This study aimed to evaluate the reliability of slaughterhouse-obtained small intestinal tissue as control material in equine colic research where molecular stress responses in small intestinal tissue are investigated. For this purpose, small intestinal samples from colic horses were collected during surgery or immediately after euthanasia at the oral border of strangulation resection sites and routinely processed for histopathology (i.c. rinsed with 4°C Krebs' solution, fixated overnight with 4% neutral buffered formaldehyde (FH) at room temperature). Control samples consisted of pieces of mid-jejunum, collected at the slaughterhouse and routinely processed for histopathology under 4 different conditions. The 4 conditions differed with regard to incubation and fixation temperature and whether or not oxygenated Krebs' solution was used. Histological scoring revealed that slaughterhouse samples had a higher mean lesion score (P<0.001) than colic samples. In addition, more slaughterhouse samples had a higher mean inflammation score than colic samples (P=0.001). The inflammatory cells in the small intestine consisted mostly of eosinophils and as such were very suggestive for parasitic infestation. Hypoxia-inducible factor-1α (HIF1α) nuclear immunoreactivity was more pronounced in slaughterhouse tissue, probably as a result of the delay between slaughter and sampling (P=0.034). The histopathological score (P=0.291), the inflammation score (P=0.248) and the HIF1α nuclear immunoreactivity (P=0.538) did not differ between the different collection protocols. It is concluded that slaughterhouse-obtained small intestinal tissue shows distinct alterations and that its use as control tissue when evaluating molecular stress responses should be applied with prudence.

Reasons for performing study: Intestinal strangulation often leads to enterectomy after which ileus can develop. This has prompted research to look into possible pathophysiological processes triggering equine ileus. However, morphological changes of the small intestinal smooth muscle in relation to equine colic have not yet been studied. Objectives: The presence of some smooth muscle proteins was morphologically assessed and quantified in control and colic horses. In addition, the up- or down-regulation of heat shock proteins (HSP20 and HSP27) influencing the contractility of smooth muscles was studied. Methods: Cranial resection margins of 18 strangulated small intestinal samples were collected. Small intestinal control samples were collected from 11 horses subjected to euthanasia for other than gastrointestinal-related reasons. Formaldehyde-fixed tissue was paraffin-embedded and processed for conventional staining and immunohistochemistry. Snap-frozen full-thickness biopsies were collected for western blot analyses. Results: Evaluating the muscle layer microscopically, colic samples showed significantly more signs of degradation than controls (P = 0.026) of which vacuolar degeneration was most prominent (P = 0.009). In colic samples, myosin protein levels were decreased (P = 0.022) whereas desmin (P = 0.049) and HSP20 protein levels (P = 0.005) were elevated. Conclusions: In colic samples, microscopic lesions at the level of the muscle layer indicate a stress response. In addition, modified amounts of structural proteins such as myosin and desmin together with increased HSP20 levels could perhaps provide a basis for explaining the malfunctioning of the intestinal muscle layer. Potential relevance: Post operative ileus, following small intestinal strangulation and resection, could be related in part to a dysfunctional muscle layer. In addition to microscopic signs of degeneration, myosin and HSP20 were affected. Pharmacological interventions might alter HSP20 expressions and thus serve a protective effect.

Strangulation colic often leads to surgery. We aimed to document the molecular response in the non-resected intestine in these horses using quantitative Western blot analysis, and immunohistochemistry. The expression of hypoxia-inducible factor 1-alpha (HIF1α) was investigated together with two molecular pathways initiated after protein destruction: proteasome degradation via ubiquitin chain formation and protein restoration via molecular chaperones such as inducible heat shock protein 70 (HSP70). In addition, the expression of c-fos and c-jun could indicate an early proinflammatory response. Ubiquitin, HSP70, c-jun and c-fos protein levels did not differ between the control and colic samples nor were they related to the clinical outcome in case of strangulation colic. However, the immunohistochemical distribution of several of these proteins (ubiquitin, HSP70 and c-jun) differed significantly between colic and control samples. The elevated presence of ubiquitin in the enterocytes’ nucleus, of HSP70 in the smooth muscle cells’ nucleus and of c-jun in enteric neurons suggest protective and degenerative pathways are activated in the apparently healthy non-resected tissue in case of strangulation obstruction, perhaps providing a molecular and morphological basis for the development of complications like post-operative ileus.

