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Morphologic pattern of myenteric neural plexus in colonic diverticular disease. A whole-mount study employing histochemical staining for acetylcholinesterase
ARTICLE IN PRESS Ann Anat 190 (2008) 525—530
www.elsevier.de/aanat
RESEARCH ARTICLE
Morphologic pattern of myenteric neural plexus in colonic diverticular disease. A whole-mount study employing histochemical staining for acetylcholinesterase Olegas Deduchovasa, Zilvinas Saladzinskasa, Algimantas Tamelisa, Dainius Pavalkisa, Neringa Pauzieneb, Dainius H. Pauzab, a
Department of General Surgery, Hospital of Kaunas University of Medicine, Eiveniu Street 2, Kaunas LT-50009, Lithuania b Institute of Anatomy, Kaunas University of Medicine, A. Mickeviciaus Street 9, Kaunas LT-44307, Lithuania Received 11 January 2008; received in revised form 6 September 2008; accepted 15 September 2008
KEYWORDS Diverticular disease; Human; Morphology; Myenteric neural plexus; Sigmoid colon
Summary Background: Diverticular disease (DD) of the colon is a frequent clinical problem because 30–50% of the population over the age of 60 years in western communities are affected by DD. Although certain clinical, physiological and biochemical studies have shown that the origin of DD may be neurogenic, the mechanism of DD pathogenesis is still not clear. Methods: The aim of the present study has been to assess the morphologic pattern of the myenteric nerve plexus (MNP) in diverticulous sigmoid colon (DSC) comparing the structural organization in DSC (n ¼ 10) to relatively normal sigmoid colon (rNSC) that had been resected from patients for rectal tumors (n ¼ 10). The histochemical method for acetylcholinesterase was utilized to visualize the MNP on pressure bloated, non-sectioned gut preparations. Results: The study revealed that the MNP of DSC was degenerated, as its interganglionic nerves were periodically interrupted and thinner than in rNSC. The number of myenteric ganglia in same-sized areas (125 mm2) as well as the average area of myenteric plexus was significantly higher in controls compared with the DD patients, (respectively, ganglion number: 163712 and 149712, po0.02; MN-plexal area: 8.170.3 mm2 and 7.270.2 mm2, po0.001).
Corresponding author. Tel.: +370 37 327313, Mobile: +370 682 39366; fax: +370 37 220733.
E-mail address: [email protected] (D.H. Pauza). 0940-9602/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.aanat.2008.09.002
ARTICLE IN PRESS 526
O. Deduchovas et al. Conclusion: The occurrence of DD in sigmoid colon is associated with morphologic alterations in MNP (i.e. the number of ganglia and plexus rarefaction, ganglion size and plexal area involution), which presumably demonstrate the failure of MNP in DD patients. & 2008 Elsevier GmbH. All rights reserved.
Introduction Both the trigger and genesis of diverticular disease (DD) in colon is unknown so far. Statistically, there is apparently a causative association between DD and low intake of dietary cereal fiber, low stool bulk and subsequent high colonic intraluminal pressure, which presumably forces the mucosa through the full thickness of the bowel wall (Painter and Burkitt, 1971). Another traditional model of DD pathogenesis suggests elastosis of the taeniae coli as the primary event (Whiteway and Morson, 1985a), causing shortening of the sigmoid colon, with relative mucosal excess and subsequent mucosal herniations. Further, a western-type diet is implicated in the increased uptake of proline from the gut, leading to elastosis of the sigmoid colon (Ludeman et al., 2002). These models, however, do not explain why the DD mainly affects the right colon in eastern countries and the sigmoid colon in western countries (Sugihara et al., 1984; Golder et al., 2003; Yap and Hoe, 1991; Rajendra and Ho, 2005). Some recent physiological, pharmacological and histological studies have revealed that abnormalities in structure and/or function of enteric nervous system (ENS) may be primary in the pathogenesis of DD (Golder et al., 2003; Iwase et al., 2005). Therefore, studies on mechanisms of the DD development are still of pivotal clinical importance. The ENS is possibly the most complex part of the peripheral nervous system. It has been estimated that the human ENS contains approximately 100 million neurons, which means as many as in the spinal cord (Grundy and Schemann, 2005). Most of the enteric neurons are organized into two ganglionated intramural plexuses: the myenteric (Auerbach’s), and the submucosal (Meissner’s). Myenteric neurons innervate the longitudinal and circular smooth muscle layers, adjacent myenteric ganglia, to some extent the submucous ganglia, mucosa, prevertebral ganglia, and even the pancreas (Grundy and Schemann, 2005). The predominant neurotransmitter of the ENS is acetylcholine which mediates contraction of bowel smooth muscles via postsynaptic muscarinic M3 receptors (Burleigh, 1990; Porter et al., 1996).
