Esophageal Myenteric Plexus Research Articles

BN SO30 - One hundred consecutive Laparoscopic Heller's Cardiomyotomy & Fundoplication – a personal series

Abstract Background Oesophageal Achalasia (OA) is an uncommon primary oesophageal motility disorder, characterised by the loss of inhibitory neurons in the oesophageal myenteric plexus. The primary goal of treating OA is to alleviate dysphagia caused by the functional obstruction of the distal oesophagus due to the inability of the lower oesophageal sphincter (LOS) to relax appropriately after swallowing. Heller's cardiomyotomy (HCM) remains a standard treatment for OA. The aim of this study was to assess the symptomatic improvement and post-operative outcomes following HCM with fundoplication at our centre. Method All patients underwent HCM through laparoscopic blunt muscle separation technique, between January 2005 and March 2024, by a single surgeon. Retrospective review of electronic and compiled paper patient records were analysed to assess patient demographics, preoperative symptoms, operative details, length of stay (LOS), complications, post-operative symptoms, gastroscopy and pneumatic dilation (PD) as well as revisional surgery. Primary outcomes assessed were binary changes in key symptoms of weight loss, dysphagia, retrosternal pain and regurgitation. Comparison of pre and post-operative outcomes were performed using paired t-test. A p-value of <0.05 was considered statistically significant. Results 100 procedures involving 99 patients (average age 50 years, 43% female), 1 conversion to open and 1 revision at 3 years. ASA grades were 1 (27%), 2 (58%), and 3 (15%). Median LOS was one day, median follow-up 99 months. Post-operatively; weight loss persisted in 11% of patients from 48%, dysphagia reduced from 98% to 42%, retrosternal chest pain from 39% to 20% and regurgitation from 57% to 24% (all p<0.05). 2 patients required (PD) ≤30 days (CD≥3). 36 patients underwent PD at median of 13 months, 20 needed repeat PD. None of the patients developed cancer. Conclusion As demonstrated in this large series of 100 consecutive patients, HCM remains one of the best treatment modalities for OA. Key symptoms of OA significantly reduced following HCM. Our technique avoided the use of energy devices, ensured a short hospital stay and durable symptom improvement, lends support to its continued application in managing this condition.

OGBN P30 Post-operative and symptomatic outcomes following Laparoscopic Hellers Cardiomyotomy- single centre experience

Abstract Background Esophageal achalasia represents a uncommon primary oesophageal motility disorder secondary to loss of inhibitory neurons in the oesophageal myenteric plexus level. The main goal of treating oesophageal achalasia is to relieve dysphagia secondary to the functional obstruction of the distal oesophagus by the inability of the Lower Oesophageal Sphincter (LOS) to relax appropriately after deglutition. The Eckardt score (ES) was developed to objectively assess the symptom severity and continues to be widely used. Aim of this study was to assess the post-operative outcomes and symptomatic improvement using ES in consecutive patients undergoing laparoscopic Hellers cardiomyotomy for achalasia in our unit. Methods Data of consecutive patients who underwent LHCM between January 2016 and December 2021 was collected. Patient demographics, surgical details, perioperative course, ES, and manometric data including Integrated Relaxation Pressure (IRP), were retrieved from a prospectively maintained database. Primary outcomes assessed were peri-operative morbidity assessed using Clavien-Dindo grade. Secondary outcomes assessed were improvement in symptoms using ES (pre- and post-operative). ES of ≤3 was considered as symptomatic improvement. Data was analysed using Microsoft Excel. Comparison of pre-operative and post-operative means was performed using paired t test. Fishers exact test was used for categorical variables. A p-value <0.05 was considered statistically significant. Results 41 patients underwent LHCM. Twenty-one were males. Median age was 56 years (20-77). 95.12% patients had dysphagia for a median duration of 24 months (2-180). Type II achalasia was most common in 87.80%. Median IRP was 22.3 mm Hg (11-78). Oesophageal dilation was performed in 2 while, 10 patients received botulin injection pre-operatively. Median hospital stay was 1 day (1-12). 92.68% had no post operative complications. None of the patients had significant complications (Clavien-Dindo >grade 2). At a median follow-up of 60 months, there was significant improvement in ES 8±1.97 to 1.82±1.16, p<0.001 and dysphagia score (p<0.001). Conclusions LHCM with fundoplication leads to significant symptomatic improvement with excellent short and medium term outcomes with low morbidity and complication rates. A detailed pre-operative discussion with patients is essential to provide tailored treatment as per individual patient symptoms.

