Subserosal localization of myenteric ganglia in normal human appendix: immunostaining with neuronal and glial markers.

OBJECTIVES: Visualization of unexpected distribution of myenteric ganglia in normal human appendiceal wall by immunofluorescence. BACKGROUND: The myenteric plexus is located between the longitudinal and circular muscle layers of the GIT. However, recently the irregular distribution of myenteric ganglia was revealed in human appendix. METHODS: The cryosections prepared from normal human appendices were examined by immunofluorescence methods using antibodies to neurofilaments (NF) and glial fibrillary acidic protein (GFAP). RESULTS: Indirect immunofluorescence revealed the positive staining of myenteric ganglia with both neuronal and glial marker antibodies. Double labeling for NF/GFAP staining showed close assotiation between glia and neurons inside ganglia. GFAP-positive cells were often observed as the cells surrounding myenteric ganglia. The staining confirmed the irregular distribution of myenteric ganglia in human appendiceal wall and revealed the small ganglia in the subserosal area. CONCLUSION: Our results showed that localization of myenteric ganglia in human appendix differs from other parts of GIT. GFAP immunostaining is available for visualization of smaller myenteric ganglia located mainly in the subserosal area. Our studies may find application in current HIV research focused on enteric neuropathogenesis and in diagnostic laparoscopy for chronic abdominal pain: to detect the irritation of subserosal ganglia (Fig. 2, Ref. 26).

A case of diffuse neuronal hypertrophy in acute appendicitis.

We report a case of neuronal hypertrophy associated with acute appendicitis in which significant neuronal fibers and the number of ganglion cells increased in the absence of inflammatory cells. Differential diagnosis from diffuse ganglioneuromatosis by the pathologic findings of resected specimen was difficult. A 33-year-old Japanese female visited our hospital complaining of acute abdominal pain. The patient underwent appendectomy upon the diagnosis of acute appendicitis on the day of admission. Postoperative examinations found no neoplastic lesions in other organs or inherited disorders such as multiple endocrine neoplasia (MEN) type 2b and von Recklinghausen's disease (VRD). The pathologic diagnosis was neuronal hypertrophy of the appendix. Because the clinical outcomes of diffuse ganglioneuromatosis and neuronal hypertrophy of the gastrointestinal tract are quite different, clinical and pathologic examination should be carefully carried out for lesions in which significant proliferation of neuronal components is seen.

Interstitial cell of Cajal loss correlates with the degree of inflammation in the human appendix and reverses after inflammation.

BACKGROUND: Normal gut motility relies on the complex interaction between the interstitial cell of Cajal (ICC) and the enteric nerve networks. Inflammation of the gastrointestinal tract adversely affects both ICC and enteric nerves. We aimed to determine the distribution of ICC and nerve networks in patients with appendicitis. METHODS: Specimens from controls and patients with appendicitis were examined with immunohistochemistry (c-Kit for ICC, beta III tubulin [Tuj-1] and neuronal nitric oxide synthase [histochemical diaphorase] for nitrergic neurons) and electron microscopy (EM). Data were quantified using image analysis. RESULTS: We found a profound decrease in c-Kit immunoreactivity (c-Kit IR) in the advanced inflammatory stages of appendicitis, which correlated with the severity of inflammation. Electron microscopy confirmed ultrastructural injury in both ICC and nerve fiber networks during acute inflammation. After the inflammation resolved, interval appendices displayed a recovery in ICC c-Kit IR to control levels and normal ultrastructure. The neuronal network also displayed ultrastructural recovery; however, neuronal nitric oxide synthase activity did not recover. CONCLUSIONS: Severe inflammation results in significant ultrastructural damage of nerves and ICC networks in appendicitis. The loss of c-Kit IR is likely due to impaired ICC cytophysiology because ICC was still present under EM. After resolution of acute inflammation, ICC recovers their normal ultrastructure and c-Kit IR.

The bases of the inflammatory acute abdomen.

Vermiform appendix is an anatomical structure, which due to its topographical relations usually causes many complications, especially in inflammation such as appendicitis. One of the manifestations of the inflammatory processes is pain, which may have different location. It could be probably a result of the neuron stimulation. The nerve formation and distribution of the vermiform appendix is still unknown in fine details and is a subject of further studies (Fig. 8, Ref.12).

