Ultrastructural differences between diabetic and idiopathic gastroparesis.

The ultrastructural changes in diabetic and idiopathic gastroparesis are not well studied and it is not known whether there are different defects in the two disorders. As part of the Gastroparesis Clinical Research Consortium, full thickness gastric body biopsies from 20 diabetic and 20 idiopathic gastroparetics were studied by light microscopy. Abnormalities were found in many (83%) but not all patients. Among the common defects were loss of interstitial cells of Cajal (ICC) and neural abnormalities. No distinguishing features were seen between diabetic and idiopathic gastroparesis. Our aim was to provide a detailed description of the ultrastructural abnormalities, compare findings between diabetic and idiopathic gastroparesis and determine if patients with apparently normal immunohistological features have ultrastructural abnormalities. Tissues from 40 gastroparetic patients and 24 age- and sex-matched controls were examined by transmission electron microscopy (TEM). Interstitial cells of Cajal showing changes suggestive of injury, large and empty nerve endings, presence of lipofuscin and lamellar bodies in the smooth muscle cells were found in all patients. However, the ultrastructural changes in ICC and nerves differed between diabetic and idiopathic gastroparesis and were more severe in idiopathic gastroparesis. A thickened basal lamina around smooth muscle cells and nerves was characteristic of diabetic gastroparesis whereas idiopathic gastroparetics had fibrosis, especially around the nerves. In conclusion, in all the patients TEM showed abnormalities in ICC, nerves and smooth muscle consistent with the delay in gastric emptying. The significant differences found between diabetic and idiopathic gastroparesis offers insight into pathophysiology as well as into potential targeted therapies.

Cellular changes in diabetic and idiopathic gastroparesis.

BACKGROUND & AIMS: Cellular changes associated with diabetic and idiopathic gastroparesis are not well described. The aim of this study was to describe histologic abnormalities in gastroparesis and compare findings in idiopathic versus diabetic gastroparesis. METHODS: Full-thickness gastric body biopsy specimens were obtained from 40 patients with gastroparesis (20 diabetic) and matched controls. Sections were stained for H&E and trichrome and immunolabeled with antibodies against protein gene product (PGP) 9.5, neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide, substance P, and tyrosine hydroxylase to quantify nerves, S100ß for glia, Kit for interstitial cells of Cajal (ICC), CD45 and CD68 for immune cells, and smoothelin for smooth muscle cells. Tissue was also examined by transmission electron microscopy. RESULTS: Histologic abnormalities were found in 83% of patients. The most common defects were loss of ICC with remaining ICC showing injury, an abnormal immune infiltrate containing macrophages, and decreased nerve fibers. On light microscopy, no significant differences were found between diabetic and idiopathic gastroparesis with the exception of nNOS expression, which was decreased in more patients with idiopathic gastroparesis (40%) compared with diabetic patients (20%) by visual grading. On electron microscopy, a markedly increased connective tissue stroma was present in both disorders. CONCLUSIONS: This study suggests that on full-thickness biopsy specimens, cellular abnormalities are found in the majority of patients with gastroparesis. The most common findings were loss of Kit expression, suggesting loss of ICC, and an increase in CD45 and CD68 immunoreactivity. These findings suggest that examination of tissue can lead to valuable insights into the pathophysiology of these disorders and offer hope that new therapeutic targets can be found.

Endoscopic full-thickness biopsy of the gastric wall with defect closure by using an endoscopic suturing device: survival porcine study.

BACKGROUND: The pathogenesis of several common gastric motility diseases and functional GI disorders remains essentially unexplained. Gastric wall biopsies that include the muscularis propria to evaluate the enteric nervous system, interstitial cells of Cajal, and immune cells can provide important insights for our understanding of the etiology of these disorders. OBJECTIVES: To determine the technical feasibility, reproducibility, and safety of performing a full-thickness gastric biopsy (FTGB) by using a submucosal endoscopy with mucosal flap (SEMF) technique; the technical feasibility, reproducibility, and safety of tissue closure by using an endoscopic suturing device; the ability to identify myenteric ganglia in resected specimens; and the long-term safety. DESIGN: Single center, preclinical survival study. SETTING: Animal research laboratory, developmental endoscopy unit. SUBJECTS: Twelve domestic pigs. INTERVENTIONS: Animals underwent an SEMF procedure with gastric muscularis propria resection. The resultant offset mucosal entry site was closed by using an endoscopic suturing device. Animals were kept alive for 2 weeks. MAIN OUTCOME MEASUREMENTS: The technical feasibility, reproducibility, and safety of the procedure; the clinical course of the animals; the histological and immunochemical evaluation of the resected specimen to determine whether myenteric ganglia were present in the sample. RESULTS: FTGB was performed by using the SEMF technique in all 12 animals. The offset mucosal entry site was successfully closed by using the suturing device in all animals. The mean resected tissue specimen size was 11 mm. Mean total procedure time was 61 minutes with 2 to 4 interrupted sutures placed per animal. Histology showed muscularis propria and serosa, confirming full-thickness resections in all animals. Myenteric ganglia were visualized in 11 of 12 animals. The clinical course was uneventful. Repeat endoscopy and necropsy at 2 weeks showed absence of ulceration at both the mucosal entry sites and overlying the more distal muscularis propria resection sites. There was complete healing of the serosa in all animals with minimal single-band adhesions in 5 of 12 animals. Retained sutures were present in 10 of 12 animals. LIMITATIONS: Animal experiment. CONCLUSIONS: FTGB by using the SEMF technique and an endoscopic suturing device is technically feasible, reproducible, and safe. Larger tissue specimens will allow improved analysis of multiple cell types.

