Geoff Pearson

Structural organization and neuropeptide distributions in the equine enteric nervous system: an immunohistochemical study using whole-mount preparations from the small intestine

The architecture and neurochemistry of the enteric nervous system was studied by use of whole-mount preparations obtained by microdissection of the horse jejunum. A myenteric plexus and two plexuses within the submucosa were identified. The external submucosal plexus lying in the outermost region of the submucosa had both neural and vascular connections with the inner submucosal plexus situated closer to the mucosa. Counts of neurones stained for NADH-diaphorase demonstrated the wide variation in size, shape and neurone content of individual ganglia in both the external and internal submucosal plexuses. The average number of cells/ganglion was similar in each plexus (about 25 cells). Immunoreactivities for galanin, vasoactive intestinal peptide and neuropeptide Y were observed in nerve cell bodies and fibres of each of the plexuses. Immunoreactivity for substance P was extensive and strong in nerve fibres of all plexuses but was weaker in cell bodies of the submucosal neurones and absent in the cell bodies of the myenteric plexus. Comparative quantitative analysis of immunoreactive cell populations with total cell numbers (enzyme staining) was indicative of neuropeptide colocalization in the external submucosal plexus.

Immunohistochemical localization of neuropeptides in bovine pancreas

The occurrence and density of distribution of nerves and endocrine cells that are immunoreactive for neuropeptides in the bovine pancreas were studied by immunohistochemistry. The six neuropeptides localized were galanin (GAL), substance P (SP), methionine‐enkephalin (MENK), neuropeptide Y (NPY), calcitonin gene‐related peptide (CGRP) and vasoactive intestinal polypeptide (VIP). The exocrine pancreas was shown to have an appreciable number of GAL‐ and SP‐immunoreactive nerve fibres but few fibres showing immunoreactivity for VIP and CGRP. Numerous MENK‐, GAL‐, SP‐, and NPY‐immunoreactive nerve fibres were seen in the endocrine portion of the pancreas. Nerve cell bodies in the intrapancreatic ganglia showed immunoreactivity for all of the neuropeptides except CGRP. Endocrine cells showing immunoreactivity for GAL and SP were observed in the large islets and islets of Langerhans, respectively. The present results indicate a characteristic distribution of neuropeptides in the bovine pancreas, which may regulate both exocrine and endocrine secretions of pancreas.

The equine enteric nervous system?neuron characterization and distribution in adults and juveniles

A study of myenteric and submucosal plexuses was undertaken in the jejunum and ileum of horses and ponies in which no clinical or pathological evidence of intestinal abnormality was apparent. Complete transverse sections of the intestine, stained by a modified haematoxylin and eosin method, were examined using up to 20 sequential sections per animal. Information was gathered from adult, juvenile and fetal equidae. In adults, the longitudinal muscle layers were thinner than the circular muscle layers and the ileum had thicker layers compared to the jejunum. In adults, the submucosal plexus had more neurons per section than the myenteric plexus by mean ratios of 1:3 in the jejunum and 1:1.9 in the ileum. In juveniles, the ratios were respectively 1:1.8 and 1:1.5 and in the fetus 1:2.5 and 1:1.3. The three-dimensional distribution of neurons in both plexuses varied from animal to animal and no consistent pattern was observed. Groups of neurons contained between one and 42 cells per section examined and their length in a cranio-caudal direction varied from 10 to over, 100 µm. There were few statistical differences observed between the cranial, middle and caudal portions of either the jejunum or the ileum when neuron groups or neuron numbers per section were examined in 10 adult animals.

Tissue culture of the enteric nervous system from equine ileum.

Ileal samples were harvested fresh from euthanized adult horses. The tissues were microdissected to prepare wholemount preparations for immunohistochemistry and for either explant or dissociated culture systems of the enteric nervous system. Explant culture systems were established using wholemounts of either the submucous plexus or the muscularis externa (including the myenteric plexus). Dissociated cell cultures could only be obtained from the submucous plexus. Culture systems were maintained for up to 5 days. Immunoreactivity for a neuronal marker (Pan-N) and for glial cell markers (GFAP and S100) indicated the presence of both neurons and enteric glia in the tissue culture preparations.This is the first report of equine enteric neurons being grown in tissue culture. Further refinements to the techniques will be required before this in vitro model can be used for quantitative analysis.

A study of the interstitial cells of Cajal in aged donkeys with and without intestinal disease

Intestinal tissue samples were collected during routine post-mortem examinations from 12 aged donkeys. Six animals were euthanised due to impaction of the pelvic flexure of the large colon, while the remaining six were euthanised for non-enteric reasons such as dental or orthopaedic disease. Immunohistochemical labelling was performed to demonstrate the gastrointestinal pacemaker cells, the interstitial cells of Cajal (ICC), with polyclonal c-Kit antibodies. The distribution and density of the cellular networks were assessed qualitatively and semi-quantitatively. ICC networks are present in the donkey, with distribution similar to that of the horse, and they remain strongly immunoreactive in the older animal. There was no difference in the density and distribution of ICC in animals with or without intestinal disease.

