Characterization Of Excitatory And Inhibitory Motor Neurons To The Human Lower Esophagus Sphincter

Objective: The lower esophagus sphincter is innervated primarily by enteric motor neurons. The morphological and positional characterization of excitatory and inhibitory motor neurons to the sling and clasp fibers of human lower esophagus sphincter were identified.Methods: 1 Tissue collection: Twenty patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in our hospital between March 2006 and November 2006 were selected in this study. Fresh specimens were obtained from the gastroesophageal junction (GEJ) in the operating room. The specimens were opened up by one of two methods. To keep the clasp fibers intact, an incision was made along the greater curvature, and a second incision was made along the lesser curvature to keep the sling fibers intact. The tissue was then pinned out flat in a Sylgard-lined Petri dish and rinsed repeatedly in fresh, sterile Krebs'solution. The mucosa was then removed by dissection from the entire preparation, and the submucosal tissue was carefully cleared away from the clasp or sling fibers. 2 Retrograde labeling:A glass bead, 200μm in diameter coated with the lipid soluble dye DiI was pressed onto the clasp or sling fiber. After 10 minutes, DiI-coated bead had adhered to the underlying tissue and remained in place for the duration of the organ culture. The preparations were covered with culture medium (DEM/F12). They were placed on a rocking tray in a humidified incubator containing 5% CO2 in air at 37°C and the medium was changed daily during the culture period. After five days in organotypic culture, the preparations were fixed for 16–24 hours in modified Zamboni's fixative at 4°C. They were then washed repeatedly in PBS and the remaining circular muscle was dissected away to make a whole mount of the myenteric plexus attached to the thin longitudinal muscle layer. Preparations were then cleared in 90% bicarbonate buffered glycerol (pH 8.6) for three days.3 Immunohistochemistry: DiI labeled preparations were incubated with a primary antiserum to ChAT or NOS. Five clasp and five sling preparations were labeled with antisera to ChAT (Mouse monoclonal) and the other were single labeled with antisera to NOS (Rabbit monoclonal) for four days at room temperature. All primary antisera were diluted in 10% normal goat serum. Tissues were then rinsed repeatedly in PBS and incubated with fluorescein isothiocyanate-conjugated goat antimouse or goat antirabbit IgG for two days. The preparation was mounted in buffered glycerol (pH 8.6) and viewed under an epifluorescence microscope fitted with appropriate filter blocks to discriminate DiI and FITC. The positions of labeled cell bodies and immunoreactivity were recorded. Proportions of immunoreactive and nonimmunoreactive neurons were compared using Wilcoxon Signed Ranks Test with a significance level of 5%. Results are expressed as mean±SEM.Results: 1 At the DiI application site, a band of intensely labeled smooth muscle cells was visible. Directly underneath the application site, there was an area of diffuse fluorescence of up to 1 mm in radius.2 When DiI was placed on the clasp fibers, the motor enteric neurons to this were labeled. In 10 preparations, total 292 neurons were labeled (range15-45;mean=29±3). Fifty two percent (SEM=5%)of the DiI-filled cell bodies were located at more than 2mm oral to the DiI application site, 45% ( SEM=6% ) were located within 2mm to the application site, only approximately 3% were aboral. Of all the cells labeled in 5 preparations, 31%(SEM=1%)were immunoreactive for ChAT. In fact, 35% (SEM=2%)of local motor neurons were immunoreactive for ChAT, whereas only 25% (SEM=7%)of descending motor neurons were ChAT positive. With NO synthase immunoreactivity in 5 preparations, an average of 65% (SEM=2%) were immunoreactive for NOS(Wilcoxon Signed Ranks Test ; P=0.039 ) . Eighty-five percent(SEM=4%) of cell bodies of descending motor neurons were immunoreactive for NOS.3 When DiI was placed on the sling fibers, the motor enteric neurons to this were labeled. In 10 preparations, a total of 433 neurons were labeled, ranging from 21 to 73 (mean=43±6). Fourty-six percent (SEM=2%) of the DiI-filled cell bodies were located at more than 2 mm anal to the DiI application site, 43% (SEM=6%) were located within 2 mm to the application site, approximately 10% were oral. Of all the cells labeled in 5 preparations, 58%(SEM=2%) were immunoreactive for ChAT(Wilcoxon Signed Ranks Test;P=0.042). Sixty-two percent (SEM=2%) of local motor neurons were immunoreactive for ChAT. With NO synthase immunoreactivity in 5 preparations, 36%(SEM=2%) were immunoreactive for NOS. Sixty-four percent(SEM=10%) cell bodies of descending motor neurons were immunoreactive for NOS.4 In labeled motor neurons, DiI showed much of the somadendritic morphology, and in more intensely labeled cells, the axon was also clearly visible. The nuclei of labeled nerve cells were stained. All of the cells that were labeled adequately had a single axon and a number of filamentous or flattened lobular dendrites and fitted into the broad category of Dogiel type I neurons.Conclusions: 1 The DiI labeled motor neurons were mostly ChAT- immunoreactive or NOS- immunoreactive, as considered excitatory or inhibitory motor neurons, respectively. 2 The majority of the motor neurons to the clasp fibers were located locally and orally, and most of them were inhibitory. Most of descending neurons were inhibitory, however, half of local neurons were immunoreactive for NOS. Most of excitatory neurons were located locally. 3 The majority of the motor neurons to the sling fibers were located locally and anally, and most of them were excitatory. Most of inhibitory neurons were located orally or locally. 4 All of the cells that were labeled adequately had a single axon and a number of filamentous or flattened lobular dendrites and fitted into the broad category of Dogiel type I neurons. However, there was considerable variability in the shape of the cells, ranging from small cells with few dendrites to large cells with many dendrites.