After birth, intestinal morphology and function have to adapt at a high pace. This remodelling is more challenging in low birth weight neonates or in preterms, where it may result in necrotizing enterocolitis (NEC). We hypothesized that in the preterm piglet, feeding induces maladaptations of the enteric nervous system (ENS) and vasculature. Using image analysis, the densities of neurons expressing VIP, glial cells containing GFAP and the endothelium containing eNOS on immunohistochemically stained small intestinal sections of preterm 1) unfed piglets, 2) piglets receiving total parenteral nutrition (TPN) for 2-3 days and 3) piglets fed 2 days sow's colostrum (SOW) or formulated milk (FOR) following TPN were estimated.After enteral feeding, the ENS and vascular endothelium grew in the same order as the intestine. However, feeding formula increased the density of VIP'ergic myenteric neurons, lowered eNOS in the endothelium and resulted in a reactive gliosis. In conclusion, formula induces destructive changes in the immature small intestine, whereas colostrum prevents their occurrence. These conditions may be among the factors that predispose to NEC. (C) 2010 Elsevier B.V. All rights reserved.

The preterm intestine is immature and responds differently to total parenteral nutrition (TPN) and enteral nutrition, compared with the term intestine. We hypothesised that in preterms, diet composition and feeding route affect mucosal morphology, enterocyte mitosis and apoptosis, and the distribution of laminin-1, fibronectin and collagen IV (extracellular matrix proteins (ECMP)). Preterm piglets (93·5 % of gestation) were delivered via caesarean section and birth weight-matched allocated to one of the four experimental groups: the piglets were either euthanised immediately after delivery, after 3 d of TPN or after 2 d enteral feeding with colostrum or milk formula, following 3 d of TPN. We combined immunohistochemistry, image analysis and stereological measurements to describe the intestinal mucosal layer. No significant changes occurred after 3 d of TPN. Feeding colostrum or milk replacer for 2 d after TPN was associated with an increased crypt depth. Only enteral feeding with colostrum resulted in an increased villus height and mitotic index. Neither TPN nor enteral feeding changed the distribution pattern of ECMP or the occurrence of bifid crypts. The immature distribution pattern of ECMP in TPN-fed piglets, coupled with unchanged enterocyte mitosis and apoptosis indices, illustrates that feeding preterm pigs 3 d TPN does not lead to mucosal atrophy. Despite the invariable distribution of ECMP, colostrum was associated with crypt hyperplasia resulting in an increased villus height. These data illustrate that some mechanisms regulating cell turnover are immature in preterms and may in part explain the abnormal gut responses to TPN and enteral feeding in prematurely born pigs.

Background: Nutrition regimens influence postnatal small intestinal development, which shows prominent changes after 6 hours of suckling. Such influences are particularly important in preterm neonates as inappropriate feeding responses may predispose to gastrointestinal disorders such as necrotizing enterocolitis (NEC). The authors investigated the early morphological responses to enteral feeding, prior to the time period when a large proportion of preterm pigs normally develop clinical NEC symptoms. Methods: Preterm piglets (106‐107 days of gestation) were fed parenteral nutrition (PN) for 2 days with or without a subsequent 8‐hour or 17‐hour period of enteral nutrition (EN) with sow's colostrum or formula. Another group of piglets was delivered at 108–109 days of gestation and used for comparison to PN pigs before enteral feeding. Stereological measurements of the mucosal surface density and the volume densities of the tunica mucosa, tunica muscularis, proliferative, and apoptotic cells were made and related to microscopical NEC‐lesion score. In addition, villus length and crypt depth were measured. Results: PN‐fed piglets showed minimal PN‐induced mucosal atrophy, although their crypts were deeper, together with lower cell proliferation and higher apoptotic indices, than newborn (NB) unfed piglets. After PN, enteral feeding with colostrum, for just 8 hours, induced a rapid increase in the mucosal volume density while formula feeding was associated with an elevated number of both proliferating and apoptotic cells and a higher NEC lesion score than PN‐ or colostrum‐fed pigs. Conclusion: Enteral feeding of formula, for only a few hours, induces rapid enterocyte turnover and mucosal structural changes that may predispose to later development of NEC.

