ganglia were examined for retrograde dye‐labeled neurons. Neurons retrogradely labeled from the bladder were found primarily in L7‐S2 spinal ganglia, spinal cord lateral zona intermedia at S1‐S3 levels, caudal mesenteric ganglia, T11‐L2 and L6‐S2 sympathetic trunk ganglia, and pelvic plexus ganglia. The mid‐bladder wall contained many intramural ganglia neurons labeled anterogradely from the pelvic nerve, and intramural ganglia retrogradely labeled from dye labeling sites surrounding ureteral orifices. Neurons retrogradely labeled from the clitoris were found only in L7 and S1 spinal ganglia, L7‐S3 spinal cord lateral zona intermedia, and S1 sympathetic trunk ganglia, and caudal mesenteric ganglia. Neurons retrogradely labeled from the EUS were found in primarily at S1 and S2 spinal ganglia, spinal cord lamina IX at S1‐S3, caudal mesenteric ganglia, and S1‐S2 sympathetic trunk ganglia. Thus, direct inputs from the spinal cord to each end organ were identified, as well as multisynaptic circuits involving several ganglia, including intramural ganglia in the bladder wall. Knowledge of this complex circuitry of afferent and efferent inputs to genitourinary structures is necessary to understand and treat genitourinary dysfunction. Anat Rec, 2018.

MP42-16 INNERVATION OF GENITOURINARY STRUCTURES: A NEURONAL TRACING STUDY IN FEMALE DOGS

identification of novel pathological regulators in this tissue. Oxidative stress is a fundamental pathological mediator; ROS generating enzyme NADPH oxidase (Nox enzyme) has attracted intense interest recently as it is the only enzyme that produces ROS as its sole function and can be targeted without compromising normal biochemical oxidation. Our recent pilot study provided initial evidence for the presence of such system in bladder urothelium and its potential functional significance. This study aimed firstly to define the importance of urothelial superoxide production in the body and secondly to dissect the enzymatic sources of superoxide production in the bladder. METHODS: C57BL/6J mice were euthanized. Bladder and other types of tissue were isolated. Lucigenin-enhanced chemiluminescence quantified superoxide production in live tissue. Western blot determined Nox subtype expressions. RESULTS: Superoxide production in bladder mucosa (RLU/mg tissue: 536.8 104.8, mean SEM) was many folds as high as those in detrusor muscle (21.8 3.8, n1⁄415, p<0.01), aorta (67.2 26.6, n1⁄47, p<0.05), brain (9.4 1.7, n1⁄46, p<0.01), kidney (84.3 23.0, n1⁄46, p<0.05), ventricle (21.8 3.8, n1⁄46, p<0.01) and liver (80.8 12.9, n1⁄47, p<0.05). NADPH oxidase inhibitor diphenyleneiodonium (DPI, 20mM) reduced superoxide production to 10.8 3.4 % of control (n1⁄46, p<0.01) in bladder mucosa and to 30.8 8.4% of control (n1⁄46, p<0.01) in detrusor. Mitochondria de-coupler FCCP (10mM) suppressed superoxide production to 51.8 10.5 % of control (n1⁄46; p<0.01) in bladder mucosa and to 59.8 10.4 % of control (n1⁄46, p<0.05) in detrusor. Xanthine oxidase inhibitor oxypurinol (100mM) produced no significant effect in bladder mucosa (87.9 17.4 % of control, n1⁄46, p>0.05) but a small inhibition in detrusor (78.0 6.6% of control). Western blot showed specific bands for Nox1, Nox2 and Nox4 but no Nox3 expression in bladder mucosa and detrusor with significantly higher expression in bladder mucosa (p<0.05, n1⁄44-6). CONCLUSIONS: These data demonstrate for the first time that the urothelium is the most active tissue for superoxide production in the body. Nox enzymes are the main enzymatic source for superoxide in bladder. The main Nox subtypes are Nox1, Nox2 and Nox4, mainly located in the urothelium. Exceptionally high levels of Nox-driven superoxide explain why bladder urothelium is prone to oxidative stress, inflammation and sensory dysfunction.

AB308. SPR-35 Sympathetic reinnervation of the urinary bladder using somatic donor nerves in a canine model of lower motoneuron lesioned bladder