The density of nerve fibers in the bowel is reduced with age and the incidence of DD increases concomitantly (Gomes et al., 1997; Phillips and Powley, 2007; Whiteway and Morson, 1985b). However, in-vitro studies on smooth bowel muscles have shown that cholinergic activity increases in DD (Huizinga et al., 1999; Kerr et al., 1995; Tomita et al., 1999). It is presumed that this hyperactivity could be secondary to amplified expression of diverticulous bowel-muscle M3 receptors that could be a compensatory response to a reduction in cholinergic innervation and a mechanism by which smooth-muscle cells increase their sensitivity to acetylcholine, in an attempt to maintain intrinsic background tone (Golder et al., 2003). The aim of the present study has been to assess the morphologic pattern of the myenteric (Auerbach’s) nerve plexus (MNP) in the diverticulous sigmoid colon (DSC) regions and to compare it with the MNP in relatively normal sigma resected from patients operated on for rectal tumors in order to determine whether the DD is associated with a structural remodeling of this nerve plexus.
Materials and methods Segments of sigmoid colon (10–15 cm from the rectosigmoid junction) about 10 cm in length were taken from 10 patients operated on for chronic DD and 10 controls (after resections for rectal tumors). The local bioethical committee (Kaunas Region Bioethical Committee) approved the study. The same coloproctological team operated all the patients from October 2004 till April 2006. We excluded patients if they had received preoperative radiotherapy, had inflammatory bowel disease, or had symptoms of diverticulitis (Golder et al., 2003) or preoperative bowel obstruction. Six women and four men underwent surgery for chronic DD (mean age 61 (range 46–73) years). The remaining five men and five women (mean age 66 (range 56–75) years) were used as controls. The major criterion for patient assignment to the DD group was the presence of multiple diverticula (over 12) in contrast to the relatively normal sigmoid colon (rNSC) group, in which patients
ARTICLE IN PRESS Myenteric neural plexus in diverticular disease
527 medium described by Karnovsky and Roots (1964). The composition of the incubation medium (pH 5.6) was (in mM): sodium acetate, 60; acetylthiocholine iodide (FLUKA), 2; sodium citrate, 15; CuSO4, 3; K3 Fe(CN)6, 0.5. Triton-X 100 supplemented the incubation medium up to 1% and hyaluronidase (0.5 mg/100 ml). Since the remaining fat patches on the gut surface were almost non-permeable for the incubation media, an additional precise dissection of those fat pads was usually performed to expose the neural structures located beneath. Following staining, the preparations were fixed in neutral 4% formalin in 0.1 M phosphate buffer. The preparations were stored in the same formalin without noticeable alterations for months.
Figure 1. Gross anatomical view of the DSC resected from a 46 years old woman operated on for DD. The handled preparation is dissected out of fatty connective tissue at the tenia mesocolica, sewn at both ends and pressure-bloated by a hand-made rubber bladder placed inside the gut lumen and filled by isotonic saline. Black arrows point to some diverticula. Asterisks indicate the Taenia coli.
lacked any diverticula. Every patient gave written informed consent before participation in the study. Resection after 1–2 h, the resected sigmoid colon specimens were transported to an anatomical laboratory, where they were cleansed of blood with isotonic saline (pH 7.3), and dissected from the fatty connective tissue at the Tenia mesocolica. Further, samples were sewn on both ends and pressure-bloated by a hand-made rubber bladder placed inside the gut lumen and filled with isotonic saline, so that the gut walls were uniformly distended at the same pressure, which was controlled using a membranous manometer connected to the rubber bladder in T-joint manner (Figure 1). We did not assess samples with scar tissue that could have originated from previous diverticulitis. Pressure-bloated samples were fixed by immersion for 30 min in 4% paraformaldehyde solution in 0.1 M phosphate buffer at room temperature. Following fixation, the preparations were washed for 1 h at 4 1C in isotonic saline containing hyaluronidase (0.005 mg ml1, FLUKA).
Staining of the MNP Enzyme histochemistry for acetylcholinesterase was used to stain the myenteric neural plexus in total gut samples as this enzyme occurs nonspecifically within intrinsic nerves and ganglia: parasympathetic, sympathetic and sensory ones (Pauza et al., 2000). The hyaluronidase-treated samples were incubated for 1–3 h at 4 1C in the
Microscopy of the MNP Total gut preparations were placed in distilled water and examined in a transient light from a fiber optic illuminator using a stereomicroscope (Stemi 2000 CS, ZEISS) at a magnification of 16–56 . Stereomicroscopically visible intrinsic nerves and ganglia were photographed using a digital camera (AxioCam MRc5, ZEISS). Stereomicroscopic pictures were taken from every sample consistently from all walls of a preparation excluding Tenia coli regions. Only images without diverticulae were analyzed for the DD group. The thickness of the intramural nerves and the density of the myenteric ganglia in the DD group versus controls were assessed on randomly selected (choosing every fifth) images using the AxioVision software (v. 4.5; ZEISS, Germany).