The effect of temperature changes on in vitro slow wave activity in the equine ileum

REASONS FOR PERFORMING STUDY Slow waves are rhythmic pacemaker currents generated by the gastrointestinal pacemaker cells, the interstitial cells of Cajal, and represent the rate-limiting step for small intestinal smooth muscle contractions. Therefore, factors that affect slow wave activity may also influence contractile activity. It is not known how temperature changes may influence slow wave activity in the horse. This could be of relevance during colic surgery if cooling of exposed intestine resulted in reduced slow wave activity potentially exacerbating post operative ileus. OBJECTIVES To evaluate the effect of temperature changes on in vitro slow wave activity of normal equine ileum using intracellular recording techniques. STUDY DESIGN In vitro experimental study. METHODS A segment of ileum was collected immediately following euthanasia from 9 horses for reasons unrelated to the gastrointestinal tract. Intracellular recordings of membrane potentials were made from individual smooth muscle cells. The temperature of the tissue bath was altered during the course of each experiment across a range of 27-41°C. All data were recorded and stored using a computer-interfaced acquisition system. A software package was used to analyse slow wave frequency, duration, amplitude and resting membrane potential. RESULTS In all 9 horses, slow wave frequency was highly temperature sensitive and approximately linearly related to the temperature over the range studied, increasing by 0.5 cycles/min for each 1°C increase in temperature (P<0.001). The initial slow wave frequency resumed when the temperature was returned to 37°C. The recovery time appeared to be directly related to the duration for which the temperature had been changed. CONCLUSIONS Slow wave frequency in the equine ileum is highly temperature sensitive. As post operative ileus is a major cause of morbidity and mortality in the horse, the negative effect of lower temperatures on slow waves, and therefore contractile activity, should be considered.

In vitro responses to noradrenaline of small intestine taken from normal and grass sickness-affected horses.

Small intestine was taken from the caudal flexure of the duodenum and the terminal ileum proximal to the ileocaecal fold of 25 horses, 9 with acute grass sickness (AGS), 12 with subacute grass sickness (SAGS) and 12 with chronic grass sickness (CGS). The motility in the samples was measured isometrically either within 1 h of death or after storage for 24 h at 4°C.In control tissue, noradrenaline produced contractions of muscle strips which did not involve a muscarinic cholinergic mechanism and which were unaffected by the α1 antagonist prazosin but were blocked by the α2 antagonist yohimbine. Pretreatment with the α antagonist phentolamine prevented the contractile response to noradrenaline and the background contractions either continued at a reduced rate and amplitude or were abolished after a few minutes. Thus, following α blockade, noradrenaline reduced the background contraction rate by an effect on inhibitory β adrenoceptors. The rate of background contractions in duodenal preparations was significantly greater than that in control ileal preparations.Although cold storage for 24 h caused a reduction in the background contraction rates of the control preparations, there was no effect on the contractile responses to noradrenaline, the associated pharmacology being similar to that of fresh tissue. This suggests that noradrenaline-evoked contraction was not dependent on enteric neural elements.The response to noradrenaline by grass sickness-affected tissue was generally similar to that of tissue from control horses, with an immediate contraction which was α2 sensitive. The contractile response to noradrenaline after propranolol was significantly reduced in the CGS group and there were significant differences between the AGS, CGS and control groups. There was a significant difference between the ileal preparations from the control and SAGS groups in their response to noradrenaline following pretreatment with propranolol.

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.

Similar challenges, different approaches: a review of student support systems in UK veterinary schools

The characteristics of individuals pursuing a veterinary career, and the negative impact of undergraduate training, have been suggested as possible contributing factors to poor mental wellbeing in the profession (Bartram and Baldwin 2010). Therefore, veterinary students may be a vulnerable student population (Collins and Foote 2005, Hafen and others 2006, Sutton 2007). On-site counselling services were offered at approximately half the American veterinary schools in 2001 (Kogan and McConnell 2001). The aim of this study was to collate information on support systems in UK veterinary schools. A web-based questionnaire (Survey Monkey, Palo Alto, California, USA; freely available at www.surveymonkey.com) was electronically delivered to the Heads of Teaching at all UK Veterinary Schools in August 2011 which contained 16 closed (10 with additional free text option) and 13 open multiple choice questions. Questions included where on campus student teaching was undertaken, the distance of the school from the main campus, and the availability and types of counselling and other support services offered. Responses were received from all seven UK schools, although details of student support were received from only six schools. Veterinary teaching was undertaken at both the main university and a separate veterinary campus at four schools, exclusively at a separate veterinary campus at two schools and solely at the main university campus by one school. Two schools shared a campus with other schools/departments. Counselling services …

In vitro electrical activity of the equine pelvic flexure

REASONS FOR PERFORMING STUDY The generation and maintenance of intestinal motility patterns involve the complex interactions of several components including the gastrointestinal pacemaker cells (interstitial cells of Cajal, ICC). Central to ICC function is the generation of rhythmic pacemaker currents, namely slow waves, which represent the rate limiting step for intestinal smooth muscle contractions. Currently, intracellular slow wave activity has not been demonstrated in the equine colon. OBJECTIVES To characterise the in vitro myoelectrical activity of the equine pelvic flexure using intracellular recording techniques. METHODS Intestinal samples were collected immediately following euthanasia from 14 normal horses. One millimetre thick tissue sections were pinned and superfused with warmed, oxygenated Krebs solution. Intracellular recordings were made from smooth muscle cells close to the submucosal border of the circular muscle layer. The L-type Ca(2+) channel blocker nifedipine was added to the superfusion fluid in 9 experiments while the Na(+) channel blocker tetrodotoxin was added to the superfusion fluid in 4 experiments. The data were recorded and stored using an acquisition system and a software package used to analyse the recordings. RESULTS In 10 of the 14 horses, electrical events consistent with slow wave patterns were recorded from individual smooth muscle cells. Surprisingly, adding nifedipine to the superfusion fluid abolished all electrical activity. In contrast, tetrodotoxin had no apparent effect on the electrical activity. CONCLUSIONS Assuming that the electrical events were slow waves, the blockade by nifedipine suggests significant and potentially important differences in the ionic mechanisms responsible for slow waves in the different regions of the equine intestinal tract, which deserve further evaluation.