Ekta Tiwari

Determining integrity of bladder innervation and smooth muscle function 1 year after lower spinal root transection in canines

To assess bladder smooth muscle function and innervation after long‐term lower spinal root transection in canines.

Lumbar to sacral root rerouting to restore bladder function in a feline spinal cord injury model: Urodynamic and retrograde nerve tracing results from a pilot study

Lumbar to sacral rerouting surgery can potentially allow voiding via a skin‐central nervous system‐bladder reflex pathway. Here, we assessed if this surgery was effective in treating neurogenic bladder dysfunction/sphincter in felines.

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.

AB314. SPR-41 Localization of neuromuscular nicotinic receptors in the functionally reinnervated canine bladder after prolonged decentralization

Objective We previously found that intravenous succinylcholine, a depolarizing neuromuscular nicotinic receptors blocker, prevents bladder contractions induced by new neuronal pathways established by nerve transfer in decentralized dogs. We studied the detrusor pressure response in vivo and contractile response of bladder smooth muscle strips in vitro from sham, decentralized and reinnervated animals to localize the neuromuscular nicotinic receptors involved. Methods Three groups of female mongrel hound dogs were used: sham (N=4), 12-month decentralized (N=3) and 6-month reinnervated (N=3). Decentralization was created by bilateral transection of all spinal roots caudal to L7, including the dorsal roots of L7 and the hypogastric nerves. Reinnervation was created by bilateral transfer of the obturator nerve to anterior vesical branches of the pelvic nerve. Two-way ANOVAs and Sidak post-hoc tests were used to determine group differences. Results In reinnervated dogs, blockade of neuromuscular nicotinic receptor with intravenous injections of the competitive antagonist atracurium besylate significantly reduced the increase in detrusor pressure induced by electrical stimulation of the transferred obturator nerve (L1 or L2). Atracurium did not block the increase in pressure induced by stimulation of sacral nerve roots in sham-operated controls. In vitro, neither the competitive neuromuscular nicotinic receptor antagonist d-tubocurarine nor the ganglionic antagonist hexamethonium inhibited electric field stimulation (EFS)-induced contractions of reinnervated or sham-operated control bladder strips. No contractile response was elicited in the presence of 1 µM tetrodotoxin (TTX) across groups. Similarly, EFS-evoked contractions were strongly reduced by 10 µM alpha, beta-methylene ATP (α,β-mATP) and 1 µM atropine in all groups relative to the vehicle (water). Conclusions In vivo blockade of nerve-evoked bladder pressure by atracurium in the reinnervated, but not sham operated controls, suggests that neuromuscular nicotinic receptors become involved in bladder contractions induced by the new neuronal pathway. Because d-tubocurarine did not block in vitro contractions induced by EFS in the reinnervated bladders, the neuromuscular nicotinic receptors involved in the new neuronal pathway must not be located in the bladder muscle or intramural ganglia and therefore, are likely in preganglionic neurons. TTX blockade validates that EFS-induced contractions at all frequencies were nerve-evoked. Both muscarinic and purinergic components contributed similarly to neurotransmission based on response to blockade of nerve evoked muscle strip contractions with a combination of atropine and α,β-mATP. Funding Source(s) NIH-NINDS NS070267