Quantitative analysis of the MNP The plexal area of the MNP and the density of myenteric ganglia stained for AChE were evaluated in same-sized areas by counting only those ganglia that were well stained and well observable (Figure 2). Plexal area is defined as the area occupied by MNP on a digital image of 125 mm2. In order to reliably assess the MNP morphology, three random images each from 10 samples in the DD group (n ¼ 30) and three of each from 10 controls (n ¼ 30) were analyzed.
Statistical analysis Results shown in the text, tables and graphs are expressed as mean plus/minus standard error. A test of the statistical significance of the difference between the means was performed with Student’s independent tests (Microcal Origin v. 4.00). Linear
ARTICLE IN PRESS 528
O. Deduchovas et al. Table 1. MNP area (mm2) and ganglion number in randomly selected three areas of 125 mm2 from diverticulated sigmoid colon (DSC) group (n ¼ 10) versus relatively normal sigmoid colon (rNSC) group (n ¼ 10). MN-plexal area (mm2)
Ganglion number
DSC
rNSC
DSC
rNSC
7,570,3 7,270,1 7,670,4 6,870,5 6,670,1 7,570,2 6,970,1 7,570,2 7,270,1 7,670,3
8.070,5 8,370,1 8,370,2 7,870,1 7,670,3 8,070,3 8,170,2 8,370,4 8,270,3 8,170,2
145715 160726 14178 16276 13478 155716 13376 142715 15278 162711
162712 16679 146716 180717 166713 155711 151722 17076 17379 16178
In average 7,270,1
8,170,1
149712
163712
Differences of means are significant at po0,001 (MN-plexal area (mm2)) and po0,02 (Ganglion number).
Figure 2. Stereo-microphotographs of the myenteric nerve plexus (MNP) stained for acetylcholinesterase from whole (non-sectioned) diverticulous (A and C) and rNSCs (B and D). The boxed areas (E) in panel A and (F) in panel B are enlarged in panels E and F, respectively. The arrowheads in E and F indicate some intensively stained ganglionic cells. Note that MNP from DSC (A and C) is extensively atrophied, because its interganglionic nerves are periodically interrupted and obviously thinner (white arrows) in comparison to the rNSC (B and D). The density of the ganglia is obviously higher in the rNSC (B and D) than in diverticulous one (A and C). Black arrows in all panels indicate the well-developed interganglionic nerves in rNSC. Arrowheads in A–D panels point into some ganglia with well-stained ganglionic cells. Abbreviations: DV-diverticula.
area the number of myenteric ganglia was also significantly (po0.001) higher in rNSC group compared to the DSC group (Table 1). The interganglionic nerves of ganglionated MNP close to diverticula were thinner than those located distant to diverticula. No neural structures (either myenteric or submucosal ones) were identified on diverticula in any examined case of DSC (Figure 2A). The largest diverticulae were mostly developed in fatty tissues in the region of the Taenia mesocolica. We found no significant correlation between the age of patients and the ganglion number and the area of the MNP (Figure 3). We also did not observe a relationship between the character of diverticula and the patient age.
Discussion regression was used to quantitatively determine the relationship between the MNP ganglion number and its area and patient’s age. Significance was accepted at po0,05.
Results In contrast to that of the rNSC group, the MNP in the DSC group was degenerated: interganglionic nerves were periodically interrupted and thinner than in rNSC (Figure 2). On an average, the number of myenteric ganglia per same-sized area was significantly (po0.02) higher in the rNSC group compared to the DSC patients. In the MN-plexal
The present findings are the first demonstration of the morphology of MNP on a total (nonsectioned) sigmoid colon from patients with DD. The technique used in this study allowed for assessment of the MNP in situ on large bowel areas highlighting the stereomicroscopic alterations in the DSC compared to controls: a substantial decrease in the number and size of myenteric ganglia as well as a rarefaction of the myenteric plexus. Several recent reports indicate that the origin of DD may be neurogenic (Bassotti et al., 2005; Golder et al., 2003; Iwase et al., 2005; Spiller, 2006; Stoss and Meier-Ruge, 1994; Tomita et al., 1999; Vuong
ARTICLE IN PRESS Myenteric neural plexus in diverticular disease
529
Figure 3. Graph demonstrating the absence of correlation between patient age and myenteric ganglion number (on left) and MNP area (on right) counted in three areas of 125 mm2 selected at random from samples of the diverticulous sigmoid colon group (DSC) versus the relatively normal sigmoid colon group (rNSC). Solid and dotted lines show correlation in DSC and rNSC groups, respectively.