The role of type II esophageal microbiota in achalasia: Activation of macrophages and degeneration of myenteric neurons

ObjectiveThe gut microbiota plays a critical role in the appropriate development and maintenance of the enteric nervous system (ENS). Esophageal achalasia (EA) is a rare motility disorder characterized by the selective degeneration of inhibitory neurons in the esophageal myenteric plexus. This study aimed to evaluate the composition of the esophageal microbiota in achalasia and explore the potential microbial mechanisms involved in its pathogenesis. DesignThe lower esophageal mucosal microbiota was analyzed in patients with achalasia and control participants using 16 S rRNA sequencing. The association between the esophageal microbiota and achalasia was validated by inducing esophageal dysbiosis in C57BL/10 J and C57BL/10ScNJ (TLR4KO) mice via chronic exposure to ampicillin sodium in their drinking water. ResultsThe esophageal microbiota in EA patients had lower diversity and a predominance of Gram-negative bacteria (Type II microbiota) compared to that in the healthy controls. Additionally, the relative abundance of Rhodobacter decreased significantly in patients with achalasia, which correlated with an enrichment of lipopolysaccharide (LPS) biosynthesis based on the COG database. Antibiotic-treated mice showed an esophageal microbiota characterized by increased abundance of Gram-negative bacteria (Type II microbiome), decreased abundance of Rhodobacter, and enriched LPS biosynthesis. Compared to the control and TLR4KO mice, the antibiotic-treated wild-type mice had higher LES resting pressure, increased LES contraction rate after carbachol stimulation, and decreased relaxation response to L-arginine. Moreover, the number of myenteric neurons decreased, while the number of lamina propria macrophages (LpMs) increased after antibiotic exposure. Furthermore, the TLR4-MYD88-NF-κB pathway was up-regulated, and the production of TNF-α, IL-1β, and IL-6 increased in the antibiotic-treated mice. ConclusionsPatients with achalasia exhibit esophageal dysbiosis, which may induce aberrant esophageal motility.

Case Report on Achalasia Cardia

Motor dysphagia has a number of frequent causes, including achalasia. The failure of the lower esophageal sphincter to relax results in achalasia, a condition of esophageal smooth muscle motility. The gastroesophageal junction becomes functionally blocked as a result of this disease. The actual pathophysiology of this disorder is still unknown, despite the fact that the disease was originally recorded more than 300 years ago. Loss of the inhibitory ganglion in the myenteric plexus of the oesophagus can be the pathophysiological aetiology of achalasia cardia. In the esophageal myenteric plexus, there is a loss of inhibitory neurons that produce VIP and nitric oxide synthase, but in extreme situations, it also affects cholinergic neurons. Despite numerous possibilities being put forth, the specific cause of this deterioration remains unknown. The autoimmune phenomenon, viral infection, and genetic predisposition are some of these hypotheses. Read more...

Chagas Disease Megaesophagus Patients Carrying Variant MRPS18B P260A Display Nitro-Oxidative Stress and Mitochondrial Dysfunction in Response to IFN-γ Stimulus.