Heme oxygenase-1 protects interstitial cells of Cajal from oxidative stress and reverses diabetic gastroparesis.

BACKGROUND & AIMS: Diabetic gastroparesis (delayed gastric emptying) is a well-recognized complication of diabetes that causes considerable morbidity and makes glucose control difficult. Interstitial cells of Cajal, which express the receptor tyrosine kinase Kit, are required for normal gastric emptying. We proposed that Kit expression is lost during diabetic gastroparesis due to increased levels of oxidative stress caused by low levels of heme oxygenase-1 (HO-1), an important cytoprotective molecule against oxidative injury. METHODS: Gastric emptying was measured in nonobese diabetic mice and correlated with levels of HO-1 expression and activity. Endogenous HO-1 activity was increased by administration of hemin and inhibited by chromium mesoporphyrin. RESULTS: In early stages of diabetes, HO-1 was up-regulated in gastric macrophages and remained up-regulated in all mice that were resistant to development of delayed gastric emptying. In contrast, HO-1 did not remain up-regulated in all the mice that developed delayed gastric emptying; expression of Kit and neuronal nitric oxide synthase decreased markedly in these mice. Loss of HO-1 up-regulation increased levels of reactive oxygen species. Induction of HO-1 by hemin decreased reactive oxygen species, rapidly restored Kit and neuronal nitric oxide synthase expression, and completely normalized gastric emptying in all mice. Inhibition of HO-1 activity in mice with normal gastric emptying caused a loss of Kit expression and development of diabetic gastroparesis. CONCLUSIONS: Induction of the HO-1 pathway prevents and reverses cellular changes that lead to development of gastrointestinal complications of diabetes. Reagents that induce this pathway might therefore be developed as therapeutics.

Endoscopic "no hole" full-thickness biopsy of the stomach to detect myenteric ganglia.

BACKGROUND: The etiology of several common gastric motility diseases remains largely unknown. Gastric wall biopsy specimens that include the muscularis propria to evaluate the enteric nervous system, interstitial cells of Cajal, and related cells are essential to promote our understanding of the pathophysiologic mechanisms. On the basis of our previous work, a double EMR technique provided sufficient tissue to identify myenteric ganglia. A serious limitation to the technique was the resultant gastric wall perforation after tissue resection. The optimal procedure would seal the gastric wall defect before tissue resection, eliminating the risk of peritonitis. OBJECTIVES: The aims of this study were to (1) determine the technical feasibility and reproducibility of a full-thickness gastric biopsy by use of a novel double EMR technique without creating a perforation ("no hole") and to (2) determine safety of the procedure. DESIGN AND INTERVENTIONS: Preclinical study of 6 pigs. Each animal underwent a "no hole" double EMR survival procedure. To prevent perforation, detachable endoloops and prototype T-tag tissue anchors were placed before resection. At 2 weeks repeat endoscopy was performed followed by necropsy. MAIN OUTCOME MEASUREMENTS: Hematoxylin-eosin staining was used to determine which muscle layers were included in the resected specimen, and an antibody to neuronal nitric oxide synthase was used to visualize myenteric ganglia in the sample. Technical feasibility, reproducibility, and safety of the procedure were evaluated. RESULTS: Full-thickness gastric biopsy specimens were obtained from all animals without overt perforation. There were no procedural complications. Histologic examination showed muscularis propria with all layers of muscle present, and immunochemical studies demonstrated myenteric ganglia in all tissue samples. Four animals had an uneventful clinical course, and repeat endoscopy at week 2 showed ulceration with stellate fibrosis. Necropsy showed mild localized adhesions. Two animals were killed at days 3 and 6, respectively, because of suspected peritonitis. At necropsy, delayed perforations at the resection sites were noted with displaced endoloops and tissue anchors. CONCLUSION: This study explored the concept of obtaining deep muscle wall biopsy specimens with use of a unique approach of resection without perforation. The novel "no hole" double EMR technique was technically feasible and reproducible with sufficient tissue obtained to identify myenteric ganglia. However, there was a high delayed perforation rate associated with displaced endoloops and tissue anchors. On the basis of this early experience, improved safety data may be anticipated with future studies using improved tissue closure devices.

Changes in the gastric enteric nervous system and muscle: a case report on two patients with diabetic gastroparesis.

