Differential Myosin 5a splice variants in innervation of pelvic organs.

Introduction: Myosin proteins interact with filamentous actin and translate the chemical energy generated by ATP hydrolysis into a wide variety of mechanical functions in all cell types. The classic function of conventional myosins is mediation of muscle contraction, but myosins also participate in processes as diverse as exocytosis/endocytosis, membrane remodeling, and cytokinesis. Myosin 5a (Myo5a) is an unconventional motor protein well-suited to the processive transport of diverse molecular cargo within cells and interactions with multiprotein membrane complexes that facilitate exocytosis. Myo5a includes a region consisting of six small alternative exons which can undergo differential splicing. Neurons and skin melanocytes express characteristic splice variants of Myo5a, which are specialized for transport processes unique to those cell types. But less is known about the expression of Myo5a splice variants in other tissues, their cargos and interactive partners, and their regulation. Methods: In visceral organs, neurotransmission-induced contraction or relaxation of smooth muscle is mediated by Myo5a. Axons within urogenital organs and distal colon of rodents arise from cell bodies located in the major pelvic ganglion (MPG). However, in contrast to urogenital organs, the distal colon also contains soma of the enteric nervous system. Therefore, the rodent pelvic organs provide an opportunity to compare the expression of Myo5a splice variants, not only in different tissues innervated by the pelvic nerves, but also in different subcellular compartments of those nerves. This study examines the expression and distribution of Myo5a splice variants in the MPG, compared to the bladder, corpus cavernosum of the penis (CCP) and distal colon using immunohistochemistry and mRNA analyses. Results/discussion: We report detection of characteristic Myo5a variants in these tissues, with bladder and CCP displaying a similar variant pattern but one which differed from that of distal colon. In all three organs, Myo5a variants were distinct compared to the MPG, implying segregation of one variant within nerve soma and its exclusion from axons. The expression of distinct Myo5a variant arrays is likely to be adaptive, and to underlie specific functions fulfilled by Myo5a in those particular locations.

Integrated omics analysis unveils a DNA damage response to neurogenic injury.

Spinal cord injury (SCI) evokes profound bladder dysfunction. Current treatments are limited by a lack of molecular data to inform novel therapeutic avenues. Previously, we showed systemic inosine treatment improved bladder function following SCI in rats. Here, we applied multi-omics analysis to explore molecular alterations in the bladder and their sensitivity to inosine following SCI. Canonical pathways regulated by SCI included those associated with protein synthesis, neuroplasticity, wound healing, and neurotransmitter degradation. Upstream regulator analysis identified MYC as a key regulator, whereas causal network analysis predicted multiple regulators of DNA damage response signaling following injury, including PARP-1. Staining for both DNA damage (γH2AX) and PARP activity (poly-ADP-ribose) markers in the bladder was increased following SCI, and attenuated in inosine-treated tissues. Proteomics analysis suggested that SCI induced changes in protein synthesis-, neuroplasticity-, and oxidative stress-associated pathways, a subset of which were shown in transcriptomics data to be inosine-sensitive. These findings provide novel insights into the molecular landscape of the bladder following SCI, and highlight a potential role for PARP inhibition to treat neurogenic bladder dysfunction.

Neuropilin 2 Is a Novel Regulator of Distal Colon Contractility.

Appropriate coordination of smooth muscle contraction and relaxation is essential for normal colonic motility. The impact of perturbed motility ranges from moderate, in conditions such as colitis, to potentially fatal in the case of pseudo-obstruction. The mechanisms underlying aberrant motility and the extent to which they can be targeted pharmacologically are incompletely understood. This study identified colonic smooth muscle as a major site of expression of neuropilin 2 (Nrp2) in mice and humans. Mice with inducible smooth muscle-specific knockout of Nrp2 had an increase in evoked contraction of colonic rings in response to carbachol at 1 and 4 weeks following initiation of deletion. KCl-induced contractions were also increased at 4 weeks. Colonic motility was similarly enhanced, as evidenced by faster bead expulsion in Nrp2-deleted mice versus Nrp2-intact controls. In length-tension analysis of the distal colon, passive tension was similar in Nrp2-deficient and Nrp2-intact mice, but at low strains, active stiffness was greater in Nrp2-deficient animals. Consistent with the findings in conditional Nrp2 mice, Nrp2-null mice showed increased contractility in response to carbachol and KCl. Evaluation of selected proteins implicated in smooth muscle contraction revealed no significant differences in the level of α-smooth muscle actin, myosin light chain, calponin, or RhoA. Together, these findings identify Nrp2 as a novel regulator of colonic contractility that may be targetable in conditions characterized by dysmotility.