et al., 1985). In spite of this, there are very few studies dealing with a morphologic pattern of MNP in DSC (Iwase et al., 2005; Vuong et al., 1985). Data of these reports, however, are fairly controversial. In an immunohistochemical study, Iwase et al. (2005) have shown differences in MNP morphology between DD patients and controls (operated on rectal tumors). The latter authors found that the sections of the diverticulous colon contains (1) significantly more plexuses (i.e. a denser neural network), (2) fewer ganglion cells and (3) a nuclear area of ganglion cells which appears to be smaller in DD patients than in controls. Vuong et al. (1985), using H&E staining and silver impregnation, did not reveal any morphological abnormalities of the MNP in patients with sigmoid DD compared to controls. Bassotti et al. (2005), using an immunohistochemical assay in sectioned specimens, also did not find significant differences in the number of neurons in DD patients with respect to controls. The present method, which aims to investigate the morphology of MNP in non-sectioned diverticulous sigma preparations, has never been used before the present study. Presumably, application of distinct morphologic techniques should be considered as the main reason for the discrepancies between findings of the above-mentioned and present studies. It seems that the whole-mount technique with histochemical staining for acetylcholinesterase used according to the prolonged protocol of the present study should demonstrate the most complete pattern of the MNP. However, this is correct only if the activity of AChE was not missing in the assessed samples of
the DD patients. On the other hand, the lack or reduction up to undetectable amount of general neural markers, immunohistochemistry of which was used to analyze samples from the DSC in studies by other authors, could be also fairly possible. Therefore, the morphologic alterations of MNP in DD patients revealed in the present study should be taken into account and verified by other sensitive methods. The atrophied MNP in DSC that we demonstrate here could be related to the age of patients, because it is known that the density of nerves in the bowel is reduced at high age and the incidence of DD increases with age (Gomes et al., 1997; Phillips and Powley, 2007). Nonetheless, the mean age of the examined patients in the DSC group was similar to the rNSC group or was even younger than in controls. We found no significant correlation between the age of patients and the ganglion number and the area of MNP. We also did not observe the relationship between the character of diverticula and patient age. Thereby, we presume that the influence of age is not pivotal for alterations of the MNP in the examined DD. Besides, we did not find any relationship between the character of diverticula (their size, quantity) and the morphology of the MNP. At the present moment, therefore, we are unable to explain how the demonstrated atrophy of the MNP could be related to the manifestation of DD, whether our findings are primary or secondary abnormalities of DD and how these alterations influence the diverticula’s development. However, the presented
ARTICLE IN PRESS 530 findings strongly imply that the occurrence of DD in sigmoid colon is associated with morphologic alterations in the MNP that may be considered as failure of the MNP in DD. The present observations deserve to be further examined immunohistochemically and electron-microscopically in order to determine the potential role of the MNP in pathogenesis of this digestive tract disease, because results of such investigations may modify both the prophylaxis and treatment of the colonic DD.
Acknowledgements This work was supported by grants from the Lithuanian State Science and Studies Foundation (T-76/07) as well as from the Science Foundation of Kaunas University of Medicine (PAR 18).
References Bassotti, G., Battaglia, E., Bellone, G., Dughera, L., Fisogni, S., Zambelli, C., Morelli, A., Mioli, P., Emanuelli, G., Villanacci, V., 2005. Interstitial cells of Cajal, enteric nerves, and glial cells in colonic diverticular disease. J. Clin. Pathol. 58, 973–977. Burleigh, D.E., 1990. Motor responsiveness of proximal and distal human colonic muscle layers to acetylcholine, noradrenaline, and vasoactive intestinal peptide. Dig. Dis. Sci. 35, 617–621. Golder, M., Burleigh, D.E., Belai, A., Ghali, L., Ashby, D., Lunniss, P.J., Navsaria, H.A., Williams, N.S., 2003. Smooth muscle cholinergic denervation hypersensitivity in diverticular disease. Lancet 361, 1945–1951. Gomes, O.A., de Souza, R.R., Liberti, E.A., 1997. A preliminary investigation of the effects of aging on the nerve cell number in the myenteric ganglia of the human colon. Gerontology 43, 210–217. Grundy, D., Schemann, M., 2005. Enteric nervous system. Curr. Opin. Gastroenterol. 21, 176–182. Huizinga, J.D., Waterfall, W.E., Stern, H.S., 1999. Abnormal response to cholinergic stimulation in the circular muscle layer of the human colon in diverticular disease. Scand. J. Gastroenterol. 34, 683–688. Iwase, H., Sadahiro, S., Mukoyama, S., Makuuchi, H., Yasuda, M., 2005. Morphology of myenteric plexuses in the human large intestine: comparison between large intestines with and without colonic diverticula. J. Clin. Gastroenterol. 39, 674–678.