Human visceral afferent recordings: preliminary report.

BACKGROUND: Conditions characterised by chronic visceral pain represent a significant healthcare burden with limited treatment options. While animal models have provided insights into potential mechanisms of visceral nociception and identified candidate drug targets, these have not translated into successful treatments in humans. OBJECTIVE: To develop an in vitro afferent nerve preparation using surgically excised freshly isolated human colon and vermiform appendix-mesentery tissues. METHODS: Non-inflamed appendix (n=18) and colon (n=9) were collected from patients undergoing right and left hemicolectomy. Electrophysiological recordings were made from mesenteric nerves and the tissue stimulated chemically and mechanically. RESULTS: Ongoing neuronal activity was sparse and where units occurred peak firing rates were: colon (2.0±0.4 spikes/s, n=4) and appendix (2.4±0.6 spikes/s, n=9). Afferent nerves innervating the appendix responded with a significant increase in activity following stimulation with inflammatory mediators (73±10.6 vs 3.0±0.3 spikes/s, n=6, p<0.001, inflammatory mediator vs baseline) and capsaicin (63±15.8 vs 2±0.3 spikes/s, n=3, p<0.001, capsaicin vs buffer). Afferent nerves innervating the colon responded with increased activity to blunt probing of the serosal surface. CONCLUSIONS: This first-in-human study demonstrates afferent nerve recordings from human gut tissue ex vivo and shows that tissue may be stimulated both chemically and mechanically to study neuronal responses. Collectively, the results provide preliminary evidence to validate this in vitro human tissue model as one that may aid future disease mechanistic studies and candidate drug testing.

Observations on a vestigial organ: a potential surrogate for enteric neuromesenchymal disease.

Abnormalities of enteric nerves, interstitial cells of Cajal (ICC) and smooth muscle are often associated with severe gastrointestinal motility disorders. In this context, full-thickness biopsy of the gut may provide important diagnostic and prognostic clues as well as some possible therapeutic implications. Nonetheless, the unavoidable risk to further worsen prognosis evoked by laparotomy, and the unclear yield of histopathological analysis has hampered full-thickness gut sampling in patients with severe dysmotility. However, recent advances in minimally invasive surgery have refuelled enthusiasm in gastrointestinal neuromuscular pathology. In this issue of Neurogastroenterology and Motility, Miller et al. provide novel and exciting evidence that the appendix might be used as a surrogate tissue to analyse changes to enteric nerves, ICC and smooth muscle cells in patients with diabetic gastroenteropathy. The objective of this short review was to place this very important work in the context of current understanding of enteric neuromuscular dysfunction.

Distribution of interstitial cells of Cajal and nitrergic neurons in normal and diabetic human appendix.

The objective of this study was to determine the distribution of enteric nerves and interstitial cells of Cajal (ICC) in the normal human appendix and in type 1 diabetes. Appendixes were collected from patients with type 1 diabetes and from non-diabetic controls. Volumes of nerves and ICC were determined using 3-D reconstruction and neuronal nitric oxide synthase (nNOS) expressing neurons were counted. Enteric ganglia were found in the myenteric plexus region and within the longitudinal muscle. ICC were found throughout the muscle layers. In diabetes, c-Kit positive ICC volumes were significantly reduced as were nNOS expressing neurons. In conclusion, we describe the distribution of ICC and enteric nerves in health and in diabetes. The data also suggest that the human appendix, a readily available source of human tissue, may be useful model for the study of motility disorders.

An "eye" in the gut: the appendix as a sentinel sensory organ of the immune intelligence network.

Neural systems are the traditional model of intelligence. Their complex interconnected network of wired neurons acquires, processes, and responds to environmental cues. We propose that the immune system is a parallel system of intelligence in which the gut, including the appendix, plays a prominent role in data acquisition. The immune system is essentially a virtual unwired network of interacting cells that acquires, processes, and responds to environmental data. The data is typically acquired by antigen-presenting cells (APCs) that gather antigenic information from the environment. The APCs chemically digest large antigens and deconstruct them into smaller data packets for sampling by other cells. The gut performs the same function on a larger scale. Morsels of environmental content that enter the gut are sequentially deconstructed by physical and chemical digestion. In addition to providing nutrients, the componentized contents offer environmental data to APCs in mucosa-associated lymphoid tissues (MALT) that relay the sampled information to the immune intelligence network. In this framework, positioning of the appendix immediately after the ileocecal valve is strategic: it is ideally positioned to sample environmental data in its maximally deconstructed state after small bowel digestion. For single-celled organisms, digestion of the environment has been the primary way to sample the surroundings. Prior to the emergence of complex sensory systems such as the eye, even multi-cellular organisms may have relied heavily on digestion to acquire environmental information. While the relative value of immune intelligence has diminished since the emergence of neural intelligence, organisms still use information from both systems in integrated fashion to respond appropriately to ecologic opportunities and challenges. Appendicitis may represent a momentary maladaptation in the evolutionary transition of sensory leadership from the gut to the eye. Relationships between immune dysfunctions and cognition are explored.

Multiple myenteric networks in the human appendix.

The general histological organization of the appendix, including its innervation, is believed to be generally similar to that of the large intestine. However, several authors described an unusual arrangement of the myenteric ganglia within the appendiceal muscle, but conflicting reports do not allow clear conclusions on this matter. The aim of this work was to examine the appendiceal innervation in detail. The myenteric plexus of the human appendix was examined using sections and whole mount preparations. Human small and large intestines were used for comparison. The nerves were stained using immunohistochemistry, enzyme histochemistry for NADPH-diaphorase, and vital staining with 4-(4-diethylaminostyryl)-methylpyridinium iodide. Appendices from rabbits were also studied. In most cases, the innervation of the external muscle of the appendix consisted of three concentric networks of ganglia. These networks were located both between the circular and longitudinal muscle layers and within them. The middle network made connections with the other two. Such arrangement was not observed in the human small and large intestines. The myenteric plexus in the rabbit appendix displayed a much smaller degree of three-dimensional distribution compared with that of the human appendix. It is concluded that the myenteric plexus in the human appendix consists of several distinct networks, and appears to be unique in comparison with the other parts of the intestine.

Nitrergic hyperinnervation in appendicitis and in appendices histologically classified as normal.

CONTEXT: The pathogenesis of appendicitis remains poorly understood. Despite new diagnostic techniques, appendices removed from patients with suspected appendicitis often appear histologically normal on conventional examination. There is increasing evidence of involvement of the enteric nervous system in immune regulation and in inflammatory responses in the gastrointestinal system. OBJECTIVE: To investigate the nitrergic innervation of (a) acutely inflamed appendices, (b) appendices classified as histologically normal from patients with a clinical diagnosis of appendicitis, and (c) normal control appendix specimens, using the whole-mount preparation technique. PATIENTS AND DESIGN: Full-thickness specimens were collected from 28 acutely inflamed appendices (age range, 3.2-13.4 years), 31 histologically normal appendices removed from patients (age range, 5.7-13.6 years) with suspected appendicitis, and 23 histologically normal appendices from patients (age range, newborn to 12.1 years) undergoing elective abdominal surgery (controls). Whole-mount preparation using nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry and neuronal nitric oxide synthase immunohistochemistry were performed. The density of myenteric plexus was measured with a computerized analysis system. RESULTS: The density of myenteric plexus in normal appendix specimens was similar to that of large bowel from the newborn period up to 3 years of age; this density decreased significantly thereafter. The myenteric plexus of normal appendix specimens from patients older than 4 years demonstrated smaller ganglia connected by thin nerve bundles, compared to larger ganglia and nerve bundles in large bowel. Significant neuronal hypertrophy was found in 55% of acutely inflamed and 41% of histologically classified normal appendix specimens. The myenteric plexus of these appendix specimens had even thicker nerve bundles connecting an increased number of ganglion cells. CONCLUSIONS: Differences in the architecture of the myenteric plexus in patients older than 3 years suggest an altered function and motility of appendix in the early years of life. The significant increase in neuronal components of the myenteric plexus in a high proportion of acutely inflamed and histologically normal appendix specimens is unlikely to have developed during a single acute inflammatory episode. This suggests an underlying chronic abnormality as a secondary response to chronic luminal obstruction or repeated inflammatory episodes in the histologically normal appendix.

