| Objective: :The existence and structural equivalent of the lower esophageal sphincter (LES) in human beings have been a matter of speculation for many years. After studying the anatomy of 32 cadavers, Liebermann-Meffert et al described the arrangement of the smooth muscles at the esophagogastric junction (EGJ) in detail.They demonstrated that the musculature of the human LES consisted of sling fibers and clasp fibers.LES clasp fibers is a semi-circular in the right side,while sling fibers is U-shaped band hoisted in the gastroesophageal junction.Both muscle muscles maintain the closure status of the LES,and form a high-pressure zone (HPZ,15-30mmHg).This theory laid the foundation for further studies on the physiology,pathology and pharmacology of the LES.From then on,some reseachers have investigated the physiology and regulation mechanism of LES from different animals,and found that LES had diffemt property different from other smooth muscle structure.And more importantly, there are also great differences in the sling fibers and clasp fibers.Therefore,the abnormal changes of LES structure and function may be the pathological basis of a variety of esophageal diseases. Acetylcholine (Ach) is the most important excitatory neurotransmitter in the lower esophageal sphincter. Sling fiber is more sensitive to Ach in vitro. Clasp fibers have greater spontaneous tension. The different mechanisms of structure and function of the sling and clasp fibers in LES are not well known yet. Many experiments have proved that Ach could induce contraction of circular muscle and longitudinal muscle of the lower esophageal sphincter either in vitro or in vivo. Acetylcholine neurons mostly in the left of the lower esophageal sphincter, and in the right is the axon. This might be the right reason for the higher pressure on left side of the lower esophageal sphincter. P substance is one of the brain-gut peptide neurotransmitters, and most of its immune nerve fibers distributed in the lower esophageal muscles, intestinal submucosal plexus and plexus, and the ring and longitudinal muscles and mucosa of cats and guinea pigs. Immunohistochemical study demonstrayed that substance P was distributed in nerve endings of the vagus. The substance P might be a chemical neurotransmitters for nociceptive stimulation of the end of esophagous to induce esophageal smooth muscle contraction. In this experiment, we measured the contraction of the clasp fibers, sling fibers, circular muscle of the gastric fundus, circular muscle of the esophagus which was induced by acetylcholine and substance P, for the puepose of understanding and future treatment of esophageal motility disorders.Methods: Smooth muscles of the sling fibers, clasp fibers, circular muscles of the esophagus and gastric fundus were obtained from 30 patients who underwent subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital,Hebei Medical University from December 2008 to December 2009. There were 18 males and 12 females with a mean age of 59.3 years.Fresh specimens of EGJ was collected from operating room. Sharp dissection of mucosal and submucosal layers were performed.The muscle strips of sling and clasp fibers were seperated and then cut to small pieces of 1mm3 ~2 mm3.Single cells was isolated using cell separation solution containing collagenase II.After more than 90 percent viability of the cells was confirmed,the cells were resuspended in Hepes buffer at 37℃for subsequent use.Five drops of cell suspention and 1 drop of 0.2%the trypan blue was mixed for 3 min for cell viability determination.Dead cells were dyed red,and living cells colorless.The percentage of cell viability was calculated with the following formula:survival percentage=number of viable cells/total number of cells (viable cell+dead cell)×100%.Cells from clasp fiber, sling fiber, circular muscles of the esophagus and gastric fundus were contracted by exposure to acetylcholine, substance P. Then we measured the contraction of the sling fibers, clasp fibers, circular muscles of the esophagus and gastric fundus which induced by acetylcholine and substance P. A drop of the cell-containing medium was placed on a glass slide and covered with a cover slip. The length of 30 consecutive intact cells encountered at random in each slide was measured with a phase-contrast microscope. The average length of 30 cells measured in the absence of agonists was taken as"control"length. In addition, average cell length was measured after addition of test agents.Results: Under photomicrograph,the smooth muscle cells were spindle and had a high density of nucleus . Its surface was smooth and transparent, and there was a round or oval nucleus in the middle of the cell . In HEPEs buffer incubation, the average length of clasp fibers was 94.09±17.53μm (n = 210) in length, sling fibers was 97.05±11.79μm (n = 210), esophageal circular muscle fibers was 97.21±10.76μm (n = 210), and gastric circular muscle fiber was 97.25±13.719μm (n = 210). There was no significant difference between the length of four groups fiber cells (F=2.67, P=0.07).Acetylcholine could induce contraction of the clasp fibers, sling fibers, circular muscles of the esophagus and gastric fundus, the percentage of muscle cell contraction was (11.94±2.21)% , (22.62±3.22)%, (16.62±9.71)% and (22.82±2.84)%, respectively. There was statistical significance of the length of four smooth musle cells groups ((F=196.97, P = 0). Substance P could induce contraction of clasp fibers, sling fibers, circular muscles of the esophagus and gastric fundus and the percentage of contraction was (22.48±3.36)%, (24.77±9.01)%, (19.01±4.35 )% and (19.11±3.21)% respectively. There was statistical significance for four groups of fiber cells (F=152.27,P = 0).Conclusions: Acetylcholine could cause the contraction of sling fibers, clasp fibers, circular muscles of the esophagus and gastric fundus, and the strongest contraction is on the sling fibers. Substance P could also cause contraction of the four kinds of fibers, and sling fibers and clasp fibers contract gtrongest.
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