Matthew S. Lurken

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, S100β 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.

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.

Effect of Age on the Enteric Nervous System of the Human Colon

Abstract  The effect of age on the anatomy and function of the human colon is incompletely understood. The prevalence of disorders in adults such as constipation increase with age but it is unclear if this is due to confounding factors or age‐related structural defects. The aim of this study was to determine number and subtypes of enteric neurons and neuronal volumes in the human colon of different ages. Normal colon (descending and sigmoid) from 16 patients (nine male) was studied; ages 33–99. Antibodies to HuC/D, choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), and protein gene product 9.5 were used. Effect of age was determined by testing for linear trends using regression analysis. In the myenteric plexus, number of Hu‐positive neurons declined with age (slope = −1.3 neurons/mm/10 years, P = 0.03). The number of ChAT‐positive neurons also declined with age (slope = −1.1 neurons/mm/10 years of age, P = 0.02). The number of nNOS‐positive neurons did not decline with age. As a result, the ratio of nNOS to Hu increased (slope = 0.03 per 10 years of age, P = 0.01). In the submucosal plexus, the number of neurons did not decline with age (slope = −0.3 neurons/mm/10 years, P = 0.09). Volume of nerve fibres in the circular muscle and volume of neuronal structures in the myenteric plexus did not change with age. In conclusion, the number of neurons in the human colon declines with age with sparing of nNOS‐positive neurons. This change was not accompanied by changes in total volume of neuronal structures suggesting compensatory changes in the remaining neurons.

Clinical-histological associations in gastroparesis: results from the Gastroparesis Clinical Research Consortium.

Background  Cellular changes associated with diabetic (DG) and idiopathic gastroparesis (IG) have recently been described from patients enrolled in the Gastroparesis Clinical Research Consortium. The association of these cellular changes with gastroparesis symptoms and gastric emptying is unknown. The aim of this study was to relate cellular changes to symptoms and gastric emptying in patients with gastroparesis.

Clinical-histological associations in gastroparesis: results from the Gastroparesis Clinical Research Consortium: Clinical-histological associations in gastroparesis

Background  Cellular changes associated with diabetic (DG) and idiopathic gastroparesis (IG) have recently been described from patients enrolled in the Gastroparesis Clinical Research Consortium. The association of these cellular changes with gastroparesis symptoms and gastric emptying is unknown. The aim of this study was to relate cellular changes to symptoms and gastric emptying in patients with gastroparesis.

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

BackgroundThe 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.MethodsFull 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.ResultsAlthough 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.ConclusionWe 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.

Regulation of interstitial cells of Cajal in the mouse gastric body by neuronal nitric oxide

Abstract  The factors underlying the survival and maintenance of interstitial cells of Cajal (ICC) are not well understood. Loss of ICC is often associated with loss of neuronal nitric oxide synthase (nNOS) in humans, suggesting a possible link. The aim of this study was to determine the effect of neuronal NO on ICC in the mouse gastric body. The volumes of ICC were determined in nNOS−/− and control mice in the gastric body and in organotypic cultures using immunohistochemistry, laser scanning confocal microscopy and three‐dimensional reconstruction. ICC numbers were determined in primary cell cultures after treatment with an NO donor or an NOS inhibitor. The volumes of myenteric c‐Kit‐immunoreactive networks of ICC from nNOS−/− mice were significantly reduced compared with control mice. No significant differences in the volumes of c‐Kit‐positive ICC were observed in the longitudinal muscle layers. ICC volumes were either decreased or unaltered in the circular muscle layer after normalization for the volume of circular smooth muscle. The number of ICC was increased after incubation with S‐nitroso‐N‐acetylpenicillamine and decreased by N(G)‐nitro‐l‐arginine. Neuronally derived NO modulates ICC numbers and network volume in the mouse gastric body. NO appears to be a survival factor for ICC.

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.

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.

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.