O. Deduchovas et al. Karnovsky, M.J., Roots, L., 1964. A ‘‘direct-coloring’’ thiocholine method for cholinesterases. J. Histochem. Cytochem. 12, 219–221. Kerr, P.M., Hillier, K., Wallis, R.M., Garland, C.J., 1995. Characterization of muscarinic receptors mediating contractions of circular and longitudinal muscle of human isolated colon. Br. J. Pharmacol. 115, 1518–1524. Ludeman, L., Warren, B.F., Shepherd, N.A., 2002. The pathology of diverticular disease. Best Pract. Res. Clin. Gastroenterol. 16, 543–562. Painter, N.S., Burkitt, D.P., 1971. Diverticular disease of the colon: a deficiency disease of Western civilization. Br. Med. J. 2, 450–454. Pauza, D.H., Skripka, V., Pauziene, N., Stropus, R., 2000. Morphology, distribution, and variability of the epicardiac neural ganglionated subplexuses in the human heart. Anat. Rec. 259, 353–382. Phillips, R.J., Powley, T.L., 2007. Innervation of the gastrointestinal tract: patterns of aging. Auton. Neurosci. 136, 1–19. Porter, A.J., Wattchow, D.A., Brookes, S.J., Schemann, M., Costa, M., 1996. Choline acetyltransferase immunoreactivity in the human small and large intestine. Gastroenterology 111, 401–408. Rajendra, S., Ho, J.J., 2005. Colonic diverticular disease in a multiracial Asian patient population has an ethnic predilection. Eur. J. Gastroenterol. Hepatol. 17, 871–875. Spiller, R., 2006. How inflammation changes neuromuscular function and its relevance to symptoms in diverticular disease. J. Clin. Gastroenterol. 40, S117–S120. Stoss, F., Meier-Ruge, W., 1994. Experience with neuronal intestinal dysplasia (NID) in adults. Eur. J. Pediatr. Surg. 4, 298–302. Sugihara, K., Muto, T., Morioka, Y., Asano, A., Yamamoto, T., 1984. Diverticular disease of the colon in Japan. A review of 615 cases. Dis. Colon. Rectum 27, 531–537. Tomita, R., Tanjoh, K., Fujisaki, S., Fukuzawa, M., 1999. Physiological studies on nitric oxide in the right sided colon of patients with diverticular disease. Hepatogastroenterology 46, 2839–2844. Vuong, N.P., Sezeur, A., Balaton, A., Malafosse, M., Camilleri, J.P., 1985. [Myenteric plexuses and colonic diverticulosis: results of a histological study]. Gastroenterol. Clin. Biol. 9, 434–436. Whiteway, J., Morson, B.C., 1985a. Elastosis in diverticular disease of the sigmoid colon. Gut 26, 258–266. Whiteway, J., Morson, B.C., 1985b. Pathology of the ageing—diverticular disease. Clin. Gastroenterol. 14, 829–846. Yap, I., Hoe, J., 1991. A radiological survey of diverticulosis in Singapore. Singapore Med. J. 32, 218–220.
www.elsevier.de/aanat
RESEARCH ARTICLE
Morphologic pattern of myenteric neural plexus in colonic diverticular disease. A whole-mount study employing histochemical staining for acetylcholinesterase Olegas Deduchovasa, Zilvinas Saladzinskasa, Algimantas Tamelisa, Dainius Pavalkisa, Neringa Pauzieneb, Dainius H. Pauzab, a
Department of General Surgery, Hospital of Kaunas University of Medicine, Eiveniu Street 2, Kaunas LT-50009, Lithuania b Institute of Anatomy, Kaunas University of Medicine, A. Mickeviciaus Street 9, Kaunas LT-44307, Lithuania Received 11 January 2008; received in revised form 6 September 2008; accepted 15 September 2008
KEYWORDS Diverticular disease; Human; Morphology; Myenteric neural plexus; Sigmoid colon
Summary Background: Diverticular disease (DD) of the colon is a frequent clinical problem because 30–50% of the population over the age of 60 years in western communities are affected by DD. Although certain clinical, physiological and biochemical studies have shown that the origin of DD may be neurogenic, the mechanism of DD pathogenesis is still not clear. Methods: The aim of the present study has been to assess the morphologic pattern of the myenteric nerve plexus (MNP) in diverticulous sigmoid colon (DSC) comparing the structural organization in DSC (n ¼ 10) to relatively normal sigmoid colon (rNSC) that had been resected from patients for rectal tumors (n ¼ 10). The histochemical method for acetylcholinesterase was utilized to visualize the MNP on pressure bloated, non-sectioned gut preparations. Results: The study revealed that the MNP of DSC was degenerated, as its interganglionic nerves were periodically interrupted and thinner than in rNSC. The number of myenteric ganglia in same-sized areas (125 mm2) as well as the average area of myenteric plexus was significantly higher in controls compared with the DD patients, (respectively, ganglion number: 163712 and 149712, po0.02; MN-plexal area: 8.170.3 mm2 and 7.270.2 mm2, po0.001).
Corresponding author. Tel.: +370 37 327313, Mobile: +370 682 39366; fax: +370 37 220733.
E-mail address: [email protected] (D.H. Pauza). 0940-9602/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.aanat.2008.09.002
ARTICLE IN PRESS 526
O. Deduchovas et al. Conclusion: The occurrence of DD in sigmoid colon is associated with morphologic alterations in MNP (i.e. the number of ganglia and plexus rarefaction, ganglion size and plexal area involution), which presumably demonstrate the failure of MNP in DD patients. & 2008 Elsevier GmbH. All rights reserved.
