Interstitial cells of cajal of the equine gastrointestinal tract: development and disease

Gastrointestinal motility disorders constitute a substantial problem in the horse both in terms of welfare and economic cost. It is often difficult to identify the underlying cause as many horses recover spontaneously or with empirical medical treatment. Recently, the gastrointestinal pacemaker cells, the interstitial cells of Cajal (ICC), were identified in the horse. These cells initiate and coordinate gastrointestinal motility patterns through the generation of slow waves. This current study investigated the ICC in the equine intestine both in health and disease using immunohistochemical, electrophysiological and molecular biological techniques. The aim of these studies was to further our knowledge on the role of ICC in equine intestinal motility disorders.Using immunohistochemistry targeting a receptor tyrosine kinase, c-Kit, of the ICC, the ontogeny of these cells in the horse was described. This demonstrated a proximal to distal, as well as a transmural developmental gradient in the large intestine with evidence of ongoing postnatal development. Additionally, the density of ICC in healthy, adult horses was compared to that in horses with obstructive intestinal disease requiring surgical correction. This demonstrated a significant reduction in ICC density in horses with obstructive disorders of the large intestine compared to the control group. In addition, ICC density and distribution was investigated in recovered chronic equine grass sickness horses as well as in normal and diseased donkeys.The c-kit gene, encoding the c-Kit receptor of the ICC, was identified in intestinal tissue samples. The transcription levels of this gene were determined and comparisons made - 1 - between healthy and diseased horses using quantitative real-time PCR analysis. A parallel immunohistochemical assessment was also performed. These studies demonstrated no significant changes in gene transcription levels, although a reduction in ICC density (using c-Kit immunohistochemistry) in horses with an obstructive disorder of the large colon was evident, suggesting that future investigations of c-kit posttranscriptional control as well as c-Kit protein pathology are warranted.Investigation of the in vitro electrical activity of the equine large colon was carried out using intracellular microelectrode recording techniques in order to characterise slow waves and other electrical activities in this anatomical region from normal and diseased horses.It is hoped that this study will help improve our knowledge ofthe involvement of ICC in equine intestinal motility in health and disease. Furthermore, it may facilitate future studies investigating the involvement and function of the ICC in the equine gastrointestinal tract

Alien Registration- Fintl, William R. (Bethel, Oxford County)

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Expression of PGP 9.5 by Enteric Neurons in Horses and Donkeys with and without Intestinal Disease

Intestinal motility disorders are an important problem in horses and donkeys and this study was carried out in order to evaluate the enteric neurons in animals with and without intestinal disease. Surplus intestinal tissue samples were collected from 28 horses undergoing exploratory laparotomy for colic. In addition, surplus intestinal samples from 17 control horses were collected immediately following humane destruction for clinical conditions not relating to the intestinal tract. Similar samples were also collected during routine post-mortem examinations from 12 aged donkeys; six animals were humanely destroyed for conditions related to the intestinal tract, while the remaining six were humanely destroyed for other reasons including dental and orthopaedic diseases. Tissue samples were fixed in formalin and immunohistochemical labelling was performed targeting the enteric neurons using a polyclonal antibody specific for the neuronal marker PGP 9.5. The distribution and density of neuronal networks were assessed qualitatively and semiquantitatively. There was strong PGP 9.5 expression in both the horse and donkey samples and labelling was detected throughout the tissue sections. In both species, PGP 9.5-immunoreactive nerve fibres were detected in all layers of the intestinal tract, both in large and small intestinal samples. Networks of enteric neurons were present in the donkey with a similar distribution to that seen in the horse. There was no demonstrable difference in enteric neuronal density and distribution in the groups of animals with intestinal disease compared with those without, apart from two (out of 28) horses with intestinal disease that showed a marked reduction in PGP 9.5 immunoreactivity. Apart from these two animals, this total cohort analysis differs from some previously observed findings in horses with intestinal disease and may therefore reflect the different pathophysiological processes occurring in varying intestinal conditions resulting in colic both in the donkey and the horse. © 2013 Elsevier Ltd

High-Resolution Genotyping of Expressed Equine MHC Reveals a Highly Complex MHC Structure

The Major Histocompatibility Complex (MHC) genes play a key role in a number of biological processes, most notably in immunological responses. The MHCI and MHCII genes incorporate a complex set of highly polymorphic and polygenic series of genes, which, due to the technical limitations of previously available technologies, have only been partially characterized in non-model but economically important species such as the horse. The advent of high-throughput sequencing platforms has provided new opportunities to develop methods to generate high-resolution sequencing data on a large scale and apply them to the analysis of complex gene sets such as the MHC. In this study, we developed and applied a MiSeq-based approach for the combined analysis of the expressed MHCI and MHCII repertoires in cohorts of Thoroughbred, Icelandic, and Norwegian Fjord Horses. The approach enabled us to generate comprehensive MHCI/II data for all of the individuals ( n = 168) included in the study, identifying 152 and 117 novel MHCI and MHCII sequences, respectively. There was limited overlap in MHCI and MHCII haplotypes between the Thoroughbred and the Icelandic/Norwegian Fjord horses, showcasing the variation in MHC repertoire between genetically divergent breeds, and it can be inferred that there is much more MHC diversity in the global horse population. This study provided novel insights into the structure of the expressed equine MHC repertoire and highlighted unique features of the MHC in horses

Comparative analysis of c-kit gene expression and c-Kit immunoreactivity in horses with and without obstructive intestinal disease

Previous immunohistochemical studies targeting the receptor tyrosine kinase (c-Kit) have demonstrated an apparent reduction in the number of gastrointestinal pacemaker cells--the interstitial cells of Cajal (ICC)--in horses with intestinal motility disorders. This study compared the level of transcription of the c-kit gene encoding this receptor in horses with and without such motility disorders. Transcription levels of this gene were also compared to the density of ICC immunohistochemically positive for the c-Kit antigen. Intestinal samples were collected from 18 horses with intestinal disease and from 15 control animals. Following gene extraction and identification, real-time quantitative analysis of c-kit and a control gene, ACTB (β-actin), was carried out on all samples and the density of the c-Kit-positive ICC compared. There was a significant reduction in c-Kit immunoreactivity in the ICC of horses with large intestinal obstructive disorders relative to controls but no significant difference in the transcription of the c-kit gene between normal and affected animals. Further studies will be required to elucidate the mechanisms regulating c-Kit expression and to assess the pathophysiological significance of these findings