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, affects 8 million people, and around 1/3 develop chronic cardiac (CCC) or digestive disease (megaesophagus/megacolon), while the majority remain asymptomatic, in the indeterminate form of Chagas disease (ASY). Most CCC cases in families with multiple Chagas disease patients carry damaging mutations in mitochondrial genes. We searched for exonic mutations associated to chagasic megaesophagus (CME) in genes essential to mitochondrial processes. We performed whole exome sequencing of 13 CME and 45 ASY patients. We found the damaging variant MRPS18B 688C > G P230A, in five out of the 13 CME patients (one of them being homozygous; 38.4%), while the variant appeared in one out of 45 ASY patients (2.2%). We analyzed the interferon (IFN)-γ-induced nitro-oxidative stress and mitochondrial function of EBV-transformed lymphoblastoid cell lines. We found the CME carriers of the mutation displayed increased levels of nitrite and nitrated proteins; in addition, the homozygous (G/G) CME patient also showed increased mitochondrial superoxide and reduced levels of ATP production. The results suggest that pathogenic mitochondrial mutations may contribute to cytokine-induced nitro-oxidative stress and mitochondrial dysfunction. We hypothesize that, in mutation carriers, IFN-γ produced in the esophageal myenteric plexus might cause nitro-oxidative stress and mitochondrial dysfunction in neurons, contributing to megaesophagus.

Infection with SARS-CoV-2 as a potential achalasia trigger.

Idiopathic achalasia is a chronic oesophageal motility disorder caused by loss of inhibitory neurons at the esophageal myenteric plexus resulting in incomplete relaxation of the lower oesophageal sphincter and abnormal peristaltism.Among the possible causes of this, an immune response secondary to infection by some viruses has been implicated. SARS-CoV-2 could be considered among them. The therapy option should be aimed at achieving the greatest clinical effectiveness according to each patient's health status.

The Number of Interstitial Cells of Cajal Differs Among Different Subtypes of Achalasia and is Related to Patients' Prognosis.

INTRODUCTION:Achalasia is a primary esophageal motility disorder with heterogeneous manometric subtypes and prognosis, characterized by degeneration of the esophageal myenteric plexus, and reduction in interstitial cells of Cajal (ICCs). This study aimed to explore the histopathologic characteristics of lower esophageal sphincter (LES) muscle from patients with achalasia with different subtypes and different prognosis.METHODS:We examined specimens of LES muscle from 122 patients with achalasia who underwent peroral endoscopic myotomy and from 10 control patients who underwent esophagectomy for esophageal cancer. Hematoxylin–eosin staining was performed to assess inflammation infiltration, fibrosis, and atrophy. Specific immunohistochemical staining was performed to identify ICCs and neuronal nitric oxide synthase (nNOS).RESULTS:The number of ICCs in patients with type I achalasia was significantly lower than that in patients with type II achalasia, followed by that in control patients (type I vs type II vs control group= 0.4 vs 1.2 vs 9.5; P < 0.001). The number of nNOS-positive cells was significantly lower in patients with achalasia than that in control patients (type I vs type II vs control group = 0.0 vs 0.0 vs 8.0; P < 0.001). Nonrecurrent group had significantly more ICCs than recurrent group (type I: nonrecurrent vs recurrent = 1.0 vs 0.1; P = 0.010; type II: nonrecurrent vs recurrent = 2.0 vs 0.4; P = 0.004).DISCUSSION:ICCs and nNOS-positive cells reduced significantly in LES muscle of patients with achalasia. The number of ICCs differed among different achalasia subtypes and was related to patients' clinical prognosis.

Management of Esophageal Dysphagia in Chagas Disease.

Chagas disease, caused by the infection of the protozoan parasite Trypanosoma cruzi, has clinical consequences in the heart and digestive tract. The most important changes in the digestive tract occur in the esophagus (megaesophagus) and colon (megacolon). Esophageal dysfunction in Chagas disease results from damage of the esophageal myenteric plexus, with loss of esophageal peristalsis, partial or absent lower esophageal sphincter relaxation, and megaesophagus, which characterizes secondary esophageal achalasia. The treatment options for the disease are similar to those for idiopathic achalasia, consisting of diet and behavior changes, drugs, botulinum toxin, peroral endoscopic myotomy (POEM), pneumatic dilation of the lower esophageal sphincter, laparoscopic Heller myotomy, and esophagectomy. Chagas disease causes a life-threatening cardiopathy, and this should be considered when choosing the most appropriate treatment for the disease. While some options are palliative, for temporary relief of dysphagia (such as drugs, botulinum toxin, and pneumatic dilation), other therapies provide a long-term benefit. In this case, POEM stands out as a modern and successful strategy, with good results in more than 90% of the patients. Esophagectomy is the option in Chagas disease patients with advanced megaesophagus, despite the increased risk of complications. In these cases, peroral endoscopic myotomy may be an option, which needs further evaluation.