AB320. SPR-47 Monitoring nerve activity during bladder filling in a rat model

Objective Surgical rerouting of neuronal pathways may allow functional reinnervation of the bladder. We aim to develop techniques to monitor afferent (sensory) nerve activity during bladder filling in normal intact bladders for eventual application to monitoring sensory reinnervation of the bladder following nerve transfer. Methods Electroneurogram recordings were performed in anesthetized rats under isoflurane (1–2% induction dose, inhalation) of: (I) sciatic nerves during stimulation of the hindpaw with Semmes-Weinstein monofilaments of varying forces (10–300 g); and (II) bladder nerves during bladder filling with infusion rate of 0.5 mL/min, in acute experiments. Bipolar cuff electrodes were wrapped around the sciatic nerve (n=7) and ureter (and associated bladder nerves) proximal to the bladder wall (n=7), to record sciatic and bladder nerve discharges, respectively. The sciatic nerve was transected between the spinal cord and the electrode, to eliminate efferent nerve signals and record afferent fibers discharge only with hindpaw stimulation. Whereas, recordings were made of the discharges of both afferent and efferent fibers from bladder nerves during bladder filling. All recordings were performed using a low noise amplifier (SR560, filtered 300 Hz–10 kHz, gain ×10k), sampled at 20 kHz using PowerLab software (AD Instruments) and displayed using LabChart software. Bladder pressure was also recorded during filling. Results Sciatic nerve recordings consistently showed increased afferent fibers discharge with increased size of monofilament used to stimulate the hindpaw, with the highest discharge observed with the 300 g monofilament and lowest with 10 g. In contrast, recording from bladder nerves showed that combined afferent and efferent discharges increased substantially in response to bladder filling in 2 of 7 rats, and increased moderately in 2 other rats. However, there was no response in the remaining rats, perhaps due to nerve damage during cuff placement. Conclusions We found an increase in afferent discharges during paw stimulation with increased monofilaments sizes. Also, recordings from the cuff around the ureter-bladder nerve complex revealed that the afferent and efferent discharges coincided with an increase in bladder pressure during bladder filling. We could not isolate single fibers (units) from these whole nerve recordings, thus we cannot report on individual nerve fiber activity. However, based on these results, we conclude that the present technique used to record nerve activity in the rat model may be suitable to record nerve activity during bladder filling in canines with surgically rerouted neural pathways. Funding Source(s) NIH 1R01NS070267

AB301. SPR-28 Determining integrity of the nerve-smooth muscle functional unit of the bladder after long-term decentralization

Objective Somatic nerve transection causes rapid loss of skeletal muscle mass and contractility. While skeletal muscle degeneration following nerve injury has been well investigated, less is known about the effects of autonomic nerve transection on smooth muscle. We explored changes in the nerve-smooth muscle functional unit following sacral root decentralization to determine integrity after decreased innervation. Methods Female mixed-breed hound dogs were surgically decentralized by bilateral transection of all spinal roots caudal to L7, including the dorsal root of L7 in a subgroup. Three weeks prior to the terminal surgery, bladders were injected cystoscopically with fluorogold around the ureterovesical junction for retrograde neuronal labeling. Tissue function was tested during the terminal procedure after 6-month (n=2) and 12-month (n=6) decentralization and compared to sham/unoperated control animals (n=13). Immediately prior to euthanasia, in vivo detrusor pressure after stimulation of nerves originating from the pelvic plexus (e.g., the anterior vesicle branch) was recorded. Collected bladder and pelvic plexus tissues from controls and 6-month decentralized dogs were harvested (n=3–6/group), cryosectioned, and examined for fluorogold labeling. Bladder tissues were stained for caspase-3 and immunostaining was quantified. Gastric tissue and red blood cells within bladder walls were used as positive controls. Data was analyzed using unpaired ANOVA. Results Nerve stimulation caused a robust increase in detrusor pressure in both control and decentralized groups. Likewise, abundant fluorogold-labeled neuronal cell bodies were observed in ganglia in the pelvic plexus of both sham and decentralized animals. Immunohistochemical stain for caspase-3 showed no difference across groups. Also, we did not observe co-localization of fluorogold-positive neuronal tissue and caspase-3, or presence of caspase-3 in smooth muscle fibers of the bladder wall. Conclusions The presence of fluorogold-labeled pelvic plexus ganglia in decentralized animals demonstrates that the ganglia remained intact up to 6 months after decentralization. Caspase-3 staining results showed no increase in apoptosis in the neuronal tissues or bladder smooth muscle in decentralized dogs, suggesting no increased apoptotic cell death. No significant difference between detrusor pressure responses across groups after nerve-evoked stimulation indicates that the nerve-smooth muscle functional unit of the bladder is intact up to 12 months after injury and therefore, nerve reinnervation strategies could be successful. Funding Source(s) NIH-NINDS NS070267