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Quantitative evaluation of myenteric ganglion cells in normal human left colon: implications for histopathological analysis

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The analysis of myenteric neurons is becoming increasingly important for the assessment of enteric nervous system injury and degeneration occurring in motor disorders of the gut. Limited information is presently available on the quantitative estimation of myenteric neurons and glial cells in paraffin-embedded colonic sections; additional data would be useful for diagnostic purposes. In this morphometric study, we performed immunohistochemistry to count myenteric neurons and glial cells in paraffin sections of human colon. Serial cross sections of formalin-fixed paraffin-embedded full-thickness normal human left colon (n = 10, age-range: 50–72 years) were examined. HuC/D and S100β antigens were found to be the best markers for the detection of neurons and glial cells, respectively. Significant correlations were noted between the numbers of neurons/glial cells and the respective myenteric ganglion areas. These findings suggest that HuC/D-S100β-immunostained paraffin cross sections of human colon can be regarded as valuable tools for the quantitative estimation of myenteric neurons and glial cells. Based on the present method, only a limited number of paraffin sections are needed for reliable quantitative assessments of myenteric ganglion cells, thus allowing fast and simple approaches in the settings of the histopathological diagnosis of colonic motility disorders and retrospective evaluations of pathological archival tissue specimens.

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  1. Aubé AC, Cabarrocas J, Bauer J, Philippe D, Aubert P, Doulay F, Liblau R, Galmiche JP, Neunlist M (2006) Changes in enteric neurone phenotype and intestinal functions in a transgenic mouse model of enteric glia disruption. Gut 55:630–637

  2. Baschong W, Suetterlin R, Laeng RH (2001) Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM). J Histochem Cytochem 49:1565–1572

  3. Bassotti G, Villanacci V, Maurer CA, Fisogni S, Di Fabio F, Cadei M, Morelli A, Panagiotis T, Cathomas G, Salerni B (2006) The role of glial cells and apoptosis of enteric neurones in the neuropathology of intractable slow transit constipation. Gut 55:41–46

  4. Bassotti G, Villanacci V, Fisogni S, Rossi E, Baronio P, Clerici C, Maurer CA, Cathomas G, Antonelli E (2007) Enteric glial cells and their role in gastrointestinal motor abnormalities: introducing the neuro-gliopathies. World J Gastroenterol 13:4035–4041

  5. Belkind-Gerson J, Graeme-Cook F, Winter H (2006) Enteric nervous system disease and recovery, plasticity, and regeneration. J Pediatr Gastr Nutr 42:343–350

  6. Bernardini N, Colucci R, Mattii L, Segnani C, Fornai M, De Giorgio R, Barbara G, Castagna M, Nardini V, Dolfi A, Del Tacca M, Blandizzi C (2006) Constitutive expression of cyclooxygenase-2 in the neuromuscular compartment of normal human colon. Neurogastroenterol Motil 18:654–662

  7. Boman F, Sfeir R, Priso R, Bonnevalle M, Besson R (2007) Advantages of intraoperative semiquantitative evaluation of myenteric nervous plexuses in patients with Hirschsprung disease. J Pediatr Surg 42:1089–1094

  8. Bondurand N, Natarajan D, Thapar N, Atkins C, Pachnis V (2003) Neuron and glia generating progenitors of the mammalian enteric nervous system isolated from foetal and postnatal gut cultures. Development 130:6387–6400

  9. Boyer L, Ghoreishi M, Templeman V, Vallance BA, Buchan AM, Jevon G, Jacobson K (2005) Myenteric plexus injury and apoptosis in experimental colitis. Auton Neurosci 117:41–53

  10. Bradley JS Jr, Parr EJ, Sharkey KA (1997) Effects of inflammation on cell proliferation in the myenteric plexus of the guinea-pig ileum. Cell Tissue Res 289:455–461