The casein peptide Asn-Pro-Trp-Asp-Gln enforces the intestinal tight junction partly by increasing occludin expression in Caco-2 cells.H. Yasumatsu & S. Tanabe 951-956 Cocoa polyphenols suppress TNF-α-induced vascular endothelial growth factor expression by

ABSTRACTObjectives:Intestinal colonization challenges the neonatal innate immune system, especially in newborns with an immature immune response lacking the supportive bioactive components from mother's milk. Accordingly, formula‐fed preterm pigs frequently show bacterial overgrowth, mucosal atrophy, and gut lesions reflecting necrotizing enterocolitis (NEC) within the first days after birth. We hypothesized that NEC development is related to a diet‐dependent bacterial adherence and a subsequent proinflammatory cytokine response in the gut mucosa immediately after introduction of enteral food.Materials and Methods:Premature piglets (92% gestation) received 2 to 3 days of total parenteral nutrition followed by 0, 8, or 17 hours of enteral formula or sow's colostrum feeding.Results:Following 8 hours, but not 17 hours, of colostrum feeding, a reduced number of intestinal samples with adherent bacteria (both Gram‐negative and Gram‐positive bacteria) was counted compared with 0 or 8 hours of formula feeding. Besides a more dense colonization, formula feeding leads to higher intestinal interleukin‐1β (IL‐1β) levels and more NEC‐like lesions from 8 hours onward. The load of adherent bacteria was especially high in NEC lesions. Toll‐like receptor 4 was detected in enteroendocrine, neuronal, and smooth muscle cells, potentially mediating the increase in IL‐1β levels by Gram‐negative bacteria.Conclusions:Formula feeding facilitates bacterial adherence and the development of a proinflammatory state of the intestine, which may be among the key factors that predispose formula‐fed preterm neonates to NEC.

The initiation of enteral feeding represents a challenge to the neonatal intestinal microcirculation, especially in preterms where it predisposes to necrotizing enterocolitis (NEC). We hypothesized that a structural microvascular deficiency may occur when enteral feeding is initiated in preterm piglets susceptible to NEC. Stereologic volume densities of a pan-endothelial marker (vWF), and the main vasodilator endothelial nitric oxide synthase (eNOS), were determined along the small intestine of 1) unfed preterm piglets, 2) piglets receiving total parenteral nutrition (TPN) for 2–3 d, and 3) piglets fed 2 d sow's colostrum (TPN+SOW) or milk formula (TPN+FOR) following TPN. In the mucosa, vWF-density decreased in a cranio-caudal direction. A corresponding mucosal eNOS gradient appeared only after initiating enteral feeding. In TPN+SOW, eNOS induction may lag behind the mucosal growth of the caudal region. In TPN+FOR, formula-related factors (i.e. bacteria, cytokines) may suppress mucosal eNOS, indicated by increased stress-sensitive nuclear HIF1α staining. The low mucosal endothelial eNOS density was related to the presence of NEC lesions, maybe via increased hypoxia-sensitivity, especially in the caudal region as indicated by nuclear HIF1α-staining. Our results suggest an insufficient structural adaptation of the microvasculature to enteral feeding, especially of mucosal eNOS, which may lead to NEC.

This study highlights the importance of intersitial cells of Cajal (ICs) in gastrointestinal disease. Human research is already considering IC pathologies but in veterinary research IC pathologies are rarely studied. Nevertheless, recent studies of ICs show a growing interest in the pathophysiology of gastrointestinal diseases and emphasize the consideration of this cell type in the pathophysiology of veterinary gastrointestinal malfunctions.

During development, gut morphology and functions change. Some of these are mediated by milk-bome factors, e.g. insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF), of which the effects are in part dictated by the distribution of their receptors (R).EGFR- and IGF-1R-immunoreactivity (IR) was evaluated in samples of the fetal, neonatal and weaned porcine small intestine.EGFR-IR first appeared in the neonatal duodenum and was located basolaterally in villar and to a lesser extent in cryptal enterocytes. In weaned pigs, EGFR appeared apically in enterocytes with a region-dependent distribution along the villus-crypt axis. IGF-1R-IR was observed in smooth muscle cells in each age group. From the last trimester of gestation onward IGF-IR-IR was seen at the apical and basolateral side of villar enterocytes and in submucosal arterioles.Thus, the age- and region-dependent presence of EGFR and IGF-1R has to be taken into account when evaluating effects of EGF and IGF-1 on growth or repair. (c) 2007 Elsevier B.V All rights reserved.

Heart valves were historically considered passive structures that function through the haemodynamic forces created by the contraction and relaxation of the myocardium. However, research into valvular structures has revealed that heart valves are very complex, specialized structures that contain both smooth muscle cells and nerve fibres. This is particularly true for the atrioventricular valves, which are by far the most well studied to date. The various heart valves have been shown to contract independently during different moments of the heart cycle, suggesting that compensatory adaptation mechanisms exist to mediate the timing and efficacy of heart valve closure. These adaptations occur via different mechanisms, including neural mechanisms that influence the heart valves. Accumulating evidence continues to improve our understanding of the nerve fibres in the heart (adrenergic, cholinergic, etc.). Future studies will no doubt add to this exciting picture. Here, we review the current morphological knowledge of human and animal heart valve innervation, including discussions of the chordae tendineae and the papillary muscles, as well as the differences between the atrioventricular (AV) valves and the semi-lunar (SL) valves.