Myosin 5a in the Urinary Bladder: Localization, Splice Variant Expression, and Functional Role in Neurotransmission.

Dysregulation of neurotransmission is a feature of several prevalent lower urinary tract conditions, but the mechanisms regulating neurotransmitter release in the bladder are not completely understood. The unconventional motor protein, Myosin 5a, transports neurotransmitter-containing synaptic vesicles along actin fibers towards the varicosity membrane, tethering them at the active zone prior to reception of a nerve impulse. Our previous studies indicated that Myosin 5a is expressed and functionally relevant in the peripheral nerves of visceral organs such as the stomach and the corpora cavernosa. However, its potential role in bladder neurotransmission has not previously been investigated. The expression of Myosin 5a was examined by quantitative PCR and restriction analyses in bladders from DBA (dilute-brown-nonagouti) mice which express a Myosin 5a splicing defect and in control mice expressing the wild-type Myosin 5a allele. Functional differences in contractile responses to intramural nerve stimulation were examined by ex vivo isometric tension analysis. Data demonstrated Myosin 5a localized in cholinergic nerve fibers in the bladder and identified several Myosin 5a splice variants in the detrusor. Full-length Myosin 5a transcripts were less abundant and the expression of splice variants was altered in DBA bladders compared to control bladders. Moreover, attenuation of neurally-mediated contractile responses in DBA bladders compared to control bladders indicates that Myosin 5a facilitates excitatory neurotransmission in the bladder. Therefore, the array of Myosin 5a splice variants expressed, and the abundance of each, may be critical parameters for efficient synaptic vesicle transport and neurotransmission in the urinary bladder.

Knockin mouse models demonstrate differential contributions of synaptotagmin-1 and -2 as receptors for botulinum neurotoxins.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are also utilized to treat a wide range of disorders including muscle spasm, overactive bladder, and pain. BoNTs' ability to target neurons determines their specificity, potency, and therapeutic efficacy. Homologous synaptic vesicle membrane proteins synaptotagmin-1 (Syt1) and synaptotagmin-2 (Syt2) have been identified as receptors for BoNT family members including BoNT/B, DC, and G, but their contributions at physiologically relevant toxin concentrations in vivo have yet to be validated and established. Here we generated two knockin mutant mouse models containing three designed point-mutations that specifically disrupt BoNT binding in endogenous Syt1 or Syt2, respectively. Utilizing digit abduction score assay by injecting toxins into the leg muscle, we found that Syt1 mutant mice showed similar sensitivity as the wild type mice, whereas Syt2 mutant mice showed reduced sensitivity to BoNT/B, DC, and G, demonstrating that Syt2 is the dominant receptor at skeletal neuromuscular junctions. We further developed an in vivo bladder injection assay for analyzing BoNT action on bladder tissues and demonstrated that Syt1 is the dominant toxin receptor in autonomic nerves controlling bladder tissues. These findings establish the critical role of protein receptors for the potency and specificity of BoNTs in vivo and demonstrate the differential contributions of Syt1 and Syt2 in two sets of clinically relevant target tissues.

Evaluation of Bi-Layer Silk Fibroin Grafts for Tubular Ureteroplasty in a Porcine Defect Model.

