Esophageal Muscle Strips Research Articles

350. FUNCTIONAL RESPONSES OF SWINE ESOPHAGUS TO FOCAL CRYOTHERAPY

Abstract Barrett’s esophagus is a premalignant condition where the squamous epithelium of the distal esophagus is replaced by columnar epithelium. Treatment involves endoscopic resection followed by ablation for complete eradication. Various ablative techniques exist including cryotherapy which utilizes a cryogen spray or balloon catheter. While efficacious, complications such as dysphagia have been reported. To better understand the underlying mechanism of how cryotherapy might damage the muscularis, isolated muscle strips were ablated and their function studied. Isolated muscle strips with the mucosa layers removed were prepared from freshly isolated swine esophagus and placed in tissue baths where they were electrically stimulated, and contractile forces were recorded. Contractile responses were allowed to stabilize before a given muscle strip was removed for focal ablation. Cryoablation was performed with a focal cardiac ablation catheter (Freezor, Medtronic). Following ablation, the given muscle strip was returned to the tissue baths. Regression analyses were performed using temperature data from a thermocouple integrated into the tip of the ablation catheter and contractile forces after the esophageal muscle strips had recovered for 3 hours. Ablations lasting less than 60 seconds were applied to 135 esophageal muscle strips: with an average mass of 94 mg. From the experimental data, a multiple linear regression model was generated: 71.93 + 0.4770x—0.5530y = z, where x is the minimum temperature (C), y is the time below 0 C, and z is the contractile force (normalized to preablative force, %). Our model has an adjusted R-squared value of 0.508. As expected, lower minimum temperatures and longer treatment times were associated with greater losses in stimulated muscle forces. We were able to reproducible ablate isolated swine esophageal strips that elicited contractile responses in vitro. Lower temperatures and longer treatment times were associated with greater losses in stimulated muscle forces. Yet, the adjusted R-squared value indicates that temperature and time only partially explain the response. Future studies could be improved by accounting for variabilities in sample sizes and additionally, regression diagnostics need to be performed to check for outliers and validate model assumptions.

Reduction of hydrogen sulfide synthesis enzymes in the esophagus of patients with achalasia: effect of hydrogen sulfide in achalasia.

The aim of the present study was to investigate whether the synthesis of endogenous hydrogen sulfide (H2 S) was altered in achalasia patients and to determine the effects of H2 S on esophageal motility. (1) Tissue samples in lower esophageal sphincter (LES) were obtained from 22 achalasia patients during peroral endoscopic myotomy. LES muscle from eight esophageal carcinoma patients was obtained as control. The expression of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) was detected by immunohistochemical staining. (2) Rabbit esophageal smooth muscle strips were used to measure isometric contractions. The effects of sodium hydrosulfide (NaHS) and L-cysteine on contractile activity and bethanechol-stimulated contractile activity were evaluated. The contraction of esophageal muscle strips was also measured after the inhibition of CBS and CSE by aminooxyacetic acid (AOA) and propargylglycine (PAG). Both CBS and CSE could be detected in biopsies from achalasia patients and controls. Compared with controls, the expression of CBS and CSE in the LES of achalasia patients was significantly reduced (p<0.001). Both NaHS and L-cysteine concentration-dependently inhibited esophageal contractile activity (both p<0.05). After inhibition of CBS and CSE by PAG and AOA, esophageal contractile activity increased significantly, and this effect could berestored by NaHS but not L-cysteine (p<0.05). H2 S synthesis enzymes are significantly reduced in patients with achalasia compared with the controls. H2 S inhibits esophageal contractile activity concentration-dependently, and the inhibition of H2 S synthesis enzymes increases esophageal contractile activity. H2 S might be involved in the development of achalasia.

