Danielle M. Salvadeo

Heat Shock Protein Response to Overuse Injuries

The heat shock protein (Hsp) response is understudied with non-exercise overuse injuries. We focused on the Hsp response in muscles and tendons undergoing such injury or cyclical loading. Hsp25 mRNA and protein levels increase in muscles undergoing functional overload, and show greater increases in fast type muscles. In an operant rat model of reaching and grasping, the inducible form of Hsp70 increased in muscles and tendons showing injury, with the greatest increase in rats performing a high repetition high force for 12 weeks, compared to easier repetition/loading paradigms. These increases were paralleled by increases in several repair-associated proteins (osteoactivin, MMPs, and TGFB1). Trapezius biopsies from patients with myalgia show increased mRNA levels of Hsp72 and decreased levels of growth and metabolism regulators. Prolonged exercise interventions in general, when provided to subjects with trapezius myalgia, decreased Hsp72 mRNA levels, while specific strength training of shoulder and neck muscles increased mRNA levels of analytes related to carbohydrate oxidation. In a rat model of supraspinatus injury, the Hsp response appeared related to the cascade of stress-related programmed cell death in torn tendons. A mild mechanical stimulation of cultured tendon fibroblasts reduced apoptosis and increased cell proliferation and may be helpful for tissue regeneration.

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.

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.

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

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

AB318. SPR-45 Decentralization reduces nicotinic receptor-mediated canine bladder contractions in vitro

Objective Bladder function depends upon several complex signaling pathways that induce either contraction or relaxation. We performed nerve re-routing surgery on bladder of decentralized dogs (reinnervated) as a model with the goal of restoring bladder function in spinal cord injured patients. The neuromuscular nicotinic receptor blocker, succinylcholine blocks spinal root-stimulated bladder contraction in vivo in reinnervated dogs, but not in sham-operated dogs. Our lab explored the function and location of nicotinic receptors involved in bladder contraction, with emphasis on their possible role in the release of other neurotransmitters such as acetylcholine or ATP in sham-operated, 12-month-decentralized and immediate-reinnervated bladders. Methods Smooth muscle strips (mucosa denuded) were isolated from the region just rostral to the trigone and suspended in muscle baths. Strips were ranked based on their contractile response to a 3-min exposure to 120 mM KCl and sorted so that the average response in each group was equal. Strips were incubated with either 1 µM atropine (ATR), 1 µM tetrodotoxin (TTX), 10 µM alpha, beta-methylene ATP (α,β-ATP) or vehicle (water) and then induced to contract with the nicotinic receptor agonists 1,1 dimethyl-4-phenyl-piperazinium iodide (DMPP, 100 µM), TC2559 (100 µM) or epibatidine (10 µM) or 1 mM nicotine itself. Results The DMPP-induced contraction was not different between sham, reinnervated or decentralized bladders (11%, 3.3%, and 3.6% of KCl contraction respectively). While the epibatidine-induced contraction in shams was not different relative to that in the reinnervated (41% vs. 27% KCl respectively), it was significantly greater than that in decentralized bladders (13% of KCl). TC2559 did not induce bladder contractions. Nicotine-induced contractions in sham-operated controls were 16% of KCl. ATR completely blocked nicotine-induced contraction while α,β-ATP had no statistically significant effect in shams. TTX had no significant inhibitory effect on DMPP, epibatidine or nicotine-induced contraction in any group. Conclusions Nicotinic receptors mediate contraction in sham, reinnervated and decentralized bladders. This nicotinic receptor-mediated contraction is decreased after decentralization. TTX does not block nicotinic receptor-mediated contractions, indicating that action potentials are not required to induce contraction. In sham-operated dog bladders, the nicotine-induced contraction is blocked by ATR, suggesting that these nicotinic receptors are located on cholinergic nerve terminals and induce the release of acetylcholine, which activates muscarinic receptors on the smooth muscle. Funding Source(s) NIH-NINDS NS070267