  11. Cabarrocas J, Savidge TC, Liblau RS (2003) Role of enteric glial cells in inflammatory bowel disease. Glia 41:81–93

  12. Cheuk W, Chan JKC (2004) Subcellular localization of immunohistochemical signals. Int J Surg Pathol 12:185–206

  13. da Silveira AB, Lemos EM, Adad SJ, Correa-Oliveira R, Furness JB, D’Avila Reis D (2007) Megacolon in Chagas disease: a study of inflammatory cells, enteric nerves, and glial cells. Hum Pathol 38:1256–1264

  14. De Giorgio R, Camilleri M (2004) Human enteric neuropathies: morphology and molecular pathology. Neurogastroenterol Motil 16:515–531

  15. De Giorgio R, Bovara M, Barbara G, Canossa M, Sarnelli G, De Ponti F, Stanghellini V, Tonini M, Cappello S, Pagnotta E, Nobile-Orazio E, Corinaldesi R (2003) Anti-Hu-induced neuronal apoptosis underlying paraneoplastic gut dysmotility. Gastroenterology 125:70–79

  16. De Giorgio R, Barbara G, Furness JB, Tonini M (2007) Novel therapeutic targets for enteric nervous system disorders. Trends Pharmacol Sci 28:473–481

  17. Di Nardo G, Blandizzi C, Volta U, Colucci R, Stanghellini V, Barbara G, Del Tacca M, Tonini M, Corinaldesi R, De Giorgio R (2008) Review article: molecular, pathological and therapeutic features of human enteric neuropathies. Aliment Pharmacol Ther 28:25–42

  18. Doleshal M, Magotra AA, Choudhury B, Cannon BD, Labourier E, Szafranska AE (2008) Evaluation and validation of total RNA extraction methods for microRNA expression analysis in formalin-fixed, paraffin-embedded tissues. J Mol Diagn 10:203–211

  19. Eaker EY (1997) Neurofilament and intermediate filament immunoreactivity in human intestinal myenteric neurons. Dig Dis Sci 42:1926–1932

  20. Ferri G-L, Probert L, Cocchia D, Michetti F, Marangos PJ, Polak JM (1982) Evidence for the presence of S-100 protein in the glial component of the human enteric nervous system. Nature 297:409–410

  21. Fornai M, Blandizzi C, Antonioli L, Colucci R, Bernardini N, Segnani C, De Ponti F, Del Tacca M (2006) Differential role of cyclooxygenase 1 and 2 isoforms in the modulation of colonic neuromuscular function in experimental inflammation. J Pharmacol Exp Ther 317:938–945

  22. Furness JB (2000) Types of neurons in the enteric nervous system. J Auton Nerv Syst 81:87–96

  23. Furness JB (2006) The enteric nervous system. Blackwell, Oxford

  24. Gabella G (1987) The number of neurons in the small intestine of mice, guinea-pigs and sheep. Neuroscience 22:737–752

  25. Gabella G, Trigg P (1984) Size of neurons and glial cells in the enteric ganglia of mice, guinea-pigs, rabbits and sheep. J Neurocytol 13:49–71

  26. Ganns D, Schrödl F, Neuhuber W, Brehmer A (2006) Investigation of general and cytoskeletal markers to estimate numbers and proportions of neurons in the human intestine. Histol Histopathol 21:41–51

  27. Gershon MD (2005) Nerves, reflexes, and the enteric nervous system: pathogenesis of the irritable bowel syndrome. J Clin Gastroenterol 39:S184–S193

  28. Gomes OA, Souza RR de, Liberti EA (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

  29. Gonzalez-Martinez T, Perez-Piñera P, Diaz-Esnal B, Vega JA (2003) S-100 proteins in the human peripheral nervous system. Microsc Res Tech 60:633–638

  30. Hanani M (2004) Multiple myenteric networks in the human appendix. Auton Neurosci 110:49–54

  31. Hanani M, Fellig Y, Udassin R, Freund HR (2004) Age-related changes in the morphology of the myenteric plexus of the human colon. Auton Neurosci 113:71–78