Achalasia Cardia: A Comprehensive Review

Achalasia cardia is the best characterised oesophageal motility disorder. It is characterised by progressive ganglion cell degeneration in the oesophageal myenteric plexus, which results in impaired lower oesophageal sphincter (LES) relaxation upon swallowing and aperistalsis in the distal smooth muscle segment of the oesophagus. The usual presenting features are dysphagia to both liquids and solids from onset, regurgitation of undigested food, retrosternal pain, heartburn, and weight loss. Initial investigations include upper gastrointestinal (GI) endoscopy and timed barium oesophagogram, whereas high resolution manometry is diagnostic. Therapy in achalasia cardia is directed towards biochemical or mechanical reduction in LES pressures. If candidates are fit for surgery, pneumatic dilatation, peroral endoscopic myotomy, and laparoscopic Heller’s myotomy are the mainstays of therapy that act by mechanical disruption of LES. On the other hand, botulinum toxin and pharmacotherapy (nitrates and calcium channel blockers) act by biochemical reduction of LES and are reserved for surgically unfit patients with limited life expectancy because of their short-lived efficacy. Oesophagectomy is reserved for treating refractory longstanding cases, who have previously failed multiple therapies.

Gelatinase B/Matrix Metalloproteinase-9 as Innate Immune Effector Molecule in Achalasia

ObjectivesAchalasia is a primary esophageal motility disorder resulting from selective loss of inhibitory neurons in the esophageal myenteric plexus, likely due to an autoimmune response with involvement of the adaptive immune system. Innate immune processes of the host constitute the bridge between environmental etiological factors and the adaptive immune system. Although these remain poorly investigated, they might be of diagnostic and therapeutic relevance. In view of the role of extracellular proteolysis in organ-specific autoimmunity, we studied gelatinases of the matrix metalloproteinase (MMP) family in achalasia patients.MethodsThe presence of MMP-2 and MMP-9 proteoforms was analyzed in sera of two cohorts of achalasia patients. Additionally, with the use of immunohistopathological analysis, in situ MMP-2 and MMP-9 expression was investigated. Finally, we tested the paradigm of remnant epitopes generating autoimmunity (REGA) for achalasia-associated autoantigens by evaluating whether autoantigenic proteins are cleaved by MMP-9 into remnant epitopes.ResultsWe showed significantly increased ratios of MMP-9/MMP-2 and activated MMP-9/proMMP-9 in sera of achalasia patients (n = 88) versus controls (n = 60). MMP-9-positive and MMP-2-positive cells were more abundant in achalasia (n = 49) versus control biopsies from transplant donors (n = 10). Furthermore, extensive damage within the plexus was found in the tissues with more MMP-9-positive cells. Additionally, we documented achalasia-associated autoantigens PNMA2, Ri, GAD65, and VIP as novel MMP-9 substrates.ConclusionsWe provide new biomarkers and insights into innate immune mechanisms in the autoimmune pathology of achalasia. Our results imply that extracellular protease inhibition is worthwhile to test as therapeutic intervention in achalasia.

Differences between idiopathic and chagasic achalasia

Idiopathic and Chagas’ disease achalasia are characterized by absent or partial lower esophageal sphincter relaxation, absence of peristaltic esophageal contraction, food retenction in the esophagus and esophageal dilatation. The most frequent symptoms are dysphagia, regurgitation, heartburn, weight loss and non-cardiac chest pain. The diagnosis is made by radiologic examination and esophageal manometry, which is considered the most accurate exam to characterized achalasia. In both diseases there is destruction of the esophageal myenteric plexus. Despite similarities in clinical and manometric presentation there is evidence of greater loss of inhibitory neurons of the myenteric plexus in idiopathic achalasia, whereas in Chagas’ disease there is a loss of both excitatory and inhibitory neurons. Such differences, though do not affect patients’ clinical presentation, and hence treatment options should be the same for both diseases.