Ureteral reconstruction with autologous tissue grafts is often limited by tissue availability and donor site morbidity. This study investigates the performance of acellular, bi-layer silk fibroin (BLSF) scaffolds in a porcine model of ureteroplasty. Tubular ureteroplasty with BLSF grafts in combination with transient stenting for 8 weeks was performed in adult female, Yucatan, mini-swine (N = 5). Animals were maintained for 12 weeks post-op with imaging of neoconduits using ultrasonography and retrograde ureteropyelography carried out at 2 and 4 weeks intervals. End-point analyses of ureteral neotissues and unoperated controls included histological, immunohistochemical (IHC), histomorphometric evaluations as well as ex vivo functional assessments of contraction/relaxation. All animals survived until scheduled euthanasia and displayed mild hydronephrosis (Grades 1-2) in reconstructed collecting systems during the 8 weeks stenting period with one animal presenting with a persistent subcutaneous fistula at 2 weeks post-op. By 12 weeks of scaffold implantation, unstented neoconduits led to severe hydronephrosis (Grade 4) and stricture formation in the interior of graft sites in 80% of swine. Bulk scaffold extrusion into the distal ureter was also apparent in 60% of swine contributing to ureteral obstruction. However, histological and IHC analyses revealed the formation of innervated, vascularized neotissues with a-smooth muscle actin+ and SM22α+ smooth muscle bundles as well as uroplakin 3A+ and pan-cytokeratin + urothelium. Ex vivo contractility and relaxation responses of neotissues were similar to unoperated control segments. BLSF biomaterials represent emerging platforms for tubular ureteroplasty, however further optimization is needed to improve in vivo degradation kinetics and mitigate stricture formation.

Expression of Myosin 5a splice variants in murine stomach.

BACKGROUND: The motor protein, Myosin 5a (Myo5a) is known to play a role in inhibitory neurotransmission in gastric fundus. However, there is no information regarding the relative expression of total Myo5a, or of its alternative exon splice variants, across the stomach. This study investigated the differential distribution of Myo5a variants expressed within distinct anatomical regions of murine stomach. METHODS: The distribution of Myo5a protein and mRNA in the stomach was assessed by immunofluorescence microscopy and fluorescent in situ hybridization. Quantitative PCR, restriction enzyme analysis, and electrophoresis were used to identify Myo5a splice variants and quantify their expression levels in the fundus, body, antrum, and pylorus. KEY RESULTS: Myo5a protein colocalized with ßIII-Tubulin in the myenteric plexus, and with synaptophysin in nerve fibers. Total Myo5a mRNA expression was lower in pylorus than in antrum, body, or fundus (p < 0.001), which expressed equivalent amounts of Myo5a. However, Myo5a splice variants were differentially expressed across the stomach. While the ABCE splice variant predominated in the antrum and body regions, the ACEF/ACDEF variants were enriched in fundus and pylorus. CONCLUSIONS AND INFERENCES: Myo5a splice variants varied in their relative expression across anatomically distinguishable stomach regions and might mediate distinct physiological functions in gastric neurotransmission.

Evaluation of Bilayer Silk Fibroin Grafts for Tubular Esophagoplasty in a Porcine Defect Model.

Surgical reconstruction of tubular esophageal defects with autologous gastrointestinal segments is the gold standard treatment to replace damaged or diseased esophageal tissues. Unfortunately, this approach is associated with adverse complications, including dysphagia, donor-site morbidity, and in some cases patient death. Bilayer silk fibroin (BLSF) scaffolds were investigated as alternative, acellular grafts for tubular esophagoplasty in a porcine defect model for 3 months of implantation. Adult Yucatan mini-swine (n = 5) were subjected to esophageal reconstruction with tubular BLSF grafts (2 cm in length) in combination with transient esophageal stenting for 2 months followed by a 1-month period, where the graft site was unstented. All animals receiving BLSF grafts survived and were capable of solid food consumption, however strictures were noted at graft regions in 60% of the experimental cohort between 2 and 3 months postop and required balloon dilation. In addition, fluoroscopic analysis showed peristaltic function in only 1/5 neotissues. Following swine harvest at 3 months, ex vivo tissue bath evaluations revealed that neoconduits exhibited contractile responses to carbachol, electric field stimulation, and KCl, whereas sodium nitroprusside and isoproterenol induced relaxation effects. Histological (Masson's Trichrome) and immunohistochemical analyses of regenerated tissue conduits showed a stratified, squamous epithelium expressing pan-cytokeratins buttressed by a vascularized lamina propria containing a smooth muscle-rich muscularis mucosa surrounded by a muscularis externa. Neuronal density, characterized by the presence of synaptophysin-positive boutons, was significantly lower in neotissues in comparison to nonsurgical controls. BLSF scaffolds represent a promising platform for the repair of tubular esophageal defects, however improvements in scaffold design are needed to reduce the rate of complications and improve the extent of constructive tissue remodeling. Impact statement The search for a superior "off-the-shelf" scaffold capable of repairing tubularesophageal defects as well as overcoming limitations associated with conventional autologous gastrointestinal segments remains elusive. The purpose of this study was to investigate the performance of an acellular, bilayer silk fibroin graft (BLSF) for tubular esophagoplasty in a porcine model. Our results demonstrated that BLSF scaffolds supported the formation of tubular neotissues with innervated, vascularized epithelial and muscular components capable of contractile and relaxation responses. BLSF scaffolds represent a promising platform for esophageal tissue engineering.