T-Helper 2 Cytokines, Transforming Growth Factor β1, and Eosinophil Products Induce Fibrogenesis and Alter Muscle Motility in Patients With Eosinophilic Esophagitis

Patients with eosinophilic esophagitis (EoE) often become dysphagic from the combination of organ fibrosis and motor abnormalities. We investigated mechanisms of dysphagia, assessing the response of human esophageal fibroblasts (HEFs), human esophageal muscle cells (HEMCs), and esophageal muscle strips to eosinophil-derived products. Biopsy specimens were collected via endoscopy from the upper, middle, and lower thirds of the esophagus of 18 patients with EoE and 21 individuals undergoing endoscopy for other reasons (controls). Primary cultures of esophageal fibroblasts and muscle cells were derived from 12 freshly resected human esophagectomy specimens. Eosinophil distribution was investigated by histologic analyses of full-thickness esophageal tissue. Active secretion of EoE-related mediators was assessed from medium underlying mucosal biopsy cultures. We quantified production of fibronectin and collagen I by HEF and HEMC in response to eosinophil products. We also measured the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by, and adhesion of human eosinophils to, HEFs and HEMCs. Eosinophil products were tested in an esophageal muscle contraction assay. Activated eosinophils were present in all esophageal layers. Significantly higher concentrations of eosinophil-related mediators were secreted spontaneously in mucosal biopsy specimens from patients with EoE than controls. Exposure of HEFs and HEMCs to increasing concentrations of eosinophil products or co-culture with eosinophils caused HEFs and HEMCs to increase secretion of fibronectin and collagen I; this was inhibited by blocking transforming growth factor β1 and p38 mitogen-activated protein kinase signaling. Eosinophil binding to HEFs and HEMCs increased after incubation of mesenchymal cells with eosinophil-derived products, and decreased after blockade of transforming growth factor β1 and p38 mitogen-activated protein kinase blockade. Eosinophil products reduced electrical field-induced contraction of esophageal muscle strips, but not acetylcholine-induced contraction. In an analysis of tissues samples from patients with EoE, we linked the presence and activation state of eosinophils in EoE with altered fibrogenesis and motility of esophageal fibroblasts and muscle cells. This process might contribute to the development of dysphagia.

Foregut smooth muscle reactivity changes in the hydrocephalus-induced infantile rats

Background An association between hydrocephalus and gastroesophageal motor abnormalities that cause gastroesophageal reflux (GER) is well known. Our aim was to investigate hydrocephalus-induced alterations in esophageal and gastric smooth muscle reactivity and their modulation by pharmacological interventions in the rat model. Materials and methods Hydrocephalus was induced in infantile rats by injection of kaolin into the cisterna magna. Hydrocephalic and sham-operated rats were exsanguinated 2 weeks after surgery. Esophageal and gastric fundus smooth muscle strips were studied in vitro for their contractile and relaxant response to receptor activation in the organ chambers set up. Additionally, esophageal and gastric tissue specimens were examined histologically for GER-induced changes. Results No histological evidence of esophageal and gastric changes reflecting GER was observed in the specimens of the control and hydrocephalus-induced rats. Maximum contractile responses of esophageal and gastric fundus smooth muscle to KCl and muscarinic receptor agonist carbachol were increased in the hydrocephalic groups compared with the control groups. These changes were statistically significant. Relaxant responses to beta adrenoceptor agonist isoprenaline were similar in the esophageal muscle strips of both hydrocephalic groups and the control groups. However, isoprenaline-induced relaxant responses of the gastric fundus muscle strips in the hydrocephalus-induced groups were significantly decreased as compared with the control groups. The relaxant responses to papaverine in the esophageal and gastric fundus smooth muscle strips were similar in the two groups. Conclusions Our study revealed alterations of receptor-dependent and receptor-independent foregut smooth muscle reactivity in the hydrocephalus-induced rat pups. Therefore, we suggest that impaired smooth muscle reactivity at least in part may contribute to abnormalities of foregut motor function seen in patients with hydrocephalus.