Prise en charge de l’achalasie

The pathophysiology of achalasia is largely unknown, and involves the destruction of ganglion cell in the esophageal myenteric plexus. High-resolution esophageal manometry is the key investigation. Endoscopic pneumodilatation and laparoscopic Heller myotomy have comparable short-term success rates, around 90%. The main complication after pneumodilatation is esophageal perforation, occurring in about 1% of cases. Peroral endoscopic myotomy is a promising treatment modality, however with frequent post-procedural gastroesophageal reflux.

Inhibitory motor neurons of the esophageal myenteric plexus are mechanosensitive.

Mechanosensitivity of enteric neurons has been reported in the small intestine and colon, but not in the esophagus. Our earlier in vivo studies show that mechanical stretch of the esophagus in the axial direction induces neurally mediated relaxation of the lower esophageal sphincter, possibly through mechanosensitive motor neurons. However, this novel notion that the motor neurons are mechanosensitive has not been examined in isolated esophageal myenteric motor neurons. The goal of our present study was to examine the mechanosensitivity of esophageal motor neurons in primary culture and elucidate the underlying molecular mechanisms. Immmunocytochemical analysis revealed that >95% cells were positive for the neuronal marker protein gene product 9.5 and that 66% of these cells costained with protein gene product 9.5 and neuronal nitric oxide (NO) synthase. Hypotonic solution induced an increase in the cytoplasm volume in all cells that was independent of extracellular Ca(2+). Hypotonic solution and mechanical stretch induced cytoplasmic free Ca(2+) signaling in ~65% of neurons in the presence, but not absence, of extracellular Ca(2+). Neurons grown on the elastic membrane responded to mechanical stretch by an increase in neuronal size and Ca(2+) signaling simultaneously. Hypotonic stretch-induced cytoplasmic free Ca(2+) signaling was not affected by extracellular Mg(2+), 5-nitro-2-(3-phenylpropylamino)benzoic acid, and nifedipine but was attenuated by 2-aminoethoxydiphenyl borate, Gd(3+), and Grammostola mechanotoxin 4, blockers of the stretch-activated ion channels. In ~57% of the neurons, hypotonic stretch also induced Ca(2+)-dependent cytoplasmic NO production, which was abolished by Grammostola mechanotoxin 4. These results prove that the esophageal inhibitory motor neurons possess a mechanosensitive property and also provide novel insights into the stretch-activated ion channel-Ca(2+)-NO signaling pathway in these neurons.

Timing of Pharyngeal Swallow Events in Chagas' Disease.

BackgroundEsophageal involvement by Chagas’ disease causes a significative decrease in the number of neurons of the esophageal myenteric plexus, causing an impairment of esophageal motility with the same alterations described in primary esophageal achalasia. There is also a longer duration of pharyngeal transit, which might be consequent of the involvement of the central control of swallowing by the disease, or an adaptation of the pharynx to the difficult bolus transit through the esophagus, which could contribute to the complaint of dysphagia.MethodsWe studied, by videofluoroscopy, the sequence and timings of pharyngeal bolus transit in 16 patients with esophageal involvement by Chagas’ disease and 12 healthy volunteers. Each subject swallowed in duplicate 5 mL and 10 mL of liquid and paste boluses.ResultsThere was no difference between Chagas’ disease patients and normal volunteers in the sequence and timing of events associated with pharyngeal bolus flow, for liquid and paste boluses, and for 5 mL and 10 mL.ConclusionThe timing and sequence of swallow pharyngeal events of patients with Chagas’ disease do not differ from that of control subjects, which suggested that the central control of swallowing is not impaired by the disease.