Introduction Both the trigger and genesis of diverticular disease (DD) in colon is unknown so far. Statistically, there is apparently a causative association between DD and low intake of dietary cereal fiber, low stool bulk and subsequent high colonic intraluminal pressure, which presumably forces the mucosa through the full thickness of the bowel wall (Painter and Burkitt, 1971). Another traditional model of DD pathogenesis suggests elastosis of the taeniae coli as the primary event (Whiteway and Morson, 1985a), causing shortening of the sigmoid colon, with relative mucosal excess and subsequent mucosal herniations. Further, a western-type diet is implicated in the increased uptake of proline from the gut, leading to elastosis of the sigmoid colon (Ludeman et al., 2002). These models, however, do not explain why the DD mainly affects the right colon in eastern countries and the sigmoid colon in western countries (Sugihara et al., 1984; Golder et al., 2003; Yap and Hoe, 1991; Rajendra and Ho, 2005). Some recent physiological, pharmacological and histological studies have revealed that abnormalities in structure and/or function of enteric nervous system (ENS) may be primary in the pathogenesis of DD (Golder et al., 2003; Iwase et al., 2005). Therefore, studies on mechanisms of the DD development are still of pivotal clinical importance. The ENS is possibly the most complex part of the peripheral nervous system. It has been estimated that the human ENS contains approximately 100 million neurons, which means as many as in the spinal cord (Grundy and Schemann, 2005). Most of the enteric neurons are organized into two ganglionated intramural plexuses: the myenteric (Auerbach’s), and the submucosal (Meissner’s). Myenteric neurons innervate the longitudinal and circular smooth muscle layers, adjacent myenteric ganglia, to some extent the submucous ganglia, mucosa, prevertebral ganglia, and even the pancreas (Grundy and Schemann, 2005). The predominant neurotransmitter of the ENS is acetylcholine which mediates contraction of bowel smooth muscles via postsynaptic muscarinic M3 receptors (Burleigh, 1990; Porter et al., 1996).
The density of nerve fibers in the bowel is reduced with age and the incidence of DD increases concomitantly (Gomes et al., 1997; Phillips and Powley, 2007; Whiteway and Morson, 1985b). However, in-vitro studies on smooth bowel muscles have shown that cholinergic activity increases in DD (Huizinga et al., 1999; Kerr et al., 1995; Tomita et al., 1999). It is presumed that this hyperactivity could be secondary to amplified expression of diverticulous bowel-muscle M3 receptors that could be a compensatory response to a reduction in cholinergic innervation and a mechanism by which smooth-muscle cells increase their sensitivity to acetylcholine, in an attempt to maintain intrinsic background tone (Golder et al., 2003). The aim of the present study has been to assess the morphologic pattern of the myenteric (Auerbach’s) nerve plexus (MNP) in the diverticulous sigmoid colon (DSC) regions and to compare it with the MNP in relatively normal sigma resected from patients operated on for rectal tumors in order to determine whether the DD is associated with a structural remodeling of this nerve plexus.
Materials and methods Segments of sigmoid colon (10–15 cm from the rectosigmoid junction) about 10 cm in length were taken from 10 patients operated on for chronic DD and 10 controls (after resections for rectal tumors). The local bioethical committee (Kaunas Region Bioethical Committee) approved the study. The same coloproctological team operated all the patients from October 2004 till April 2006. We excluded patients if they had received preoperative radiotherapy, had inflammatory bowel disease, or had symptoms of diverticulitis (Golder et al., 2003) or preoperative bowel obstruction. Six women and four men underwent surgery for chronic DD (mean age 61 (range 46–73) years). The remaining five men and five women (mean age 66 (range 56–75) years) were used as controls. The major criterion for patient assignment to the DD group was the presence of multiple diverticula (over 12) in contrast to the relatively normal sigmoid colon (rNSC) group, in which patients
ARTICLE IN PRESS Myenteric neural plexus in diverticular disease
527 medium described by Karnovsky and Roots (1964). The composition of the incubation medium (pH 5.6) was (in mM): sodium acetate, 60; acetylthiocholine iodide (FLUKA), 2; sodium citrate, 15; CuSO4, 3; K3 Fe(CN)6, 0.5. Triton-X 100 supplemented the incubation medium up to 1% and hyaluronidase (0.5 mg/100 ml). Since the remaining fat patches on the gut surface were almost non-permeable for the incubation media, an additional precise dissection of those fat pads was usually performed to expose the neural structures located beneath. Following staining, the preparations were fixed in neutral 4% formalin in 0.1 M phosphate buffer. The preparations were stored in the same formalin without noticeable alterations for months.
