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Background: Cryotherapy is a method of endoscopic mucosal ablation that involves delivery of a cryogen to result in tissue destruction by extreme low temperature. Its effects are influenced by the dosage of cryogen and thawing of ice. There are limited data on the tissue effects of multiple freeze and thaw cycles of carbon dioxide (CO2) cryotherapy on GI tissues. Objective: To investigate the extent of tissue injury due to escalating doses of CO2 cryotherapy on the esophagus, stomach, and colon of pigs. Design: Animal experiment. Intervention: Varying doses of CO2 cryotherapy with increasing number of freeze-thaw cycles were applied to each site. The animals were allowed to survive for 48 hours. Main Outcome Measurements: Depth of tissue injury assessed in blinded fashion by varying doses of cryotherapy on a defined area of porcine esophagus, stomach, and colon. Results: There was a dose-dependent relationship of CO2 cryogen and depth of injury (P = .0001 and P = .002, respectively). In the stomach, the dose-response relationship was significant (P = .007), but the average grades of injury across the various doses were lower when compared with the esophagus at comparable cryogen doses (P = .0004). The estimated depth of tissue injury from the mucosal surface in the porcine esophagus and colon tissue ranged from 1.2 to 2.5 mm and 1.3 to 2.5 mm, respectively. Limitations: The study was performed in a normal porcine model. Conclusion: There was a dose-dependent relationship between the dose of CO2 cryotherapy and the depth of tissue injury in the porcine esophagus, stomach, and colon. [Copyright &y& Elsevier]

Background: Closure of the transgastric access to the peritoneal cavity is a critical step in natural orifice transluminal endoscopic surgery (NOTES). Objective: To perform a direct comparison of the histological healing post clips and threaded tags (T-tags) closure after transgastric NOTES procedures. Setting: Design and Intervention: Twelve survival porcine experiments. After standardized endoscopic gastric wall puncture, balloon-dilation, and transgastric peritoneoscopy, closure of the gastric wall was performed with either clips or T-tags. Necropsy at 14 days was performed for histological evaluation of 2-mm interval transversal cross sections of the gastrotomy site. Main Outcome Measurements: Histological healing of the gastric wall opening. Results: Endoscopic closure of the gastrotomy was successfully achieved in all 12 animals, followed by an uneventful 2-week clinical follow-up. Transmural healing was seen in 3 (75%) animals after clip closure compared with only 1 (12.5%) in the group with T-tag closure (P = .06). Gastric wall muscular bridging was observed in 4 (100%) animals with clip closure compared with only 1 (12.5%) in the group with T-tag closure (P = .01). Limitations: Animal model with short-term follow-up. Conclusions: Endoscopic clip closure results in a layer-to-layer transmural healing of the gastric wall. In contrast, T-tag gastric wall plication impairs gastric layer bridging. These findings might guide the future design of new endoscopic devices and techniques for gastrotomy closure after NOTES procedures. [ABSTRACT FROM AUTHOR]

Introduction: The utility of the greater omentum has not been assessed in transluminal access closure after natural orifice transluminal endoscopic surgery (NOTES) procedures. Objective: Our purpose was to evaluate the feasibility, efficacy, and safety of omentoplasty for gastrotomy closure. Methods and Procedures: Survival experiments in 9 female 40-kg pigs were randomly assigned to 3 groups: group A, endoscopic full-thickness resection (EFTR) for transgastric access and peritoneoscopy without closure; group B, ETFR and peritoneoscopy with omentoplasty (flap of omentum is pulled into the stomach and attached to the gastric mucosa with clips but no clips are used for gastrotomy closure itself); group C, balloon dilation for opening and peritoneoscopy followed by omentoplasty for closure. The animals were observed for 2 weeks and then underwent endoscopy and necropsy with histologic evaluation. Results: Transgastric opening and peritoneoscopy were achieved in all pigs. In groups B and C, a flap of omentum was easily placed to seal the gastrotomy and then attached to the gastric mucosa with 2 to 5 clips (median 4) in 7 to 20 minutes (median 15 minutes). In group A, peritonitis developed in all animals. In both groups B and C, all animals survived 15 days with no peritonitis and minimal adhesions outside the gastrotomy site. In addition, all achieved complete healing (transmural, n = 4; mucosal ulceration, n = 2) of the gastrotomy site. One animal in group B had an 18-mm abscess in the omental flap. Limitations: Animal model, small sample size, lack of appropriate controls for group C. Conclusions: Omentoplasty of the gastrotomy site is a technically feasible method to seal balloon-created transgastric access to the peritoneal cavity after NOTES procedures. [Copyright &y& Elsevier]