The gastrointestinal tract of pig is a good and available (slaughterhouse material) model to study the enteric nervous system in relation to nutrition, inflammation, development and disease in general. In order to investigate the responses of the enteric nervous network to a variety of stimuli, e.g. growth factors, hormones, extracellular matrix components, but also noxious compounds in a controlled manner, an in vitro experimental set‐up is most appropriate. Methods to obtain in vitro cultures of enteric neurons of rodents, chicken and human are well described. This study attempted to use these methods on pig material. The muscle layer containing the myenteric plexus was dissected from pieces of fetal, neonatal and adult pig jejunum. They were rinsed in Hanks basal salt solution (BSS) in which antibiotics were added. Pieces of ±25 mm2 were transferred to BSS with 1 mg/ml collagenase and 1 mg/ml trypsin inhibitor and incubated for 60 min (5% CO2, 90% RH). Subsequently, they were vortexed for 20 s and ganglia were selected. The procedure was repeated ±4 times. Myenteric ganglia could then be plated or further dissociated in 1 mg/ml trypsin in BSS for 30 min in the incubator. Afterwards, the dissociated ganglia were centrifuged (5 min 1500 rpm) and the trypsin‐solution was replaced with Dulbecco's minimal essential medium (DMEM). The explants or dissociated myenteric ganglia were transferred on non‐coated or coated (poly‐L‐lysine, laminin, fibronectin, collagen or extracellular matrix gel) cover slips. After incubating for 45 min they were topped with 1 ml of DMEM with antibiotics and with or without fetal calf serum (5%). Medium was replaced twice a week. Using immunohistochemistry, both neurons (PGP9.5) and glial cells (S100) could be identified in both culture types. When cultivated under harsh conditions, the dissociated cultures gave rise to neurosphere‐like bodies, containing neurons and glial cells. Thus, the digestion and dissociation technique is applicable to pig material.

Today's students belong to an interactive generation and receive information through multiple channels. Curricula in veterinary medicine are changing due to trends such as ‘student centred education’ and ‘competence based learning’. Taking in account these changes, we were encouraged to rethink the way in which we teach and assess the knowledge of veterinary anatomy. The amount of dissection (90 h) and lecture hours (90 h) has not changed, but the way they are used and how the ‘knowledge’ is assessed has been revised. First, goals were defined: students have to acquire knowledge and insight in the structure, function and relationships of anatomical structures of various species. They have to be able to observe, palpate and expose the structures properly. Additionally, they must attain general skills, e.g. communication. Secondly a new learning environment was developed so students could accomplish these goals. The three main components, constituting this new environment were: the introduction of an assessment and development centre (ADC) as a means of assessing students as well as giving them feedback, the incorporation of e‐learning and the raise of student‐lecturer interactions during lecture hours. During the ADC the students have to go through three set‐ups: they demonstrate structures, they name structures and they ‘solve’ a problem (e.g. cut tendon). They report their findings both verbally and written. Using checklists the student's practical skills are assessed during the dissections and during the ADC. Students receive feedback at four different times. An E‐learning environment was devised, giving the students the possibility to consult photographs of the dissections, to discuss the course on a discussion board and to take self‐assessment tests to monitor their progress. During the student‐lecturer interactions, photographs, clinical examples, are discussed using various teaching methods, resulting in a more ‘active’ approach to the learning content. Students are enthusiastic about this way of teaching although they experience it as difficult. Student scores seem not different from previously. In the future the practical checklists and the ADC will be optimized.

Stereological methods were used to quantify secretin and gastric inhibitory peptide (GIP)-immunoreactivity (GIP-IR) in paraffin sections of the duodenum, jejunum and ileum of fetal and neonatal piglets. In addition, sections were processed for GLP-1-immunohistochemistry. The volume density of the tunica mucosa increased after birth, giving rise to a decreased volume density of the tela submucosa and tunica muscularis. Generally known region-specific morphological distinctions were reflected in differing volume densities of the various layers. The highest volume density of GIP-IR epithelial cells was observed in the jejunum of the neonate. In contrast, the volume density of secretin-IR epithelial cells was highest in the duodenum of both fetal and neonatal piglets. The volume occupied by GIP-IR and secretin-IR epithelial cells increased in the jejunum after birth. Additionally, ileal secretin-IR epithelial cells were more numerous in the neonatal piglet. In conclusion, the quantitative and qualitative presence of GIP-IR and secretin-IR epithelial cells agree with earlier reports of their presence and co-localization between GIP-IR and GLP-1-IR, in the pig small intestine. Furthermore, the differences suggest that age- and region-related functional demands are temporally and probably causally related with the morphological diversification of the intestine and its endocrine cells.

