Myenteric plexus injury and apoptosis in experimental colitis Lee Boyer a , Mehran Ghoreishi a , Valerie Templeman a , Bruce A. Vallance a,b , Alison M. Buchan b,c , Gareth Jevon d , Kevan Jacobson a,b,c,e, * a British Columbia Research Institute, Vancouver, British Columbia, Canada V6H 3V4 b University of British Columbia, Vancouver, British Columbia, Canada V6H 3V4 c Department of Physiology, Vancouver, British Columbia, Canada V6H 3V4 d Division of Pathology, B.C. Children’s Hospital, Vancouver, British Columbia, Canada V6H 3V4 e Division of Gastroenterology, B.C. Children’s Hospital, Vancouver, British Columbia, Canada V6H 3V4 Received 3 April 2004; received in revised form 8 September 2004; accepted 27 October 2004 Abstract Intestinal inflammatory conditions are associated with structural and functional alterations of the enteric nervous system (ENS). While injury to the enteric nervous system is well described, the mechanisms of neuronal injury and neuronal cell loss remain unclear. The aim of the present study was to examine the neural consequences of distal colitis and to assess the role of neutrophil granulocytes in mediating these changes. Colitis was induced in C3H/HEN female mice with dinitrobenzene sulfonic acid. The mice were then sacrificed at 0.5, 1, 1.5, 2, 3, 4, 6, 12, 24, 120 h post instillation of dinitrobenzene sulfonic acid. The inflammatory response was assessed by macroscopic damage score, myeloperoxidase activity and histology. HuC/D and PGP 9.5 immunostaining was used to examine myenteric plexus density and structure, neural cell body numbers and distribution in cross-section and whole mount preparations. Apoptosis was investigated in whole mount preparations double stained with HuC/D and activated caspase-3 or cleaved poly (ADP-ribose) polymerase (PARP). Dinitrobenzene sulfonic acid-induced colitis was associated with a rapid and significant loss of HuC/D immunoreactive myenteric plexus neuronal cell bodies (42% decrease relative to control) that remained unchanged between 6 and 120 h. No change in myenteric plexus density was observed with PGP 9.5 immunostaining. Neuronal apoptosis was evident between 0.5 and 3 h. PARP immunoreactive neurons ranged between 1% and 2.5%. Colitis was associated with significant impairment in colonic propulsive function. Pre-treatment of mice with anti-neutrophil serum attenuated the inflammatory response and partially reduced the extent of myenteric plexus neuronal cell loss. Taken together, these data suggest that acute colitis is associated with loss of myenteric plexus neurons that is partly mediated by neutrophil granulocyte infiltration and is accompanied by impairment of colonic motility. D 2004 Elsevier B.V. All rights reserved. Keywords: Inflammatory bowel disease; Colitis; Neutrophils; Enteric nerves; Apoptosis 1. Introduction Inflammatory bowel disease (IBD) is a chronic and recurrent intestinal inflammatory disorder of unknown aetiology that is characterised by frequent relapses of disease activity and periods of remission. In inflammatory bowel disease, the enteric nervous system (ENS) is subject to injury, leading to the genesis of symptoms that may persist even in the quiescent phase of the disease. Little is known about the mechanisms that underlie enteric nervous system injury in inflammatory bowel disease. The enteric nervous system resides within the intestinal wall as ganglionated and non-ganglionated plexuses of considerable structural complexity and functional integrity that controls the physiologic functions of the gastrointestinal tract (Goyal and Hirano, 1996). That transient or even permanent structural and functional alterations in the enteric nervous system occur in inflammatory bowel disease is supported by a number of observations. To date the 1566-0702/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2004.10.006 * Corresponding author. Division of Gastroenterology, B.C. Children’s Hospital, 4480 Oak Street, Room K4-181, Vancouver, B.C., Canada V6H 3V4. Tel.: +1 604 875 2332; fax: +1 604 875 3244. E-mail address: kjacobson@cw.bc.ca (K. Jacobson). Autonomic Neuroscience: Basic and Clinical 117 (2005) 41– 53 www.elsevier.com/locate/autneu