Background: Transluminal access site closure remains a major challenge in natural orifice transluminal endoscopic surgery (NOTES). Objective: Our purpose was to develop in vivo leak tests for evaluation of the integrity of transgastric access closure. Settings: Survival experiments on 12 50-kg pigs. Design and Interventions: After a standardized transgastric approach to the peritoneal cavity and peritoneoscopy, the gastric wall incision was closed with T-bars (Wilson-Cook Medical, Winston-Salem, NC) deployed on both sides of the incision and then cinched together. Gastrotomy closure was assessed with air and fluid leak tests. The animals were observed for 1 week and then underwent endoscopic evaluation and necropsy. Main Outcome Measurements: (1) Leak-proof closure of the gastric wall incision. (2) Gastric incision healing 1 week after the procedure. Results: The mean intraperitoneal pressure increased 10.7 ± 3.7 mm Hg during gastric insufflation when the air leak test was performed before closure compared with 0.9 ± 0.8 mm Hg after transmural closure of the transgastric access site with T-bars (P < .001). Fluid leak tests demonstrated no leakage of liquid contrast from the stomach into the peritoneal cavity after closure. Necropsy in 1 week confirmed completeness of the gastric closure in all animals with full-thickness healing and no spillage of the gastric contents into the peritoneal cavity. Limitations: Leak tests were only evaluated on an animal model. Conclusions: Fluid and air leak tests are simple techniques to evaluate in vivo the adequacy of the transluminal access site closure after NOTES procedures. Leak-proof gastric closure resulted in adequate tissue approximation and full-thickness healing of the gastric wall incision. [Copyright &y& Elsevier]

Background: Endosonography (EUS) is widely used for locoregional staging of malignant GI tumors. Delineation of a tumor''s margins with a long-lasting fluoroscopically visible material will facilitate subsequent surgical and radiation therapy. Objective: To assess the feasibility of EUS-guided submucosal implantation of a radiopaque marker in a porcine model. Setting: Survival experiments on four 50-kg pigs. Methods: A linear array echoendoscope was introduced into the esophagus and advanced to the stomach. With a 19-gauge FNA needle, a submucosal bleb was created by injecting 3 mL of normal saline solution into the gastric and esophageal wall followed by injection of 1 mL of tantalum suspension under fluoroscopic observation. Fluoroscopy was repeated after 1, 2, and 4 weeks followed by euthanasia and necropsy. Main Outcome Measurements: Long-term depositions of the marker in the injection sites. Results: Submucosal injections of tantalum were easily performed through the 19-gauge FNA needle, resulting in good fluoroscopic opacification of injected material. Follow-up fluoroscopy in 1, 2, and 4 weeks demonstrated stable deposition of the tantalum at the sites of injection. There were no complications during and after the tantalum implantation. Histologic examination of the injection sites demonstrated submucosal tantalum depositions without signs of infection, inflammation, tissue damage, or necrosis. Limitations: Animal experiments with 4 weeks'' follow-up. Conclusions: EUS-guided implantation of tantalum as a radiopaque marker into the submucosal layer of the GI tract in a porcine model is technically feasible and safe. Long-lasting fluoroscopically visible tantalum markings could facilitate subsequent surgical and radiation therapy. [Copyright &y& Elsevier]