Rapid gastric emptying in diabetes mellitus: Pathophysiology and clinical importance.

Although slow gastric emptying (gastroparesis) is a well-known complication of chronic hyperglycemia in diabetes mellitus (DM), it recently has become clear that rapid gastric emptying also is a frequent and important diabetic complication. In contrast, acute hyperglycemia causes slow gastric emptying, and acute hypoglycemia causes rapid gastric emptying. Rapid gastric emptying is frequent in T2DM; however, it may also occur in T1DM, particularly in the early stages of the disease, but may persist even into late stages. Recent studies suggest that usually, the stomach restricts the emptying of nutrients to 1-4 kcals/min. This restriction is due to the action of the gastric 'braking' hormones such as GLP-1, leptin, and amylin acting via the gastric inhibitory vagal motor circuit (GIVMC). Disruption of this braking system leads to rapid gastric emptying. Acute hyperglycemia also slows gastric emptying by stimulating the GIVMC, while acute hypoglycemia causes rapid gastric emptying by stimulating the gastric excitatory vagal motor circuit (GEVMC). In contrast, chronic hyperglycemia causes rapid gastric emptying by inducing oxidative stress in the stomach wall that disrupts inhibitory neuromuscular transmission and increases the contractility of the smooth muscle, while chronic hyperglycemia may also cause slow gastric emptying via severe inflammatory stress caused by proinflammatory macrophages and reduce contractility of the smooth muscle. There is a bidirectional relationship between blood glucose and gastric emptying. Thus, rapid gastric emptying may lead to a sizeable postprandial spike, and slow gastric emptying may blunt it. Postprandial hyperglycemia is involved in the development, progression, and complications of DM. Correction of fast gastric emptying involves agents that activate GIVMC and the use of gastric 'braking' hormones or their analogs. Recognition and treatment of rapid gastric emptying may contribute to better management of postprandial hyperglycemia and prevention of some diabetic complications.

Augmentation Cystoplasty of Diseased Porcine Bladders with Bi-Layer Silk Fibroin Grafts.

IMPACT STATEMENT: The search for an ideal "off-the-shelf" biomaterial for augmentation cystoplasty remains elusive and current scaffold configurations are hampered by mechanical and biocompatibility restrictions. In addition, preclinical evaluations of potential scaffold designs for bladder repair are limited by the lack of tractable large animal models of obstructive bladder disease that can mimic clinical pathology. The results of this study describe a novel, minimally invasive, porcine model of partial bladder outlet obstruction that simulates clinically relevant phenotypes. Utilizing this model, we demonstrate that acellular, bi-layer silk fibroin grafts can support the formation of vascularized, innervated bladder tissues with functional properties.

Inosine - a Multifunctional Treatment for Complications of Neurologic Injury.

Spinal cord injury (SCI) caused by trauma or disease leads to motor and sensory abnormalities that depend on the level, severity and duration of the lesion. The most obvious consequence of SCI is paralysis affecting lower and upper limbs. SCI also leads to loss of bladder and bowel control, both of which have a deleterious, life-long impact on the social, psychological, functional, medical and economic well being of affected individuals. Currently, there is neither a cure for SCI nor is there adequate management of its consequences. Although medications provide symptomatic relief for the complications of SCI including muscle spasms, lower urinary tract dysfunction and hyperreflexic bowel, strategies for repair of spinal injuries and recovery of normal limb and organ function are still to be realized. In this review, we discuss experimental evidence supporting the use of the naturally occurring purine nucleoside inosine to improve the devastating sequelae of SCI. Evidence suggests inosine is a safe, novel agent with multifunctional properties that is effective in treating complications of SCI and other neuropathies.