Figure 1. Gross anatomical view of the DSC resected from a 46 years old woman operated on for DD. The handled preparation is dissected out of fatty connective tissue at the tenia mesocolica, sewn at both ends and pressure-bloated by a hand-made rubber bladder placed inside the gut lumen and filled by isotonic saline. Black arrows point to some diverticula. Asterisks indicate the Taenia coli.
lacked any diverticula. Every patient gave written informed consent before participation in the study. Resection after 1–2 h, the resected sigmoid colon specimens were transported to an anatomical laboratory, where they were cleansed of blood with isotonic saline (pH 7.3), and dissected from the fatty connective tissue at the Tenia mesocolica. Further, samples were sewn on both ends and pressure-bloated by a hand-made rubber bladder placed inside the gut lumen and filled with isotonic saline, so that the gut walls were uniformly distended at the same pressure, which was controlled using a membranous manometer connected to the rubber bladder in T-joint manner (Figure 1). We did not assess samples with scar tissue that could have originated from previous diverticulitis. Pressure-bloated samples were fixed by immersion for 30 min in 4% paraformaldehyde solution in 0.1 M phosphate buffer at room temperature. Following fixation, the preparations were washed for 1 h at 4 1C in isotonic saline containing hyaluronidase (0.005 mg ml1, FLUKA).
Staining of the MNP Enzyme histochemistry for acetylcholinesterase was used to stain the myenteric neural plexus in total gut samples as this enzyme occurs nonspecifically within intrinsic nerves and ganglia: parasympathetic, sympathetic and sensory ones (Pauza et al., 2000). The hyaluronidase-treated samples were incubated for 1–3 h at 4 1C in the
Microscopy of the MNP Total gut preparations were placed in distilled water and examined in a transient light from a fiber optic illuminator using a stereomicroscope (Stemi 2000 CS, ZEISS) at a magnification of 16–56 . Stereomicroscopically visible intrinsic nerves and ganglia were photographed using a digital camera (AxioCam MRc5, ZEISS). Stereomicroscopic pictures were taken from every sample consistently from all walls of a preparation excluding Tenia coli regions. Only images without diverticulae were analyzed for the DD group. The thickness of the intramural nerves and the density of the myenteric ganglia in the DD group versus controls were assessed on randomly selected (choosing every fifth) images using the AxioVision software (v. 4.5; ZEISS, Germany).
Quantitative analysis of the MNP The plexal area of the MNP and the density of myenteric ganglia stained for AChE were evaluated in same-sized areas by counting only those ganglia that were well stained and well observable (Figure 2). Plexal area is defined as the area occupied by MNP on a digital image of 125 mm2. In order to reliably assess the MNP morphology, three random images each from 10 samples in the DD group (n ¼ 30) and three of each from 10 controls (n ¼ 30) were analyzed.
Statistical analysis Results shown in the text, tables and graphs are expressed as mean plus/minus standard error. A test of the statistical significance of the difference between the means was performed with Student’s independent tests (Microcal Origin v. 4.00). Linear
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O. Deduchovas et al. Table 1. MNP area (mm2) and ganglion number in randomly selected three areas of 125 mm2 from diverticulated sigmoid colon (DSC) group (n ¼ 10) versus relatively normal sigmoid colon (rNSC) group (n ¼ 10). MN-plexal area (mm2)
Ganglion number
DSC
rNSC
DSC
rNSC
7,570,3 7,270,1 7,670,4 6,870,5 6,670,1 7,570,2 6,970,1 7,570,2 7,270,1 7,670,3
8.070,5 8,370,1 8,370,2 7,870,1 7,670,3 8,070,3 8,170,2 8,370,4 8,270,3 8,170,2
145715 160726 14178 16276 13478 155716 13376 142715 15278 162711
162712 16679 146716 180717 166713 155711 151722 17076 17379 16178
In average 7,270,1
8,170,1
149712
163712
Differences of means are significant at po0,001 (MN-plexal area (mm2)) and po0,02 (Ganglion number).
Figure 2. Stereo-microphotographs of the myenteric nerve plexus (MNP) stained for acetylcholinesterase from whole (non-sectioned) diverticulous (A and C) and rNSCs (B and D). The boxed areas (E) in panel A and (F) in panel B are enlarged in panels E and F, respectively. The arrowheads in E and F indicate some intensively stained ganglionic cells. Note that MNP from DSC (A and C) is extensively atrophied, because its interganglionic nerves are periodically interrupted and obviously thinner (white arrows) in comparison to the rNSC (B and D). The density of the ganglia is obviously higher in the rNSC (B and D) than in diverticulous one (A and C). Black arrows in all panels indicate the well-developed interganglionic nerves in rNSC. Arrowheads in A–D panels point into some ganglia with well-stained ganglionic cells. Abbreviations: DV-diverticula.
area the number of myenteric ganglia was also significantly (po0.001) higher in rNSC group compared to the DSC group (Table 1). The interganglionic nerves of ganglionated MNP close to diverticula were thinner than those located distant to diverticula. No neural structures (either myenteric or submucosal ones) were identified on diverticula in any examined case of DSC (Figure 2A). The largest diverticulae were mostly developed in fatty tissues in the region of the Taenia mesocolica. We found no significant correlation between the age of patients and the ganglion number and the area of the MNP (Figure 3). We also did not observe a relationship between the character of diverticula and the patient age.