Neurotransmitters in parasympathetic ganglionic neurons and nerves in mouse lower airway smooth muscle

In most species, including humans, lower airway smooth muscle (ASM) contains nerve terminals from two distinct populations of parasympathetic ganglionic neurons based on neurotransmitter phenotype: cholinergic and non-adrenergic non-cholinergic (NANC), causing contraction and relaxation, respectively, of ASM. Using immunohistological staining, the density and distribution of NANC-associated neurotransmitters, vasoactive intestinal peptide (VIP) and nitric oxide synthase were 6% of total nerve profiles compared to 19% cholinergic nerves in ASM in mouse (C57BL/6) central airways. The location of the NANC parasympathetic neurons innervating the tracheal ASM, as determined by retrograde neuronal tracer from the trachealis muscle, was the myenteric plexus of the esophagus, closely associated with the outer striated longitudinal muscle layers; the majority of the retrograde-labeled neurons were VIP- and NOS-IR. The results of these experiments provide the first direct evidence that VIP-IR and NOS-IR neurons intrinsic to the mouse esophagus project axons to the adjacent trachealis muscle.

Achalasia: A review of clinical diagnosis, epidemiology, treatment and outcomes

Achalasia is a neurodegenerative motility disorder of the oesophagus resulting in deranged oesophageal peristalsis and loss of lower oesophageal sphincter function. Historically, annual achalasia incidence rates were believed to be low, approximately 0.5-1.2 per 100000. More recent reports suggest that annual incidence rates have risen to 1.6 per 100000 in some populations. The aetiology of achalasia is still unclear but is likely to be multi-factorial. Suggested causes include environmental or viral exposures resulting in inflammation of the oesophageal myenteric plexus, which elicits an autoimmune response. Risk of achalasia may be elevated in a sub-group of genetically susceptible people. Improvement in the diagnosis of achalasia, through the introduction of high resolution manometry with pressure topography plotting, has resulted in the development of a novel classification system for achalasia. This classification system can evaluate patient prognosis and predict responsiveness to treatment. There is currently much debate over whether pneumatic dilatation is a superior method compared to the Heller's myotomy procedure in the treatment of achalasia. A recent comparative study found equal efficacy, suggesting that patient preference and local expertise should guide the choice. Although achalasia is a relatively rare condition, it carries a risk of complications, including aspiration pneumonia and oesophageal cancer. The risk of both squamous cell carcinoma and adenocarcinoma of the oesophagus is believed to be significantly increased in patients with achalasia, however the absolute excess risk is small. Therefore, it is currently unknown whether a surveillance programme in achalasia patients would be effective or cost-effective.

Achalasia With Dense Eosinophilic Infiltrate Responds to Steroid Therapy

A patient presented with chronic substernal discomfort and intermittent dysphagia for solids. High-resolution impedance manometry (HRIM) of the esophagus showed that there was no peristalsis in the esophageal body but incomplete relaxation of the lower esophageal sphincter and incomplete bolus transit, so the patient was diagnosed with achalasia. Moreover, probably because of esophageal stasis, eosinophilic infiltration that mimicked a pattern of eosinophilic esophagitis was observed, on the basis of multiple biopsies of the esophagus. The patient was given 50 mg prednisolone once daily; the symptoms improved dramatically, and HRIM showed complete recovery of esophageal peristalsis, deeper relaxation of the lower esophageal sphincter, and complete bolus transit profile. HRIM can therefore be used to assess dysmotility abnormalities in patients with achalasia and eosinophilic-like esophagitis, and steroids relieve these symptoms. Treatment with a high dose of prednisolone resulted in a complete disappearance of dysphagia because of improved esophageal motility and reduced eosinophilic infiltrate. It is therefore important to control the inflammatory process in patients with idiopathic achalasia, which is likely to result from an autoimmune reaction.

The prevalence and patterns of pharyngoesophageal dysmotility in patients with early stage Parkinson's disease