The role of the mucosa in modulation of evoked responses in the spinal cord injured rat bladder.

AIMS: Mounting evidence indicates that a variety of factors released from the urothelium or suburothelium can modulate smooth muscle activity. Although the relationship between the mucosa and smooth muscle has been investigated, little is known about the pathophysiologic changes in detrusor-mucosa interactions in neurogenic bladders. The goal of the study was to determine the impact of the mucosa on evoked responses in spinal cord injured (SCI) bladders. METHODS: Urinary bladders were obtained from 6wk SCI rats or age-matched uninjured controls. Ex vivo isometric tension studies were performed and muscarinic receptor expression was measured in bladder tissue with and without mucosa. RESULTS: The magnitude and area of nerve evoked responses in SCI tissue with mucosa was higher than without mucosa. The duration and decay time of nerve-evoked responses were longer in SCI than control tissue irrespective of the mucosa. The level of the muscarinic M2 receptor was decreased in the mucosa of SCI bladders. CONCLUSIONS: Detrusor-mucosa interactions are substantially altered in the neurogenic bladder. After spinal cord injury, an excitatory modulation of smooth muscle contraction by the mucosa emerges, and could be targeted via intravesical treatment in the context of neurogenic bladder dysfunction.

Bilayer silk fibroin grafts support functional oesophageal repair in a rodent model of caustic injury.

Surgical repair of caustic oesophageal injuries with autologous gastrointestinal segments is often associated with dysmotility, dysphagia and donor site morbidity, and therefore alternative graft options are needed. Bilayer silk fibroin (BLSF) scaffolds were assessed for their ability to support functional restoration of damaged oesophageal tissues in a rat model of onlay oesophagoplasty. Transient exposure of isolated oesophageal segments with 40% NaOH led to corrosive oesophagitis and a 91% reduction in the luminal cross-sectional area of damaged sites. Oesophageal repair with BLSF matrices was performed in injured rats (n = 27) as well as a nondiseased cohort (n = 12) for up to 2 months after implantation. Both implant groups exhibited >80% survival rates, displayed similar degrees of weight gain, and were capable of solid food consumption following a 3-day liquid diet. End-point µ-computed tomography of repaired sites demonstrated a 4.5-fold increase in luminal cross-sectional area over baseline injury levels. Reconstructed oesophageal conduits from damaged and nondiseased animals produced comparable contractile responses to KCl and electric field stimulation while isoproterenol generated similar tissue relaxation responses. Histological and immunohistochemical evaluations of neotissues from both implant groups showed formation of a stratified, squamous epithelium with robust cytokeratin expression as well as skeletal and smooth muscle layers positive for contractile protein expression. In addition, synaptophysin positive neuronal junctions and vessels lined with CD31 positive endothelial cells were also observed at graft sites in each setting. These results provide preclinical validation for the use of BLSF scaffolds in reconstructive strategies for oesophageal repair following caustic injury.

Bi-layer silk fibroin grafts support functional tissue regeneration in a porcine model of onlay esophagoplasty.