  32. Hoff S, Zeller F, Weyhern CW von, Wegner M, Schemann M, Michel K, Rühl A (2008) Quantitative assessment of glial cells in the human and guinea pig enteric nervous system with an anti-Sox8/9/10 antibody. J Comp Neurol 509:356–371

  33. Holst M-C, Powley TL (1995) Cuprolinic blue (quinolinic phthalocyanine) counterstaining of enteric neurons for peroxidase immunocytochemistry. J Neurosci Methods 62:121–127

  34. Hu HZ, Gao N, Lin Z, Gao C, Liu S, Ren J, Xia Y, Wood JD (2002) Chemical coding and electrophysiology of enteric neurons expressing neurofilament 145 in guinea pig gastrointestinal tract. J Comp Neurol 442:189–203

  35. Huizinga JD, White EJ (2008) Progenitors cells of interstitial cells of Cajal: on the road to tissue repair. Gastroenterology 134:1252–1254

  36. Jessen KR, Mirsky R (1980) Glial cells in the enteric nervous system contain glial fibrillary acidic protein. Nature 286:736–737

  37. Krammer HJ, Karahan ST, Sigge W, Kühnel W (1994) Immunohistochemistry of markers of the enteric nervous system in whole-mount preparations of the human colon. Eur J Pediatr Surg 4:274–278

  38. Kruger GM, Mosher JT, Bixby S, Joseph N, Iwashita T, Morrison SJ (2002) Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness. Neuron 35:657–669

  39. Linden DR, Couvrette JM, Ciolino A, McQuoid C, Blaszyk H, Sharkey KA, Mawe GM (2005) Indiscriminate loss of myenteric neurones in the TNBS-inflamed guinea-pig distal colon. Neurogastroenterol Motil 17:751–760

  40. Lindley RM, Hawcutt DB, Connell MG, Almond SN, Vannucchi MG, Faussone-Pellegrini MS, Edgar DH, Kenny SE (2008) Human and mouse enteric nervous system neurosphere transplants regulate the function of aganglionic embryonic distal colon. Gastroenterology 135:205–216

  41. Miettinen M, Lehto V-P, Virtanen I (1984) Antibodies to intermediate filament proteins in the diagnosis and classification of human tumors. Ultrastruct Pathol 7:83–107

  42. Murphy EM, Defontgalland D, Costa M, Brookes SJ, Wattchow DA (2007) Quantification of subclasses of human colonic myenteric neurons by immunoreactivity to Hu, choline acetyltransferase and nitric oxide synthase. Neurogastroenterol Motil 19:126–134

  43. Neunlist M, Aubert P, Toquet C, Oreshkova T, Barouk J, Lehur PA, Schemann M, Galmiche JP (2003) Changes in chemical coding of myenteric neurones in ulcerative colitis. Gut 52:84–90

  44. Nishiyama H, Knöpfel T, Endo S, Itohara S (2002) Glial protein S100B modulates long-term neuronal synaptic plasticity. Proc Natl Acad Sci USA 99:4037–4042

  45. Phillips RJ, Hargrave SL, Rhodes BS, Zopfs DA, Powley TL (2004) Quantification of neurons in the myenteric plexus: an evaluation of putative pan-neuronal markers. J Neurosci Methods 133:99–107

  46. Porter AJ, Wattchow DA, Brookes SJ, Costa M (2002) Cholinergic and nitrergic interneurones in the myenteric plexus of the human colon. Gut 51:70–75

  47. Roberts PJ, Morgan K, Miller R, Hunter JO, Middleton SJ (2001) Neuronal COX-2 expression in human myenteric plexus in active inflammatory bowel disease. Gut 48:468–472

  48. Roberts RR, Bornstein JC, Bergner AJ, Young HM (2008) Disturbances of colonic motility in mouse model of Hirchsprung’s disease. Am J Physiol Gastrointest Liver Physiol 294:G996–G1008

  49. Robertson D, Savage K, Reis-Filho JS, Isacke CM (2008) Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue. BMC Cell Biology 9:13–22