Background: EUS is the preferred modality for local staging of esophageal cancer. The presence of a long-lasting fluoroscopically visible marker of malignant lymph nodes would facilitate subsequent radiation and surgical therapy. Objective: To assess the feasibility of EUS-guided implantation of a radiopaque marker (tantalum) into mediastinal and celiac lymph nodes in a porcine model. Setting: Survival experiments on six 50-kg pigs. Design and Interventions: A linear-array echoendoscope was advanced into the esophagus and the stomach. Mediastinal and celiac lymph nodes were identified and injected with 1 mL tantalum suspension by using 19- and 22-gauge FNA needles under fluoroscopy. The pigs were recovered. Fluoroscopy was repeated after 1, 2, and 4 weeks, then a postmortem examination was performed. Main Outcome Measurements: Long-term opacification of lymph nodes. Results: It was not possible to inject tantalum through the 22-gauge FNA needle because of its rapid precipitation inside the needle, which caused needle occlusion. Intranodal injection with the 19-gauge FNA needle was easily accomplished and resulted in excellent fluoroscopic opacification of injected lymph nodes. Repeat fluoroscopy at 1, 2, and 4 weeks demonstrated stable tantalum deposition at the injection site. There were no complications. Histologic examination of harvested lymph nodes revealed intranodal tantalum depositions without signs of infection, inflammation, tissue damage, or necrosis. Conclusions: EUS-guided implantation of tantalum as a radiopaque marker into mediastinal and celiac lymph nodes in a porcine model is technically feasible, safe, and results in long-lasting intranodal depositions to facilitate subsequent surgical and radiotherapeutic interventions. [Copyright &y& Elsevier]

PURPOSE: Current animal hindlimb ischemia models involve surgical ligation of the femoral artery and delivery of therapeutic angiogenic agents into the adductor compartment. The authors hypothesize that an endovascular model of hindlimb ischemia would be a more appropriate platform, closely resembling atherosclerosis by occluding the vessel from within, causing less inflammation, wound healing and subsequent collateralization. MATERIALS AND METHODS: The left superficial femoral artery in 17 rabbits was occluded by endovascular coil embolization (n = 9) or surgical ligation (n = 8). Animals (n = 3; in each group) were sacrificed on day 3 to determine the arteriolar luminal area, number of arterioles, microsphere determined perfusion, and degree of inflammation. On day 28, the remaining animals underwent calf blood pressure measurements and angiography to determine the number of collaterals and diameter of vessels supplying the hindlimb. RESULTS: Immediate postprocedure (day 0) and presacrifice (day 3 or 28) occlusion rates were 89% (eight of nine rabbits) and 100% for the endovascular model; 100% and 100% for the surgical model, respectively. Hindlimb paralysis and muscle atrophy was found in one surgical animal. On day 3, there was an increase in hindlimb perfusion (surgery, 0.04 ± 0.01; endovascular, 0.02 ± 0.01; P = .02), an increase in arteriolar luminal area (surgery, 481 μm ± 240; endovascular, 345 μm ± 151; P = .04), and a trend toward more inflammation (surgery, 5.5 ± 3.8; endovascular, 2.5 ± 3.0; P = .08) in the surgical group. There was no difference in number of vessels between both groups. On day 28 there was no difference in the calf blood pressure ratios or in the number of collaterals. However, there was enlargement of the distal profunda femoris artery, the vessel closest to the surgical incision, in the surgical group (L/R ratio: immediate post-occlusion, 1.06 ± 0.11; day 28, 1.27 ± 0.08; P = .02). CONCLUSION: The endovascular model was efficacious in providing occlusion of the superficial femoral artery, and induced less of an arteriogenic response compared with the surgical model. The authors believe that this endovascular model is a superior platform for studying therapeutic angiogenic agents. [Copyright &y& Elsevier]

Objectives: The aim of this study was to determine whether activation of β3-adrenergic receptor (AR) and downstream signaling of nitric oxide synthase (NOS) isoforms protects the heart from failure and hypertrophy induced by pressure overload. Background: β3-AR and its downstream signaling pathways are recognized as novel modulators of heart function. Unlike β1- and β2-ARs, β3-ARs are stimulated at high catecholamine concentrations and induce negative inotropic effects, serving as a “brake” to protect the heart from catecholamine overstimulation. Methods: C57BL/6J and neuronal NOS (nNOS) knockout mice were assigned to receive transverse aortic constriction (TAC), BRL37344 (β3 agonist, BRL 0.1 mg/kg/h), or both. Results: Three weeks of BRL treatment in wild-type mice attenuated left ventricular dilation and systolic dysfunction, and partially reduced cardiac hypertrophy induced by TAC. This effect was associated with increased nitric oxide production and superoxide suppression. TAC decreased endothelial NOS (eNOS) dimerization, indicating eNOS uncoupling, which was not reversed by BRL treatment. However, nNOS protein expression was up-regulated 2-fold by BRL, and the suppressive effect of BRL on superoxide generation was abrogated by acute nNOS inhibition. Furthermore, BRL cardioprotective effects were actually detrimental in nNOS –/– mice. Conclusions: These results are the first to show in vivo cardioprotective effects of β3-AR–specific agonism in pressure overload hypertrophy and heart failure, and support nNOS as the primary downstream NOS isoform in maintaining NO and reactive oxygen species balance in the failing heart. [ABSTRACT FROM AUTHOR]