Partial circumferential, full thickness defects of the esophagus can occur as a result of organ perforation or tumour resection, or during surgical reconstruction of strictured segments. Complications associated with autologous tissue flaps conventionally utilized for defect repair necessitate the development of new graft options. In this study, bi-layer silk fibroin (BLSF) scaffolds were investigated for their potential to support functional restoration of partial circumferential defects in a porcine model of esophageal repair. Onlay thoracic esophagoplasty with BLSF matrices (~3 x 1.5 cm) was performed in adult swine (N = 6) for 3 months of implantation. All animals receiving BLSF grafts survived with no complications and were capable of solid food consumption. Radiographic esophagrams revealed preservation of organ continuity with no evidence of contrast extravasation or strictures. Fluoroscopic analysis demonstrated peristaltic contractions. Ex vivo tissue bath studies displayed contractile responses to carbachol, electric field stimulation, and KCl while isoproterenol produced tissue relaxation. Histological and immunohistochemical evaluations of neotissues showed a stratified, squamous epithelium, a muscularis mucosa composed of smooth muscle bundles, and a muscularis externa organized into circular and longitudinal layers, with a mix of striated skeletal muscle fascicles interspersed with smooth muscle. De novo innervation and vascularization were observed throughout the graft sites and consisted of synaptophysin-positive neuronal boutons and vessels lined with CD31-positive endothelial cells. The results of this study demonstrate that BLSF scaffolds can facilitate constructive remodeling of partial circumferential, full thickness esophageal defects in a large animal model. Copyright © 2017 John Wiley & Sons, Ltd.

Inosine attenuates spontaneous activity in the rat neurogenic bladder through an A2B pathway.

Neurogenic detrusor overactivity (NDO) is among the most challenging complications of spinal cord injury (SCI). A recent report by us demonstrated an improvement in NDO in SCI rats following chronic systemic treatment with the purine nucleoside inosine. The objective of this study was to investigate the mechanism of action of inosine underlying improvement of NDO. Male Sprague-Dawley rats underwent complete spinal cord transection at T8. Inosine (1 mM) delivered intravesically to SCI rats during conscious cystometry significantly decreased the frequency of spontaneous non-voiding contractions. In isolated tissue assays, inosine (1 mM) significantly decreased the amplitude of spontaneous activity (SA) in SCI bladder muscle strips. This effect was prevented by a pan-adenosine receptor antagonist CGS15943, but not by A1 or A3 receptor antagonists. The A2A antagonist ZM241385 and A2B antagonist PSB603 prevented the effect of inosine. The effect of inosine was mimicked by the adenosine receptor agonist NECA and the A2B receptor agonist BAY60-6583. The inhibition of SA by inosine was not observed in the presence of the BK antagonist, iberiotoxin, but persisted in the presence of KATP and SK antagonists. These findings demonstrate that inosine acts via an A2B receptor-mediated pathway that impinges on specific potassium channel effectors.

Deletion of neuropilin 2 enhances detrusor contractility following bladder outlet obstruction.

Chronic urethral obstruction and the ensuing bladder wall remodeling can lead to diminished bladder smooth muscle (BSM) contractility and debilitating lower urinary tract symptoms. No effective pharmacotherapy exists to restore BSM contractile function. Neuropilin 2 (Nrp2) is a transmembrane protein that is highly expressed in BSM. Nrp2 deletion in mice leads to increased BSM contraction. We determined whether genetic ablation of Nrp2 could restore BSM contractility following obstruction. Partial bladder outlet obstruction (pBOO) was created by urethral occlusion in mice with either constitutive and ubiquitous, or inducible smooth muscle-specific deletion of Nrp2, and Nrp2-intact littermates. Mice without obstruction served as additional controls. Contractility was measured by isometric tension testing. Nrp2 deletion prior to pBOO increased force generation in BSM 4 weeks following surgery. Deletion of Nrp2 in mice already subjected to pBOO for 4 weeks showed increased contractility of tissues tested 6 weeks after surgery compared with nondeleted controls. Assessment of tissues from patients with urodynamically defined bladder outlet obstruction revealed reduced NRP2 levels in obstructed bladders with compensated compared with decompensated function, relative to asymptomatic controls. We conclude that downregulation of Nrp2 promotes BSM force generation. Neuropilin 2 may represent a novel target to restore contractility following obstruction.

Inosine Improves Neurogenic Detrusor Overactivity following Spinal Cord Injury.