BACKGROUND: The pathophysiological basis of diabetic gastroparesis is poorly understood, in large part due to the almost complete lack of data on neuropathological and molecular changes in the stomachs of patients. Experimental models indicate various lesions affecting the vagus, muscle, enteric neurons, interstitial cells of Cajal (ICC) or other cellular components. The aim of this study was to use modern analytical methods to determine morphological and molecular changes in the gastric wall in patients with diabetic gastroparesis. METHODS: Full thickness gastric biopsies were obtained laparoscopically from two gastroparetic patients undergoing surgical intervention and from disease-free areas of control subjects undergoing other forms of gastric surgery. Samples were processed for histological and immunohistochemical examination. RESULTS: Although both patients had severe refractory symptoms with malnutrition, requiring the placement of a gastric stimulator, one of them had no significant abnormalities as compared with controls. This patient had an abrupt onset of symptoms with a relatively short duration of diabetes that was well controlled. By contrast, the other patient had long standing brittle and poorly controlled diabetes with numerous episodes of diabetic ketoacidosis and frequent hypoglycemic episodes. Histological examination in this patient revealed increased fibrosis in the muscle layers as well as significantly fewer nerve fibers and myenteric neurons as assessed by PGP9.5 staining. Further, significant reduction was seen in staining for neuronal nitric oxide synthase, heme oxygenase-2, tyrosine hydroxylase as well as for c-KIT. CONCLUSION: We conclude that poor metabolic control is associated with significant pathological changes in the gastric wall that affect all major components including muscle, neurons and ICC. Severe symptoms can occur in the absence of these changes, however and may reflect vagal, central or hormonal influences. Gastroparesis is therefore likely to be a heterogeneous disorder. Careful molecular and pathological analysis may allow more precise phenotypic differentiation and shed insight into the underlying mechanisms as well as identify novel therapeutic targets.

Evaluation of endoscopic approaches for deep gastric-muscle-wall biopsies: what works?

BACKGROUND: A major barrier to furthering our understanding of the pathophysiology of neuromuscular GI diseases, including functional GI disorders, is the inability to obtain deep gastric-wall biopsy specimens that include both layers of the muscularis propria, which allows evaluation of specific cell types, including myenteric ganglia. OBJECTIVES: The aims of this preclinical study were to (1) evaluate different endoscopic approaches for obtaining deep gastric-muscle-wall biopsy specimens and (2) determine if myenteric ganglia were present in the tissue samples. DESIGN AND INTERVENTIONS: This was a preclinical acute study by using a pig model. Multiple samples were obtained from 4 pigs. The endoscopic techniques evaluated were (1) EUS-guided tru-cut biopsy of the gastric wall, (2) jumbo biopsy of the post-EMR site, (3) jumbo biopsy of the gastrotomy margin, (4) serosal-side biopsy through a gastrotomy, and (5) double-EMR resection. MAIN OUTCOME MEASUREMENTS: Resected tissue was submitted for histology to determine which wall layers were included in the resected specimen. Hematoxylin and eosin staining was used to determine which muscle layers were biopsied, and an antibody to protein gene product 9.5 was used to determine if myenteric ganglia were present in the sample. RESULTS: Seventy-two tissue samples were obtained: EUS-guided tru-cut biopsy (n=16), jumbo biopsy of the post-EMR site (n=16), jumbo biopsy of the gastrotomy (n=16), serosal-side biopsy (n=16), and double-EMR resection (n=8). Only the double-EMR resection tissues showed the presence of longitudinal muscle, indicating the presence of both muscle layers and the myenteric plexus. Immunofluorescence studies demonstrated the presence of myenteric ganglia only in the double-EMR tissues and in none of the other gastric samples. No adjacent organs were included in the resection. CONCLUSIONS: The double-EMR technique was the only studied technique that resulted in a deep gastric-wall sample and provided sufficient tissue to evaluate both muscle layers and the intermuscular layer that contain myenteric ganglia. Further studies are needed to verify the efficacy and to assess the safety of this approach.

Computer aided classification of cell nuclei in the gastrointestinal tract by volume and principal axis.

Normal function of the gastrointestinal tract involves the coordinated activity of several cell types Human disorders of motor function of the gastrointestinal tract are often associated with changes in the number of these cells. For example, in diabetic patients, abnormalities in gastrointestinal transit are associated with changes in nerves and interstitial cells of Cajal (ICC), two key cells that generate and regulate motility. ICC are cells of mesenchymal origin that function as pacemakers and amplify neuronal signals in the gastrointestinal tract. Quantifying the changes in number of specific cell types in tissues from patients with motility disorders is challenging and requires immunolabeling for specific antigens. The shape of nuclei differs between the cell types in the wall of the gastrointestinal tract. Therefore the objective of this study was to determine whether cell nuclei can be classified by analyzing the 3D morphology of the nuclei. Furthermore, the orientation of the long axis of nuclei changes within and between the muscle layers. These features can be used to classify and differentially label the nuclei in confocal volume images of the tissue by computing the principal axis of the coordinates of the set of voxels forming each nucleus and thereby to identify cells by their nuclear morphology. Using this approach, we were able to separate and quantify nuclei in the smooth muscle layers of the tissue. Therefore we conclude that computer-aided classification of cell nuclei can be used to identify changes in the cell types expressed in gastrointestinal smooth muscle.