Objectives: We tested the hypothesis that bi-directional, gene-targeted regulation of cardiomyocyte cyclic guanosine monophosphate–selective phosphodiesterase type 5 (PDE5) influences maladaptive remodeling in hearts subjected to sustained pressure overload. Background: PDE5 expression is up-regulated in human hypertrophied and failing hearts, and its inhibition (e.g., by sildenafil) stimulates protein kinase G activity, suppressing and reversing maladaptive hypertrophy, fibrosis, and contractile dysfunction. Sildenafil is currently being clinically tested for the treatment of heart failure. However, researchers of new studies have questioned the role of myocyte PDE5 and protein kinase G (PKG) to this process, proposing alternative targets and mechanisms. Methods: Mice with doxycycline-controllable myocyte-specific PDE5 gene expression were generated (medium transgenic [TG] and high TG expression lines) and subjected to sustained pressure overload. Results: Rest myocyte and heart function, histology, and molecular profiling were normal in both TG lines versus controls at 2 months of age. However, upon exposure to pressure overload (aortic banding), TG hearts developed more eccentric remodeling, maladaptive molecular signaling, depressed function, and amplified fibrosis with up-regulation of tissue growth factor signaling pathways. PKG activation was inhibited in TG myocytes versus controls. After establishing a severe cardiomyopathic state, high-TG mice received doxycycline to suppress PDE5 expression/activity only in myocytes. This in turn enhanced PKG activity and reversed all previously amplified maladaptive responses, despite sustained pressure overload. Sildenafil was also effective in this regard. Conclusions: These data strongly support a primary role of myocyte PDE5 regulation to myocardial pathobiology and PDE5 targeting therapy in vivo and reveal a novel mechanism of myocyte-orchestrated extracellular matrix remodeling via PDE5/cyclic guanosine monophosphate–PKG regulatory pathways [Copyright &y& Elsevier]

Objective: This study sought to test the efficacy of phosphodiesterase type 5A (PDE5A) inhibition for treating advanced hypertrophy/remodeling caused by pressure overload, and to elucidate cellular and molecular mechanisms for this response. Background: Sildenafil (SIL) inhibits cyclic guanosine monophosphate–specific PDE5A and can blunt the evolution of cardiac hypertrophy and dysfunction in mice subjected to pressure overload. Whether and how it ameliorates more established advanced disease and dysfunction is unknown. Methods: Mice were subjected to transverse aortic constriction (TAC) for 3 weeks to establish hypertrophy/dilation, and subsequently treated with SIL (100 mg/kg/day) or placebo for 6 weeks of additional TAC. Results: The SIL arrested further progressive chamber dilation, dysfunction, fibrosis, and molecular remodeling, increasing myocardial protein kinase G activity. Isolated myocytes from TAC-SIL hearts showed greater sarcomere shortening and relaxation, and enhanced Ca2+ transients and decay compared with nontreated TAC hearts. The SIL treatment restored gene and protein expression of sarcoplasmic reticulum Ca2+ uptake adenosine triphosphatase (SERCA2a), phospholamban (PLB), and increased PLB phosphorylation (S16), consistent with improved calcium handling. The phosphatase calcineurin (Cn) and/or protein kinase C-α (PKCα) can both lower phosphorylated phospholamban and depress myocyte calcium cycling. The Cn expression and PKCα activation (outer membrane translocation) were enhanced by chronic TAC and reduced by SIL treatment. Expression of PKCδ and PKCε also increased with TAC but were unaltered by SIL treatment. Conclusions: SIL treatment applied to well-established hypertrophic cardiac disease can prevent further cardiac and myocyte dysfunction and progressive remodeling. This is associated with improved calcium cycling, and reduction of Cn and PKCα activation may be important to this improvement. [Copyright &y& Elsevier]