Neurogenic detrusor overactivity and the associated loss of bladder control are among the most challenging complications of spinal cord injury (SCI). Anticholinergic agents are the mainstay for medical treatment of detrusor overactivity. However, their use is limited by significant side effects such that a search for new treatments is warranted. Inosine is a naturally occurring purine nucleoside with neuroprotective, neurotrophic and antioxidant effects that is known to improve motor function in preclinical models of SCI. However, its effect on lower urinary tract function has not been determined. The objectives of this study were to determine the effect of systemic administration of inosine on voiding function following SCI and to delineate potential mechanisms of action. Sprague-Dawley rats underwent complete spinal cord transection, or cord compression by application of an aneurysm clip at T8 for 30 sec. Inosine (225 mg/kg) or vehicle was administered daily via intraperitoneal injection either immediately after injury or after a delay of 8 wk. At the end of treatment, voiding behavior was assessed by cystometry. Levels of synaptophysin (SYP), neurofilament 200 (NF200) and TRPV1 in bladder tissues were measured by immunofluorescence imaging. Inosine administration decreased overactivity in both SCI models, with a significant decrease in the frequency of spontaneous non-voiding contractions during filling, compared to vehicle-treated SCI rats (p<0.05), including under conditions of delayed treatment. Immunofluorescence staining demonstrated increased levels of the pan-neuronal marker SYP and the Adelta fiber marker NF200, but decreased staining for the C-fiber marker, TRPV1 in bladder tissues from inosine-treated rats compared to those from vehicle-treated animals, including after delayed treatment. These findings demonstrate that inosine prevents the development of detrusor overactivity and attenuates existing overactivity following SCI, and may achieve its effects through modulation of sensory neurotransmission.

Acellular bi-layer silk fibroin scaffolds support functional tissue regeneration in a rat model of onlay esophagoplasty.

Surgical management of long-gap esophageal defects with autologous gastrointestinal tissues is frequently associated with adverse complications including organ dysmotility, dysphagia, and donor site morbidity. In order to develop alternative graft options, bi-layer silk fibroin (SF) scaffolds were investigated for their potential to support functional tissue regeneration in a rodent model of esophageal repair. Onlay esophagoplasty was performed with SF matrices (N = 40) in adult rats for up to 2 m of implantation. Parallel groups consisted of animals implanted with small intestinal submucosa (SIS) scaffolds (N = 22) or sham controls receiving esophagotomy alone (N = 20). Sham controls exhibited a 100% survival rate while rats implanted with SF and SIS scaffolds displayed respective survival rates of 93% and 91% prior to scheduled euthanasia. Animals in each experimental group were capable of solid food consumption following a 3 d post-op liquid diet and demonstrated similar degrees of weight gain throughout the study period. End-point µ-computed tomography at 2 m post-op revealed no evidence of contrast extravasation, fistulas, strictures, or diverticula in any of the implant groups. Ex vivo tissue bath studies demonstrated that reconstructed esophageal conduits supported by both SF and SIS scaffolds displayed contractile responses to carbachol, KCl and electrical field stimulation while isoproterenol produced tissue relaxation. Histological (Masson's trichrome and hematoxylin and eosin) and immunohistochemical (IHC) evaluations demonstrated both implant groups produced de novo formation of skeletal and smooth muscle bundles positive for contractile protein expression [fast myosin heavy chain (MY32) and α-smooth muscle actin (α-SMA)] within the graft site. However, SF matrices promoted a significant 4-fold increase in MY32+ skeletal muscle and a 2-fold gain in α-SMA+ smooth muscle in comparison to the SIS cohort as determined by histomorphometric analyses. A stratified squamous, keratinized epithelium expressing cytokeratin 5 and involucrin proteins was also present at 2 m post-op in all experimental groups. De novo innervation and vascularization were evident in all regenerated tissues indicated by the presence of synaptophysin (SYP38)+ boutons and vessels lined with CD31 expressing endothelial cells. In respect to SIS, the SF group supported a significant 4-fold increase in the density of SYP38+ boutons within the implant region. Evaluation of host tissue responses revealed that SIS matrices elicited chronic inflammatory reactions and severe fibrosis throughout the neotissues, in contrast to SF scaffolds. The results of this study demonstrate that bi-layer SF scaffolds represent promising biomaterials for onlay esophagoplasty, capable of producing superior regenerative outcomes in comparison to conventional SIS scaffolds.

Myosin Va plays a role in nitrergic smooth muscle relaxation in gastric fundus and corpora cavernosa of penis.

The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction.