Nitrergic Hyperinnervation in Appendicitis and in Appendices Histologically Classified as Normal

Transcription

1 Nitrergic Hyperinnervation in Appendicitis and in Appendices Histologically Classified as Normal Laszlo Nemeth, MD; Udo Rolle, MD; Denis J. Reen, MSc, PhD; Prem Puri, MS, FRCS 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, years), 31 histologically normal appendices removed from patients (age range, years) with suspected appendicitis, and 23 histologically normal appendices from patients (age range, newborn to 12.1 years) undergoing elective abdominal surgery (controls). Wholemount 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. (Arch Pathol Lab Med. 2003;127: ) Appendicitis is the most common surgical emergency in children. It is generally accepted that delay in appendectomy is associated with increased risk of perforation and significant morbidity. 1 The practice of performing appendectomy early for clinically suspected appendicitis has resulted in decreased morbidity, but the price for this approach is a relatively high incidence of histologically normal appendices removed at surgery from patients presenting with signs and symptoms of acute appendicitis. 2,3 Both surgeons and pathologists are aware of the significant proportion of appendices removed for clinical symptoms of appendicitis that show no histologic evidence of inflammation. 4 6 This discrepancy between clinical presentation and the lack of definite morphologic changes is confounding. Since appendectomy relieves symptoms in the vast majority of patients, it is likely that an as yet un- Accepted for publication November 25, From Children s Research Centre, Our Lady s Hospital for Sick Children, University College, Dublin, Ireland. Reprints: Prem Puri, MS, FRCS, FRCS(Ed), Children s Research Centre, Our Lady s Hospital for Sick Children, Crumlin, Dublin 12, Ireland ( ppuri@crumlin.ucd.ie). known causative pathology exists in the appendices of these patients. 7,8 Recently, much attention has been paid to the ways in which the immune and enteric nervous systems interact to regulate the physiological functions of the intestine, including the epithelium and smooth muscle Some authors have suggested that inflammatory reactions might also impair the regular function of local endocrine cells and induce the hyperplasia of the enteric nervous system observed in patients with inflammatory bowel diseases. 13,14 In addition, the expression of neuropeptides appears to be altered under conditions of chronic inflammation of the gastrointestinal tract. 15,16 Some researchers have suggested that inflammatory reactions, which might also involve local endocrine cells and neuroproliferation, may cause chronic or repeated attacks of acute pain. 7,17,18 Recent investigations have provided data that indicate a possible role for neuroimmune interaction in the pathogenesis of chronic and painful inflammation in both animal models and humans. 19,20 It has also been shown that increased expression of substance P, vasoactive intestinal peptide, and growth-associated protein 43 is present in the myenteric and submucosal plexuses of inflamed appen- Arch Pathol Lab Med Vol 127, May 2003 Hyperinnervation in Appendicitis Nemeth et al 573

2 dices. 21 A recent study at our laboratory found evidence of inflammatory pathology in a subgroup within the socalled histologically normal appendices. 22 Nitric oxide, the formation of which is catalyzed by nitric oxide synthase (NOS) from L-arginine, has been recognized as an inhibitory neurotransmitter mediating smooth muscle relaxation in the intestine. Enzymes responsible for nitric oxide synthesis constitute a family with at least 3 distinct isoforms, namely, inducible, endothelial, and neuronal NOS (nnos). Neuronal NOS is the isoform expressed in the myenteric plexus of the gastrointestinal tract. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemical reaction has been widely used to detect NOS, as has detection of NOS by immunohistochemistry. 22 Recently, several studies have investigated NOS distribution in the intestine of patients with motility disorders using NOS immunohistochemistry or NADPH diaphorase histochemistry Gut innervation has a complex 3-dimensional structure, which is difficult to appreciate in thin sections. 26 The whole-mount preparation technique produces a 3-dimensional picture that very elegantly demonstrates the intestinal neuronal network and the relationship of branching and interconnecting nerve fibers to each other and to the neighboring tissues. 24,25,27 This method is extremely useful for morphologic analysis of nerve distribution in the gastrointestinal tract of both healthy subjects and patients with motility disorders. The aim of this study was to investigate the 3-dimensional morphology and distribution of the nitrergic innervation of normal (control), acutely inflamed, and histologically classified normal appendix (HCNA) specimens using a whole-mount preparation technique to test our hypothesis that a proportion of appendices removed from patients with suspected appendicitis have changes in their neuronal plasticity. MATERIALS AND METHODS Appendix Specimens We prospectively selected patients admitted with a clinical diagnosis of acute appendicitis for this study. Each patient had to meet all of the following criteria for acute appendicitis: abdominal pain, local abdominal defense in the right lower abdomen, positive rebound sign, and positive McBurney sign. Elevated axillary and rectal temperature and pathologic white blood cell count ( / L) served as additional parameters. Appendix specimens obtained at surgery were cut and fixed in Zamboni solution (4% formalin and 0.2% picric acid in 0.1mM phosphate-buffered saline [PBS], ph 7.3 [900 mosm]) overnight at 4 C. For elimination of the fixative, the specimens were repeatedly washed in 0.1mM PBS and divided into 4 4-mm pieces. A part of each small specimen was investigated using the standard method of paraffin-embedded conventional sections using hematoxylin-eosin staining. Sections were analyzed to determine the histological grading of appendicitis by 2 independent observers who were unaware of the source of the tissue. Gangrenous and perforated appendices were not included in this study because of major structural damage making the analysis impractical. Acute appendicitis was defined as signs of acute inflammation, including vasodilatation, edema, infiltration of the different layers of the appendix wall with polymorphonuclear leukocytes, ulcers of the epithelium, local abscesses, and fibrinous exudates on the serosa of the appendix. Twenty-eight appendices were randomly selected from the group of patients (age range, years; mean, 8.6 years) with histologically proven appendicitis. Thirty-one appendices from patients (age range, years; mean, 9.6 years) with clinical suspicion of acute appendicitis were classified as histologically normal. Twenty-three histologically normal appendices removed from patients (age range, newborn to 12.1 years; mean, 9.1 years) who underwent elective abdominal surgery (fundoplication for gastroesophageal reflux, bladder augmentation) acted as controls. Large bowel specimens were also collected as normal control tissues at the time of bladder augmentation from an age-matched group of patients. Whole-Mount Preparation Whole-mount preparations were made of each specimen using fine-pointed forceps, microsurgical scissors, and a dissection microscope. Initially, the mucosa-submucosa was removed, followed by separation of muscular fibers from the submucosal layer. Subsequently, the circular muscle layer was peeled off meticulously, fiber by fiber, from the longitudinal muscle layer to which the myenteric plexus is adherent. The separated tissue layers were fixed without stretching with fine-pointed pins on a Sylgard silicone elastomer tray (Dow Corning Europe, La Hulpe, Belgium) with the myenteric plexus on the surface of the longitudinal muscle layer. NADPH Diaphorase Histochemistry of the Whole-Mount Preparation Tissues For histochemical staining with NADPH diaphorase, the specimens were incubated in 1 mg/ml -NADPH (Sigma, Dorset, United Kingdom), 0.25 mg/ml nitroblue tetrazolium (Sigma), and 0.3% Triton-X in 0.05mM Tris-HCl buffer (ph 7.6) at 37 C. When the specimens were observed to have robust staining of the myenteric plexus, they were rinsed in PBS and mounted in Glycergel mounting medium (Dako, Glostrup, Denmark), covered by glass, and investigated by conventional light microscopy. Neuronal NOS Immunofluorohistochemistry For immunofluorohistochemistry, specimens were incubated at 4 C overnight with nnos monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif) with 10% normal calf serum in PBS solution to prevent nonspecific binding. After rinsing twice in PBS, fluorescein isothiocyanate labeled anti-mouse immunoglobulin (Dako) secondary antibody was used for 3 hours. The specimens were rinsed and embedded with fluorescence mounting medium (Dako). Image Analysis The percentage of the area occupied by the myenteric plexus in the microscopic visual field was measured as an objective parameter of neural density, using a complex computerized graphic analyzer system (ALCATEL TITN Answer Immuno 4.0, ALCATEL TITN Answere, Massy Cedex, France). The system was calibrated for a 10 objective, which allowed optimal conditions for a rapid and accurate identification of nerve fibers and neurons. The computerized system analyzes the slides, measuring the area occupied by the stained myenteric plexus as a percentage of the whole investigated microscopic visual field. The myenteric plexuses were measured separately in at least 10 nonoverlapping microscopic fields in each specimen at a 100 magnification. The diameter of nerve bundles was measured by the same system using its special program for microscopic measuring. The results obtained were expressed in 2 values: (1) the percent area of the myenteric plexus scanned from the whole visualized bowel wall and (2) the thickness of the interconnecting nerve fibers between the myenteric ganglia, expressed in micrometers. RESULTS Nitrergic Innervation of Normal Appendix Strong NADPH diaphorase staining of whole-mount preparations was found in the myenteric plexus of each appendix specimen. Strong nnos immunofluorohistochemical staining was also seen in the myenteric plexus of each investigated appendix specimen using the wholemount preparation technique. The myenteric plexus 574 Arch Pathol Lab Med Vol 127, May 2003 Hyperinnervation in Appendicitis Nemeth et al

3 Figure 1. Neuronal nitric oxide synthase (nnos) immunofluorohistochemistry (fluorescein isothiocyanate labeling) of whole mount preparation shows a meshlike neuronal network of normal colon (A) and normal appendix (B) from a newborn. The architecture and density of the myenteric plexus is similar in colon and appendix at this age (nnos, original magnification 100). Figure 2. Density of myenteric plexus measured by computerized image analyzer. Myenteric plexus density is given as a percentage of the investigated visual field occupied by the myenteric plexus of normal colon and normal appendix. formed a 3-dimensional meshlike neuronal network of neurons and nerve fibers. The density of the myenteric plexus in appendices was high in the newborn period, and this density was similar to that found in colon from the same patients (Figure 1). Neuronal density decreased with age in both the appendix and colon specimens. This decreasing density with age was most apparent in the appendix during the first 3 years of life (Figure 2). The myenteric plexus of appendices after the age of 3 years covered only 20% of the whole investigated area, but the myenteric plexus of colon covered around 40% of the bowel surface between the 2 muscle layers (Figure 2). The myenteric plexus of appendices in children older than 3 years had thinner nerve branches and smaller ganglia than the colon specimens of the same age. Nitrergic Innervation in Acutely Inflamed and HCNA Specimens Strong NADPH diaphorase and nnos staining was found in all appendix specimens (control, acutely inflamed, and HCNA) in the myenteric plexus. In HCNA appendix specimens, the myenteric plexus showed the typical 3-dimensional meshlike neuronal network. In normal control appendix specimens, the myenteric plexuses showed relatively thin branches and networks, because all of the children were older than 3 years in this control group (Figure 3, A). Two groups of patients were identified within both the acutely inflamed and HCNA specimens with respect to myenteric plexus density. One group (46.5% [13/28] of acutely inflamed appendices and 58% [18/31] of 31 HCNAs) showed architecture and neuronal density similar to that of the control specimens. A second group of acutely inflamed appendices (Figure 3, B) and HCNA specimens (Figure 3, C) demonstrated thick nerve bundles and a high-density neuronal network (53.5% [15/28] of acutely inflamed appendices and 42% [13/31] of HCNAs) (Figure 4). Nerve hypertrophy was present throughout the affected specimens in both acutely inflamed and HCNA specimens, and was also seen in the submucosal plexus of the appendices (Figure 5). Nerve fiber thickness was measured in normal control appendices and in the hypertrophied groups (area of myenteric plexus greater than 25%) of the acutely inflamed and HCNA specimens. These results are summarized in Figure 5. In control appendix specimens, the average thickness of the measured nerve fibers (interconnecting the ganglions) was 58.4 m. The diameter of the hypertrophied group appendices in acutely inflamed appendi- Arch Pathol Lab Med Vol 127, May 2003 Hyperinnervation in Appendicitis Nemeth et al 575

4 Figure 3. NADPH diaphorase enzyme histochemical staining of whole-mount preparation from a 10-year-old patient. Myenteric plexus of normal appendix (control, A), nitrergic hyperinnervation in acutely inflamed appendix (B), and nitrergic hyperinnervation in histologically classified normal appendix of a symptomatic patient (C) (original magnification 40). Figure 4. NADPH diaphorase enzyme histochemical staining of whole-mount preparation from a 10-year-old patient. Submucosal plexus of normal appendix (control, A) and nitrergic hyperinnervation in histologically classified normal appendix (B) of a symptomatic patient (original magnification 40). ces and HCNA specimens was 96.7 m and m, respectively (Figure 6). COMMENT The existence and distribution pattern of nerve fibers in human appendix has been demonstrated in previous studies. 8,28 However, using a new 3-dimensional morphologic technique, the whole-mount method, combined with computerized image analysis, the present study demonstrates significant differences in total innervation of human appendix in different age groups and in different inflammatory conditions. Any study describing changes in mor- 576 Arch Pathol Lab Med Vol 127, May 2003 Hyperinnervation in Appendicitis Nemeth et al

5 Figure 5. Area of myenteric plexus measured by computerized image analysis system. Myenteric plexus density is given as a percentage of the investigated visual field occupied by the myenteric plexus in normal (control), acutely inflamed (AI), and histologically classified normal appendices (HCNA). Figure 6. Diameter of nerve bundles ( m) measured by computerized analysis system in normal (control), acutely inflamed (AI) appendices showing nitrergic hyperinnervation, and histologically classified normal appendices (HCNA) with nitrergic hyperinnervation. phologic features, such as nerve fiber density, has to be based on the definition of its pattern in the normal organ. 7,24,25 Histologically, the intestinal and the appendix wall are usually examined on traditional sections vertical to the wall. This approach provides only a transsectional view of the myenteric plexus, which in reality is a flat structure extending in the intermuscular septum along a plane parallel to the intestinal wall. To visualize the overall structure of the 3-dimensional meshlike network of myenteric plexus, we must use another angle, which we call the flat view, that allows us to understand into what network system the plexus is integrated. We can obtain this view only by a 3-dimensional preparation technique, such as the whole-mount technique used by several investigators to examine normal bowel wall and motility disorders. 23,25,27 Our study started from the clinical observation that appendectomy removes the pain experienced by patients whose removed appendices showed no classical signs of acute inflammation. 7,8 To establish the basic pattern of innervation, we choose patients who underwent elective abdominal surgery, assuming that these individuals had not suffered abdominal pain in the recent past. All other patients in our study suffered from acute abdominal pain. The enteric nervous system contains a large number of neurons organized in intricate neuronal circuits. The morphology of the gut wall varies along different regions of the gut, and these variations are related to the different regional functions of the gut. 26,29 The 5 primary targets of the enteric nervous system are smooth muscle cells, mucosal secretory cells, gastrointestinal neuroendocrine cells, the gastrointestinal microvasculature that maintains mucosal blood flow during intestinal secretion, and inflammatory cells of the gut that are involved in mucosal immunologic, allergic, and inflammatory responses. 30 Enteric ganglia can maintain integrated functions, such as the peristaltic reflex, in the absence of input from the central nervous system, which has a modulatory role. Several clinical and experimental observations suggest that homeostatic control of gut function in a changing environment may be achieved through adaptive changes occurring in the enteric ganglia. 9 12,17 A recent study in infants and children indicated that the density of ganglion cells in the myenteric plexus decreases significantly with age during the first 3 to 4 years of life. 24,25 This finding supports the notion that development is an ongoing process in the first years of life. We found that the morphology of the primary myenteric plexus meshwork, when stained with NADPH diaphorase and nnos, was very similar in colon and appendix in the newborn period. As several investigators have already described, we noted decreasing density of the myenteric plexus in the first years of life. We found similar changes in the myenteric plexus of normal colon, but in the appendices, this process was more intensive, resulting a less dense myenteric plexus with thinner nerve branches and fewer numbers of ganglion cells at 3 years of age. Several possibilities could explain the decreasing density of the myenteric plexus of appendices. Growth of the bowel with increasing surface area probably explains part of decreasing neuron density. The reduction of cell number may occur by apoptosis, which is a normal means of correction of errors in nervous system development and of removal of redundant neurons. The decrease in myenteric plexus density might be an aspect of a process of reshaping, not necessarily associated with malfunction, which is triggered simply to maintain homeostasis in a different microenvironment. The diagnosis of acute appendicitis is still difficult, and very probably this is the most common abdominal diagnostic problem in general medical practice. It has been estimated that up to 12% of the population of Western countries will suffer an attack of acute appendicitis at some time during their lives, and appendectomy accounts for 1% to 2% of all surgical procedures. 1,6,21,31 These data alone indicate that determining the etiology and pathophysiology of appendicitis is of more than merely academic interest. Despite advances in fluid replacement techniques and antibiotic therapies, appendicitis is still associated with significant morbidity. Because of the high rate of complications associated with perforation, a general feeling has developed in the surgical world that in the vast majority of patients who have appendicitis, the appendix must be surgically removed or the appendix will go on to rupture. This attitude results in early appendectomy in patients with clinical symptoms of appendicitis. The price for decreased morbidity is the relatively high incidence of histologically normal appendices removed at surgery from patients presenting with signs and symptoms of appendicitis. Since appendectomy relieves symptoms in the vast Arch Pathol Lab Med Vol 127, May 2003 Hyperinnervation in Appendicitis Nemeth et al 577

6 majority of these patients, it is likely that an as yet unidentified causative pathology exists in the appendices of these patients. There has been increasing evidence for a functional role of the enteric nervous system in inflammatory bowel diseases. 26 Some authors have suggested that inflammatory reactions might also impair the regular function of local endocrine cells and induce hyperplasia of the enteric nervous system. 11,30,32,33 Changes in the pattern of innervation and morphology of nerve fibers have been observed in the enteric nervous system of patients who suffer from inflammatory bowel diseases like Crohn disease. 9,10,12 14 Recent investigations have provided data that indicate a possible role of neuroimmune interaction in the pathogenesis of chronic and painful inflammation both in animal models and humans. 20,34,35 Some researchers suggest that inflammatory reactions, which might also involve local endocrine cells and neuroproliferation, may cause chronic or repeated attacks of acute pain. 15,17,21 Furthermore, the involvement of some classes of neuropeptides, such as substance P and vasoactive intestinal peptide, the expression of which seems to be changed in inflammatory conditions, including inflammatory bowel disease, is well known. 9,12,21 Increased expression of COX-2, major histocompatibility complex class II, and inducible NOS in a subgroup of histologically normal appendices has been shown, which suggests an inflammatory response in these cases. 22 These results are similar to pathologic changes with inflammatory bowel diseases. Some authors, using protein gene product 9.5 as a general neuronal marker, have described a significant increase of nerve fibers staining in the mucosa of nonacutely inflamed appendices. 36 The existence and distribution pattern of nerve fibers in the human appendix have been previously demonstrated in retrospective studies based on subjective analysis of pathologic specimens. 2,21,31,34 By use of image analysis, this prospective study demonstrates significant differences in the innervation pattern of the human appendix using whole-mount methodology. The analysis of a subgroup of appendices by digitized morphometry confirmed the nitrergic upregulation in both acute and nonacute appendicitis. Similar changes in the pattern of innervation and morphology of nerve fibers have been observed in the enteric nervous system of patients who suffer from inflammatory bowel diseases like Crohn disease. 37 Overall, this study provides further insights into the pathogenesis of appendicitis, whereby a major subgroup of appendices classified as histologically normal by conventional methodology was shown to have significant neuronal hypertrophy, similar to that of acutely inflamed appendices. While this study does not directly address the issue of causative pain in children presenting with appendicitis, it does provide evidence for a neuropathology in many patients with what are currently classified as normal appendices. This study was supported in part by Hungarian Scientific Research grant OTKA T (Budapest). References 1. Miettinen P, Pasanen P, Lahtinen J, et al. The long-term outcome after negative appendix operation. Ann Chir Gynaecol. 1995;84: Falk S, Schutze U, Guth H, et al. 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