Discussion regression was used to quantitatively determine the relationship between the MNP ganglion number and its area and patient’s age. Significance was accepted at po0,05.
Results In contrast to that of the rNSC group, the MNP in the DSC group was degenerated: interganglionic nerves were periodically interrupted and thinner than in rNSC (Figure 2). On an average, the number of myenteric ganglia per same-sized area was significantly (po0.02) higher in the rNSC group compared to the DSC patients. In the MN-plexal
The present findings are the first demonstration of the morphology of MNP on a total (nonsectioned) sigmoid colon from patients with DD. The technique used in this study allowed for assessment of the MNP in situ on large bowel areas highlighting the stereomicroscopic alterations in the DSC compared to controls: a substantial decrease in the number and size of myenteric ganglia as well as a rarefaction of the myenteric plexus. Several recent reports indicate that the origin of DD may be neurogenic (Bassotti et al., 2005; Golder et al., 2003; Iwase et al., 2005; Spiller, 2006; Stoss and Meier-Ruge, 1994; Tomita et al., 1999; Vuong
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Figure 3. Graph demonstrating the absence of correlation between patient age and myenteric ganglion number (on left) and MNP area (on right) counted in three areas of 125 mm2 selected at random from samples of the diverticulous sigmoid colon group (DSC) versus the relatively normal sigmoid colon group (rNSC). Solid and dotted lines show correlation in DSC and rNSC groups, respectively.
et al., 1985). In spite of this, there are very few studies dealing with a morphologic pattern of MNP in DSC (Iwase et al., 2005; Vuong et al., 1985). Data of these reports, however, are fairly controversial. In an immunohistochemical study, Iwase et al. (2005) have shown differences in MNP morphology between DD patients and controls (operated on rectal tumors). The latter authors found that the sections of the diverticulous colon contains (1) significantly more plexuses (i.e. a denser neural network), (2) fewer ganglion cells and (3) a nuclear area of ganglion cells which appears to be smaller in DD patients than in controls. Vuong et al. (1985), using H&E staining and silver impregnation, did not reveal any morphological abnormalities of the MNP in patients with sigmoid DD compared to controls. Bassotti et al. (2005), using an immunohistochemical assay in sectioned specimens, also did not find significant differences in the number of neurons in DD patients with respect to controls. The present method, which aims to investigate the morphology of MNP in non-sectioned diverticulous sigma preparations, has never been used before the present study. Presumably, application of distinct morphologic techniques should be considered as the main reason for the discrepancies between findings of the above-mentioned and present studies. It seems that the whole-mount technique with histochemical staining for acetylcholinesterase used according to the prolonged protocol of the present study should demonstrate the most complete pattern of the MNP. However, this is correct only if the activity of AChE was not missing in the assessed samples of
the DD patients. On the other hand, the lack or reduction up to undetectable amount of general neural markers, immunohistochemistry of which was used to analyze samples from the DSC in studies by other authors, could be also fairly possible. Therefore, the morphologic alterations of MNP in DD patients revealed in the present study should be taken into account and verified by other sensitive methods. The atrophied MNP in DSC that we demonstrate here could be related to the age of patients, because it is known that the density of nerves in the bowel is reduced at high age and the incidence of DD increases with age (Gomes et al., 1997; Phillips and Powley, 2007). Nonetheless, the mean age of the examined patients in the DSC group was similar to the rNSC group or was even younger than in controls. We found no significant correlation between the age of patients and the ganglion number and the area of MNP. We also did not observe the relationship between the character of diverticula and patient age. Thereby, we presume that the influence of age is not pivotal for alterations of the MNP in the examined DD. Besides, we did not find any relationship between the character of diverticula (their size, quantity) and the morphology of the MNP. At the present moment, therefore, we are unable to explain how the demonstrated atrophy of the MNP could be related to the manifestation of DD, whether our findings are primary or secondary abnormalities of DD and how these alterations influence the diverticula’s development. However, the presented
ARTICLE IN PRESS 530 findings strongly imply that the occurrence of DD in sigmoid colon is associated with morphologic alterations in the MNP that may be considered as failure of the MNP in DD. The present observations deserve to be further examined immunohistochemically and electron-microscopically in order to determine the potential role of the MNP in pathogenesis of this digestive tract disease, because results of such investigations may modify both the prophylaxis and treatment of the colonic DD.
Acknowledgements This work was supported by grants from the Lithuanian State Science and Studies Foundation (T-76/07) as well as from the Science Foundation of Kaunas University of Medicine (PAR 18).
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