  50. Rühl A, Nasser Y, Sharkey KA (2004) Enteric glia. Neurogastroenterol Motil 16:44–49

  51. Sandgren K, Larsson LT, Ekblad E (2002) Widespread changes in neurotransmitter expression and number of enteric neurons and interstitial cells of Cajal in lethal spotted mice. An explanation for persisting dysmotility after operation for Hirschsprung’s disease? Dig Dis Sci 47:1049–1064

  52. Sanovic S, Lamb DP, Blennerhassett MG (1999) Damage to the enteric nervous system in experimental colitis. Am J Pathol 155:1051–1057

  53. Savidge TC, Sofroniew MV, Neunlist M (2007) Starring roles for astroglia in barrier pathologies of gut and brain. Lab Invest 87:731–736

  54. Smith VV (1993) Intestinal neuronal density in childhood: a baseline for the objective assessment of hypo- and hyperganglionosis. Pediatr Pathol 13:225–237

  55. Steiner J, Bernstein HG, Bielau H, Berndt A, Brisch R, Mawrin C, Keilhoff G, Bogerts B (2007) Evidence for a wide extra-astrocytic distribution of S100B in human brain. BMC Neurosci 8:2

  56. Streutker CJ, Huizinga JD, Campbell F, Ho J, Riddell RH (2003) Loss of CD117 (c-kit)- and CD34-positive ICC and associated CD34-positive fibroblasts defines a subpopulation of chronic intestinal pseudo-obstruction. Am J Surg Pathol 27:228–235

  57. Van Ginneken CJ, De Smet MJ, Van Meir FJ, Weyns AA (1999) Microwave staining of enteric neurons using cuprolinic blue (quinolinic phthalocyanin) combined with enzyme histochemistry and peroxidase immunohistochemistry. J Histochem Cytochem 47:13–21

  58. Vasina V, Barbara G, Talamonti L, Stanghellini V, Corinaldesi R, Tonini M, De Ponti F, De Giorgio R (2006) Enteric neuroplasticity evoked by inflammation. Auton Neurosi 126–127:264–272

  59. Wedel T, Roblick U, Gleiss J, Schiedeck T, Bruch HP, Kühnel W, Krammer HJ (1999) Organization of the enteric nervous system in the human colon demonstrated by wholemount immunohistochemistry with special reference to the submucous plexus. Ann Anat 181:327–337

  60. Wedel T, Roblick UJ, Ott V, Eggers R, Schiedeck TH, Krammer HJ, Bruch HP (2002) Oligoneuronal hypoganglionosis in patients with idiopathic slow-transit constipation. Dis Colon Rectum 45:54–62

  61. Wexler EM, Stanton PK, Nawy S (1998) Nitric oxide depresses GABAA receptor function via coactivation of cGMP-dependent kinase and phosphodiesterase. J Neurosci 18:2342–2349

  62. Wislet-Gendebien S, Hans G, Leprince P, Rigo JM, Moonen G, Rogister B (2005) Plasticity of cultured mesenchymal stem cells: switch from nestin-positive to excitable neuron-like phenotype. Stem Cells 23:392–402

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The authors thank Drs. Simon Gibbons and Gianrico Farrugia, Enteric Neuroscience Program, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minn., USA, for insightful discussions and critical review of this work.

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Correspondence to Nunzia Bernardini.

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R. De Giorgio is the recipient of grants from the Fondazione Del Monte di Bologna e Ravenna and from the Fondazione Cassa di Risparmio, Bologna, Italy. The authors declare no conflicting interests.

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Ippolito, C., Segnani, C., De Giorgio, R. et al. Quantitative evaluation of myenteric ganglion cells in normal human left colon: implications for histopathological analysis. Cell Tissue Res 336, 191–201 (2009).

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  • Enteric nervous system
  • Colon
  • Myenteric neurons
  • Myenteric glial cells
  • Paraffin-embedded tissue
  • Human