75 EFFECTS OF PEROXYNITRITE ON ESOPHAGEAL MOTOR FUNCTION.

Introduction: Nitric oxide (NO)·) is the primary mediator of nerve-induced esophageal peristalsis, and the lower esophageal sphincter (LES) relaxation. Disordered esophageal motor function accompanies esophageal inflammation. Superoxide radicals are formed during all inflammatory states and are known to scavange NO·. The reaction of NO· with superoxide generates peroxynitrite (ONOO-). Peroxynitrite may alter cellular functions in several ways: it is a potent oxidant and it nitrates tyrosine residues of cellular proteins. It is generated in or near myenteric ganglion neurons in the inflamed small bowel and is known to produce neuronal injury. It causes relaxation of vascular smooth muscle. Hypothesis: Peroxynitrite alters esophageal motor function in vitro. Methods: Circular esophageal muscle strips from opossum were placed in tissue baths containing oxygenated Krebs solution maintained at 37°C. The tissues were stimulated by an electrical field (4-s trains of 50V, 0.5ms square-wave pulses at 5 Hz) previously shown to activate intrinsic esophageal nerves. Responses to nerve stimulation were observed in the presence of a) peroxynitrite (1×10-6M to 5×10-4M) with or without superoxide dismutase (SOD), Nw-nitro-L-arginine (L-NNA) or oxyhemoglobin (Hb); b) decomposed peroxynitrite (1×10-6M to 5×10-3M) with or without Hb; or c) sodium nitrite (NaNO2)(1×10-6M to 5×10-3M) with or without Hb. Head space gas in tissue baths was analyzed for NO· by chemiluminescense assay. Results: ONOO-, its decomposed form, or its major breakdown product NaNO2 relax the LES, and attenuate esophageal contraction in a concentration-dependent manner. ONOO- is the most potent. The effects of ONOO- are not inhibited by a superoxide scavanger, SOD, or a nitric oxide synthase inhibitor, L-NNA. The effects of ONOO-, decomposed ONOO-, or NaNO2 are inhibited by a NO· scavanger, Hb. The inhibition is most pronounced with ONOO-. NO· is produced from ONOO-, but not from its decomposed form or NaNO2. Conclusions: Disordered esophageal motility may be related to the formation of ONOO- and its breakdown products during inflammation. NO· may be generated directly from ONOO- under physiologic conditions.

Oxygen uptake and mechanical tension in esophageal smooth muscle from opossums and cats.

In 44 opossums and 4 cats weighing 1.5-2.5 kg, measurements of O2 consumption were made on esophageal muscle strips using a modified Clark-type polarograph. All strips were cut in the transverse axis of the esophagus and comprised the circular and longitudinal muscle layers and intrinsic nerves. Strips were 20 mm long, 2 mm wide, and less than 0.5 mm thick. The average uptake by all strips was less than 5 microliters O2 . mg dry muscle-1.h-1 at 37 degrees C and was temperature dependent (Q10, 2.2). There was no difference in base-line O2 consumption between strips coming from the proximal, middle, or distal third of the esophageal body and strips coming from the lower esophageal sphincter. Increases in length or tension increased the O2 consumption by sphincter strips but had no effect on body strips. Carbachol (10(-5) M) led to increases of base-line muscle tension and O2 consumption in both sphincter and body strips. Isoproterenol (10(-6) M) decreased the baseline tension and O2 consumption in sphincter strips only. The maintenance of a high basal tension by the lower esophageal sphincter is oxygen dependent and therefore an active muscular process. Also, no gradient of metabolic activity can be demonstrated along the smooth muscle segment of the esophagus.

Lower Sphincter of the Opossum Esophagus in Pseudopregnancy

To seek a possible role of estrogen and progesterone in the development of changes in esophageal function during pregnancy, we produced a state of pseudopregnancy by administration of hormones to a suitable animal model. Twenty-two female opossums weighing an average of 2.5 kg, were divided into two groups. The treated group received intramuscular injections of 100 microgram of estradiol valerate daily from day 1 to day 12 and 15 mg of medroxyprogesterone acetate from day 7 to day 12. The control group received no injections. Both groups underwent manometry on days 1, 7, and 12. Lower esophageal sphincter pressure decreased (P less than 0.05) in treated animals from 58 +/- 13 mm Hg and 57 +/- 11 on days 1 and 7, respectively, to 44 +/- 10 mm Hg on day 12. The lower esophageal sphincter pressure remained unchanged in control animals. In both groups, there was no change in peristaltic wave pressure, duration, or velocity in the distal 6 cm of the esophagus. No abnormal peristaltic phenomena were observed. Esophageal muscle strips prepared from treated animals showed responses to electrical field stimulation of intrinsic nerves that were like those from control animals. The same was true for responses to acetylcholine and pentagastrin. Total tissue water and total tissue potassium content did not differ in treatment and control animals.