Neurogenic appendicopathy: a clinical disease entity?

BACKGROUND AND AIMS: This study compared two histopathological examinations for the diagnosis of neurogenic appendicopathy (NA), assessed the frequency of NA, and evaluated whether it is a clinical disease entity distinct from acute appendicitis. PATIENTS AND METHODS: In a prospective observational multicenter study (surgical departments of five hospitals with one reference pathology) we evaluated 282 patients who underwent appendectomy for suspected appendicitis; we examined the frequency of NA in acute appendicitis and in the negative appendectomy group. For the diagnosis two staining methods were compared. We also attempted to determine clinical features of NA. RESULTS: We observed 93% accuracy for hematoxylin-eosin staining compared with S-100 staining (reference standard) in the diagnosis of NA. There was NA in 3.8% of patients with acute appendicitis and in 47% of those with negative appendectomy. We observed significant differences between the three groups (NA without appendicitis, acute appendicitis, and negative appendectomy without neurogenic appendicopathy) only for sex, age, vomiting, similar previous complaints, rebound tenderness, guarding, rigidity, leukocytes (univariate analysis) and sex (multivariate analysis). CONCLUSION: Neurogenic appendicopathy is a histopathological entity that can be identified by hematoxylin-eosin staining. History and clinical examination do not enable us preoperatively to differentiate between acute appendicitis, NA, and negative appendectomy.

Mast cell density, neuronal hypertrophy and nerve growth factor expression in patients with acute appendicitis.

In acute appendicitis, although the relationship between the enteric nervous system (ENS) and mast cells (MCs) has been described in a few studies, neither the expression of nerve growth factor (NGF) nor its relation to mast cell density (MCD) and ENS has been delineated yet in this disease. The aim of this study was to immunohistochemically investigate the relationship between MCD, nervous system and NGF expression in the appendices of cases with clinically and histopathologically diagnosed acute appendicitis and of normal controls. Twenty-five patients with acute appendicitis and twelve normal controls were included in our study. Mast cell tryptase, PGP 9.5 and anti-NGF immunostained tissue sections were subjected to quantitative image analysis. Our results showed that MCD, the number of Schwann cells, the number and size of ganglia and NGF staining were significantly greater in acute appendicitis than in the control group (p < 0.01). A strong correlation between MCD and NGF staining was detected (r = 0.92) only in cases with acute appendicitis. Similarly MCD was also related to neuronal proliferation and hypertrophy in this group. We failed to detect any relationship between NGF staining and neural components either in the acute appendicitis or control groups. Our findings indicate that mast cells could be one of the important cell populations responsible for nerve proliferation and hypertrophy in acute appendicitis. The relationship between NGF staining and MCD and the lack of correlation between NGF staining and changes in neural components suggest that, in acute appendicitis, NGF might be responsible for the increased number of MCs, but not for neuronal proliferation and hypertrophy.

[Neurogenic appendicopathy: a frequent, almost unknown disease picture. Evaluation of 816 appendices and review of the literature]. / Neurogene Appendicopathie: Ein häufiges, fast unbekanntes Krankheitsbild. Auswertung von 816 Appendices und Literaturübersicht.

BACKGROUND: Neurogenic appendicopathy (NA) represents an almost unknown pathology which clinically cannot be differentiated from acute appendicitis. The diagnosis can only be established histologically. Nerve proliferation and an increased number of endocrine cells are typical for NA. This study characterizes the epidemiology, histology, clinical appearance and therapy of NA. We evaluated the incidence of NA in macroscopically normal specimens from patients presenting the symptoms of acute appendicitis and in incidental appendectomies. PATIENTS AND METHODS: 816 routine appendix specimens were examined at the Institute of Pathology, University of Basel, for the presence of NA. We analyzed the indication for appendectomy, the histological form and the age and sex of the patients. RESULTS: 140 appendices (17.1%) showed the histological criteria for NA. 25% of incidental appendectomies were positive for NA, as opposed to 53% of the macroscopically normal specimens of patients presenting the symptoms of acute appendicitis (P < 0.0001). NA is more frequent in men than in women (19.5% versus 14.5%, P = 0.057). Out of the total of 140 NA, 105 (12.9%) were classified as central, 12 (1.5%) as intramucosal and 5 (0.6%) as submucosal forms of NA. 18 times (2.2%) the histologic subtype of NA was not specified. CONCLUSIONS: This study establishes that NA is a frequent, often asymptomatic pathology. In more than half of the macroscopically normal specimens of patients presenting symptoms of acute appendicitis NA can be diagnosed, significantly more than in incidental appendectomies. Therefore it is imperative to remove and analyze a macroscopically normal appendix in a patient presenting symptoms of acute appendicitis if no other intraabdominal pathology can be found.