The extent of clinical or subclinical infection associated with Lawsonia intracellularis within Dutch pig herds was uncertain. A case-control study of slaughter age pigs was used to study natural infection within Dutch herds and to compare diagnostic methods. From six case herds where clinical disease had been identified recently, and six disease-free herds, 40 pigs of slaughter-age were examined postmortem. The diagnostic methods used were: serology, gross examination, Haematoxylin and Eosin stain (HE), Warthin-Starry silver stain, Lawsonia-specific indirect immunoperoxidase of the ileum, and PCR of ileum mucosa and colon contents. There were 59% seropositive pigs in case herds and 26% seropositive pigs in control herds. Using immunohistochemistry, 57% of case herds and 46% of control herds were bacteria positive in the ileum mucosa. It was concluded that a majority of Dutch herds contain L. intracellularis infected finisher pigs. In some herds this is associated with clinical outbreaks of acute haemorrhagic enteropathy but in other herds no clinical disease is apparent. Many seropositive pigs in herds without clinical disease had evidence of Lawsonia antigen in sites other than the apical cytoplasm of proliferating epithelial cells, particularly the supranuclear region. It was uncertain whether to classify these pigs as having “recovered” from an infection or whether they have a sub-clinical or chronic form of the disease. We concluded that PCR examination of faeces and serology probably provide more specific results than gross examinations at slaughter, and that a monoclonal antibody-based examination of ileum mucosa should be the accepted screening method for this infection.

Recent reports on the distribution of nNOS in the airways have focused on the neuronal cell bodies in intrinsic ganglia. Adult human and porcine lungs show a similar pattern of NOS-containing neurons while small laboratory animals such as the guinea pig are devoid of NOS-immunoreactivity in the peripheral airways. The present study reports on the occurrence of NOS-containing neurons in porcine airway intrinsic ganglia during development using NADPH-diaphorase staining. 14 fetal and 3 neonatal pigs were used in this study. Whole mount preparations of the trachea and the 2nd lateral segmental bronchus were incubated in the NADPH-diaphorase medium containing 10 mg NADPH, 5 mL 0.01 mPBS with 0.3% Triton-X-100, 1.25 mg nitro blue tetrazolium for 15 min at 37 °C. After rinsing in PBS the tissue was mounted in a 3 : 1 glycerine/0.01 mPBS mixture. The trachea shows a nerve plexus where nerve bundles interconnect different ganglia. During development the density of this network decreased indicating that ganglia become more separated when the trachea grows in diameter and length. These ganglia show NADPH-d positive neurons with different degree of staining intensity. With age more neurons show the NADPH-d staining with a maximum intensity and presence just before birth. In comparison with the trachea the innervation of the lateral segmental bronchus is characterised by nerve bundles interconnecting smaller ganglia. These ganglia also show NADPH-d positive neurons. Preliminary quantitative observations demonstrated the increase in numerical density in NADPH-d positive neurons in the ganglia with fetal age. For the same age group it was observed that the density of NADPH-d stained neurons is higher in trachea than in the segmental bronchus. In neonatal animals the number of positive cells still seems to increase in the peripheral airway ganglia while the tracheal ganglia shows some saturation. The distribution of NADPH-d neurons in the airways of developing pigs confirms the importance of their neuromodulating capacities both in trachea and peripheral airways in the perinatal period.

Studies that investigate possible developmental changes in gastrointestinal hormones in the pig are sparse and contradictory. Therefore, a quantitative morphological study using stereologic methods on paraffin sections of the different small intestinal regions (cranial and caudal duodenum, jejunum and ileum) of the developing pig (second half of the gestation, neonatal and weaning period) has been conducted. The sections were processed for GLP-1-immunohistochemistry. During the investigated time span, the volume of GLP-1-IR cells increased approximately 40-fold in both the jejunum and ileum, notwithstanding a decrease of their volume density. The ileal small intestinal segment contained the highest volume density of GLP-1-IR cells. In contrast, GLP-1-IR cells were only occasionally encountered in the duodenum. The development-related changes of the investigated parameters coincide with dietary changes and the regional differences are in accordance with functional reports that point to GLP-1 as a gastrointestinal hormone that ediates the 'ileal brake' and stimulates glucose-dependent pancreatic insulin secretion.