Objective Preganglionic sympathetic axons to pelvic viscera originate from lumbar preganglionic neurons and contribute to sympathetic chain ganglia and lumbar splanchnic nerves to the inferior mesenteric ganglia (many via the hypogastric nerve). Postganglionic sympathetic axons to the urinary bladder originate mostly from L7–S2 sympathetic chain ganglia and inferior mesenteric ganglion. We tested if after bladder decentralization and transfer of a lumbar originating nerve, postganglionic sympathetic axons will sprout from the lumbar nerves to reinnervate the urinary bladder. Methods A canine lower motoneuron lesioned bladder model was created by transecting sacral nerve roots that increased detrusor pressure using intraoperative electrical stimulation, and all roots caudal to S1. Female hounds underwent bladder decentralization and then femoral or genitofemoral nerve transfer (FNT, n=10, and GFNT, n=17, respectively) to the anterior vesicle branch of the pelvic nerve, or remained decentralized (n=3). Six sham/unoperated controls were included. Bladder emptying in animals without vesicostomies was accomplished by the Credé maneuver during the 8-month recovery period (242±6.2 days). Three weeks prior to euthanasia, a retrograde dye, Fluorogold, was injected into the bladder wall lateral to ureteral orifices. At euthanasia, T10 through coccygeal sympathetic ganglia and the inferior mesenteric ganglia were collected and examined for retrogradely labeled neuronal cell bodies. Multifactoral ANOVAs were performed (with the factors segment and group). The Bonferroni method of post hoc analysis for multiple comparisons with adjusted P values (adjusted down from P<0.05) was used to compare group differences. Results Increased numbers of fluorogold labeled cell bodies were observed in sympathetic ganglia of appropriate segments (mainly L4 and L5 in FNT animals; mainly L2 and L4 in GFNT animals), compared to sham/unoperated controls which showed fluorogold labeled cell bodies in S1–S3 sympathetic ganglia (P<0.01 each segment). Numbers of fluorogold labeled cells were also higher in sympathetic ganglia at vertebral levels L5 and L3 in FNT and GFNT animals, respectively, compared to decentralized controls (P<0.05 each). Low numbers of fluorogold labeled cells were visible in S1–S3 sympathetic ganglia of decentralized and FNT animals. The inferior mesenteric ganglia contained increased fluorogold labeled cells from the urinary bladder in both FNT and GFNT animals, compared to sham/unoperated controls (P<0.01 each). Conclusions Transfer of the femoral nerve to the anterior vesicle branch of the pelvic nerve lead to increased innervation of the bladder from lumbar postganglionic sympathetic ganglia, segments that did not contribute innervation in sham/unoperated controls. Functional consequences of this new innervation pattern should be explored in future studies. Funding Source(s) NIH-NINDS NS070267

PD7-05 VIRAL CYSTITIS INDUCED BY CROSS-INFECTION FROM THE COLON – POTENTIAL MECHANISM FOR INTERSTITIAL CYSTITIS

INTRODUCTION AND OBJECTIVES: Many urologic conditions are associated with dysfunction of autonomic bladder innervation. A precise understanding of the anatomical relationship of the bladder neck and its autonomic nervous system could substantially improve treatment modalities. Currently there is no comprehensive model that provides a detailed three-dimensional (3D) view of bladder neck innervation. Therefore, we sought to create a 3D reconstruction of autonomic nervous tissue innervating the bladder neck using male and female cadaver histopathology. METHODS: We obtained intact pelvic tissues from a male and female cadaver. Axial cross-sections of the bladder neck were generated at 3e5mm intervals and stained for S100 protein. Distances between autonomic nerves and bladder mucosa were recorded. Nerve tracings were manually demarcated and imported into SolidWorks (Waltham, MA, USA) and Blender (Amsterdam, Netherlands) software programs to generate 3D reconstructions of autonomic nerve anatomy. RESULTS: Longitudinal and circular nerve tracings were successfully demarcated and converted into a 3D image reconstruction. We successfully precisely characterized anatomic nerve distributions. Axial cross-sections and 3D images showed that autonomic innervation was highly concentrated in the posterior aspect of the bladder neck in both male and female bladder specimens (Figures 1 and 2). The mean distances between autonomic nerve tissue and the bladder mucosa was 1.15mm posteriorly and 4.0mm anteriorly in the male bladder (0.27-2.87 vs. 2.03-6.20, p<0.001) and 1.51mm posteriorly and 1.83mm anteriorly in the female bladder (0.50-2.91 vs. 0.553.07, p1⁄40.027). CONCLUSIONS: Novel 3D reconstruction of the bladder is feasible and may help re-define our understanding of human bladder innervation. Autonomic innervation of the bladder is highly focused in the posterior aspect of the bladder neck in both male and female bladders. Source of Funding: None

Neural Reconstruction Methods of Restoring Bladder Function: A Critical Review

During the last century, diverse studies have focused on the development of surgical strategies for restoration of function of a decentralized bladder after spinal cord or spinal root injury via repair of the original roots or by transferring in new axon sources. These techniques have included end-to-end sacral root repairs, transfer of roots from other segments to sacral roots, transfer of intercostal nerves to sacral roots, transfer of various somatic nerves to the pelvic or pudendal nerve, direct neurotization of the detrusor muscle, and creation of an artificial “skin-CNS-bladder” reflex pathway. We have comprehensively reviewed these surgical techniques, the strengths and limitations of each, and have discussed the most appropriate patient population for each procedure. Our purpose is to critically assess the potential efficacy of nerve transfer techniques as options for restoring urinary function after bladder decentralization.