Neuroimmune appendicitis.

BACKGROUND: 15-25% of appendices removed from patients with suspected appendicitis appear normal on histological examination. The cause of pain in such patients is unknown. Since the content of neuropeptides seems to be altered in chronic inflammation, we investigated possible changes in peptidergic innervation for substance P (SP), vasoactive intestinal peptide (VIP), and growth-associated protein-43 (GAP-43). METHODS: Appendices classified as showing acute appendicitis, non-acute appendicitis (clinical signs of acute appendicitis, but histologically not inflamed), or normal were processed for SP, VIP, and GAP-43 immunocytochemistry. The density of SP immunostaining was assessed by digitised morphometry. FINDINGS: 31 appendix specimens were studied (16 acute, 15 non-acute). 16 specimens were used as controls. Expression of GAP-43 was increased in the non-acute appendices. We observed larger amounts of SP-immunoreactive and VIP-immunoreactive nerves in the mucosal layer of the appendix in patients with non-acute appendicitis than in controls and patients with acute appendicitis (mean % area SP-immunoreactive 0.0496 [SD 0.0113] non-acute, 0.0221 [0.0049] acute, 0.0229 [0.0068] controls). In addition, a close spatial relation between SP-immunoreactive and VIP-immunoreactive nerve fibres and lymphoid cells was detected in the outer zone of lymph follicles. INTERPRETATION: Neuroproliferation in the appendix, in association with an increase in neurotransmitters SP and VIP, may be involved in the pathophysiology of acute right abdominal pain in the absence of an acute inflammation of the appendix. Our data, together with increasing knowledge about the way in which the nervous system and immune cells interact, suggest that neuroimmune appendicitis is a distinct pathological entity.

Relation of mast cell, nerve and fibrosis in appendix.

The relationship of mast cells, nerves and fibrosis was studied in 50 cases of Appendix using simple histochemical technique. In the mucosa, the mast cell number was greater in the early stages of fibrosis. Both in the mucosa and submucosa mast cells were found closely associated with neural tissue. As the fibrosis increased this association between mast cells and neural tissue was retained only in the submucosa.

Neuronal intestinal dysplasia presenting as an abdominal mass: report of a case.

An unusual clinical presentation of a patient with neuronal intestinal dysplasia is presented. A 46-year-old male noted a palpable mass in the right lower quadrant of his abdomen for two months. A computed axial tomographic scan showed a thickened wall of the cecum with a tumor-like appearance. The excised specimen consisted of a mass caused by the thickened, edematous wall of the dilated cecum and appendix. The wall of the cecum and appendix measured up to 2.5 and 0.8 cm, respectively, in thickness. Microscopic studies showed extensive hyperplasia and hypertrophy of the ganglia and nerve plexuses and hypertrophy of the muscularis propria, consistent with neuronal intestinal dysplasia.

Neural hyperplasia in appendix.

One hundred fifty histologically non-inflamed appendices from patients with acute appendicular pain were studied for mucosal and submucosal neurogenous hyperplasia using simple histochemical stain. Mucosal and submucosal neural hyperplasia was found in 69.34% of cases. The severity of submucosal hyperplasia was more compared to mucosal. The neural hyperplasia of higher grade was found mainly in the age group between 21 to 40 years. The appendicular pain in three unwarranted surgical cases may be either due to neural hyperplasia or substances liberated from closely associated neuroendocrine/mast cells.