Dysphagia occurs in the majority of patients with Parkinson's disease (PD) and is known to correlate with abnormalities of oropharyngeal function. The aim of this study was to evaluate pharyngoesophageal activity in patients with early-stage PD. Newly diagnosed PD patients with a symptom duration not exceeding 3 years were included. All PD patients were questioned about symptoms of dysphagia and underwent combined multichannel intraluminal impedance manometry and multiple rapid swallow tests. Fifty-four patients (22 men and 32 women, 67.1 ± 10.3 years) were enrolled. The duration of Parkinsonian motor symptoms was 11.5 ± 8.8 months, the Hoehn and Yahr stage was 1.6 ± 0.4, and the total Unified Parkinson's Disease Rating Scale was 25.1 ± 18.6. Esophageal manometry in the liquid swallow and viscous swallow tests was abnormal in 22 (40.7%) and 31 (67.4%) patients, respectively. Although manometric abnormalities were more common in patients with more severe dysphagia symptoms, many patients with no or minimal symptoms also had manometric abnormalities. Repetitive deglutition significantly correlated with failed peristalsis and incomplete bolus transit. Abnormal responses to multiple rapid swallow tests were found in 33 out of 54 patients; 29 with incomplete inhibition (repetitive contraction) and 4 with failed peristalsis. These results suggest that the majority of patients with early-stage PD showed pharyngeal and esophageal dysfunction even before clinical manifestations of dysphagia, which may reflect selective involvement of either the brain stem or the esophageal myenteric plexus in early-stage PD.

Innervation of tracheal parasympathetic ganglia by esophageal cholinergic neurons: evidence from anatomic and functional studies in guinea pigs

In the present study, we describe a subset of nerve fibers, characterized by their immunoreactivity for the calcium-binding protein calretinin, that are densely and selectively associated with cholinergic postganglionic neurons in the guinea pig tracheal ganglia. Retrograde neuronal tracing with cholera toxin B, combined with immunohistochemical analyses, showed that these nerve fibers do not originate from sensory neurons in the nodose, jugular, or dorsal root ganglia or from motor neurons in the nucleus ambiguus, dorsal motor nucleus of the vagus nerve, spinal cord, stellate ganglia, or superior cervical ganglia. Calretinin-immunoreactive nerve fibers disappeared from tracheal segments after 48 h in organotypic culture, indicating that the fibers were of extrinsic origin. However, calretinin-positive nerve fibers persisted in tracheal ganglia when tracheae were cocultured with the adjacent esophagus intact. Immunohistochemical analysis of the esophagus revealed a population of cholinergic neurons in the esophageal myenteric plexus that coexpressed calretinin. In functional studies, electrical stimulation of the esophagus in vitro evoked measurable contractions of the trachea. These contractions were not altered by prior organotypic culture of the trachea and esophagus to remove the extrinsic innervation to the airways but were significantly (P < 0.05) inhibited by the ganglionic blocker hexamethonium or by physical disruption of the tissue connecting the trachea and esophagus. These data suggest that a subset of esophageal neurons, characterized by the expression of calretinin and acetylcholine, provide a previously unrecognized excitatory input to tracheal cholinergic ganglia in guinea pigs.

Effects of perinatal protein deprivation and recovery on esophageal myenteric plexus

To evaluate effects of pre- and postnatal protein deprivation and postnatal recovery on the myenteric plexus of the rat esophagus. Three groups of young Wistar rats (aged 42 d) were studied: normal-fed (N42), protein-deprived (D42), and protein-recovered (R42). The myenteric neurons of their esophagi were evaluated by histochemical reactions for nicotinamide adenine dinucleotide (NADH), nitrergic neurons (NADPH)-diaphorase and acetylcholinesterase (AChE), immunohistochemical reaction for vasoactive intestinal polypeptide (VIP), and ultrastructural analysis by transmission electron microscopy. The cytoplasms of large and medium neurons from the N42 and R42 groups were intensely reactive for NADH. Only a few large neurons from the D42 group exhibited this aspect. NADPH detected in the D42 group exhibited low reactivity. The AChE reactivity was diffuse in neurons from the D42 and R42 groups. The density of large and small varicosities detected by immunohistochemical staining of VIP was low in ganglia from the D42 group. In many neurons from the D42 group, the double membrane of the nuclear envelope and the perinuclear cisterna were not detectable. NADH and NADPH histochemistry revealed no group differences in the profile of nerve cell perikarya (ranging from 200 to 400 microm(2)). Protein deprivation causes a delay in neuronal maturation but postnatal recovery can almost completely restore the normal morphology of myenteric neurons.