In vitro investigation of the interaction between nitric oxide and cyclo-oxygenase activity in equine ventral colon smooth muscle

The objective of this study was to determine if a correlation exists between the presence of nitric oxide and prostaglandin release in the equine ventral colon smooth muscle, since this relationship may accentuate the inflammatory process during intestinal injury. Tissue was collected from the ventral colon, cut into muscle strips oriented along the circular, longitudinal and taenial layers, and mounted in a tissue bath system. Samples of the bath fluid were collected before, following electrical field stimulation (EFS), and following EFS in the presence of L-NAME, a nitric oxide synthase inhibitor. Muscle strips were also obtained following systemic administration of a cyclo-oxygnease inhibitor and samples were collected using the previously described protocol. Concentrations of prostaglandins were determined in the fluid samples using an ELISA. Electrical field stimulated release of nitric oxide produced a significant increase in prostaglandin production which did not occur in the presence of L-NAME. Systemic administration of flunixin meglumine reduced prostaglandin levels at all sampling periods, although a small increase was present following EFS. The results of this study support the hypothesis that there is a correlation between the release of nitric oxide and the production of prostaglandins in the smooth muscle of the large colon. This association between nitric oxide and prostaglandin may act as an important regulatory mechanism for various physiological mechanisms, such as vascular smooth muscle tone, and may contribute to amplified tissue injury when the induced forms of both enzymes are activated during an inflammatory insult. This suggests that the use and development of COX2 and iNOS inhibitors may help attenuate the inflammatory response following intestinal injury.     

Distribution of the neurokinin-1 receptor in equine intestinal smooth muscle

REASON FOR PERFORMING STUDY: Tachykinins have profound effects on equine intestinal motility, but the distribution of the neurokinin receptors (NKRs) through which they act is unknown. This study reports the distribution of one of these receptors, the neurokinin-1 receptor (NK1R), in smooth muscle throughout the equine intestinal tract. OBJECTIVES: To quantify the distribution of the NK1R, based upon mRNA expression, in smooth muscle of different regions of the equine intestinal tract. METHODS: Nine regions of the intestinal tract were sampled in 5 mature horses. Total RNA was isolated from smooth muscle and reverse transcribed; NK1R mRNA was then quantified using real-time PCR. RESULTS: NK1R mRNA was found at all levels of the sampled intestinal tract. The smooth muscle of the proximal small intestine and the ventral colon exhibited the highest level of NK1R mRNA expression in the equine intestinal tract. CONCLUSIONS: Tachykinins probably affect intestinal contractility and propulsion in the proximal small intestine and in the ventral colon.     

The equine teniae coli: An electrophysiological study

Previous morphological studies of the equine teniae coli (intestinal bands) have shown them to be highly innervated. In this study, EMG electrodes were placed in the wall of the left ventral colon in order to determine whether intestinal bands serve as major conduits of myo-electrical activity. Specifically, electrodes were implanted in the lateral mesocolic band and the adjacent tenia-free bowel of 6 horses. In 3 of these horses, a 1 cm length of the intestinal band was excised to determine if a lesion of this size would ablate local waves of depolarization. Our results indicate that sequential EMG activity persisted despite this small, focal excision. The persistence of sequential EMG activity might reflect the importance of constantly regenerating stimuli to the intestinal motility of the horse. Whether making similar or somewhat larger lesions in all four teniae of the left ventral, colon would more definitively disrupt normal pelvic flexure peristalsis will require further research.     

In vitro investigation of the effect of prostaglandins and nonsteroidal anti-inflammatory drugs on contractile activity of the equine smooth muscle of the dorsal colon, ventral colon, and pelvic flexure

OBJECTIVES: To determine the in vitro effect of prostaglandin E2 (PGE2), PGF2alpha, PGI2; and nonsteroidal anti-inflammatory drugs (NSAID; ie, flunixin meglumine, ketoprofen, carprofen, and phenylbutazone) on contractile activity of the equine dorsal colon, ventral colon, and pelvic flexure circular and longitudinal smooth muscle. ANIMALS: 26 healthy horses. PROCEDURE: Tissue collected from the ventral colon, dorsal colon, and pelvic flexure was cut into strips and mounted in a tissue bath system where contractile strength was determined. Incremental doses of PGE2, PGF2alpha,, PGI2, flunixin meglumine, carprofen, ketoprofen, and phenylbutazone were added to the baths, and the contractile activity was recorded for each location and orientation of smooth muscle. RESULTS: In substance P-stimulated tissues, PGE2 and PGF2alpha enhanced contractility in the longitudinal smooth muscle with a decrease or no effect on circular smooth muscle activity. Prostaglandin I2 inhibited the circular smooth muscle response with no effect on the longitudinal muscle. The activity of NSAID was predominantly inhibitory regardless of location or muscle orientation. CONCLUSIONS AND CLINICAL RELEVANCE: In the equine large intestine, exogenous prostaglandins had a variable effect on contractile activity, depending on the location in the colon and orientation of the smooth muscle. The administration of NSAID inhibited contractility, with flunixin meglumine generally inducing the most profound inhibition relative to the other NSAID evaluated in substance P-stimulated smooth muscle of the large intestine. The results of this study indicate that prolonged use of NSAID may potentially predispose horses to develop gastrointestinal tract stasis and subsequent impaction.     

Enterotomy Technique in the Descending Colon of the Horse Effect of Location and Suture Pattern

To compare the effects of placing enterotomy incisions on or off the antimesenteric teniae and closing the intestinal mucosa as a separate layer, four longitudinal enterotomies were performed in the descending colon of each of six horses by the following techniques: incision through the antimesenteric teniae with one- and two-layer closure, and incision adjacent to the teniae with one- and two-layer closure. The horses were necropsied at day 33 for evidence of obstruction, adhesions, and ultrasonographic determination of the percent reduction in lumen diameter. Histologic and histomorphometric evaluations included: inflammatory response in the mucosal and seromuscular layers, mucosal atrophy or degeneration, alignment of the incision edges, area of fibrosis, and distance between the incised muscle edges. Adhesions were present in 5 of 24 enterotomies. Incisions adjacent to the teniae resulted in narrower lumen diameters than incisions through the teniae. Inflammatory response was greatest in incisions adjacent to the teniae with two-layer closure. Closure of the mucosa as a separate layer had no effect on any of the parameters evaluated. Enterotomies through the antimesenteric teniae were more easily performed, resulted in less hemorrhage, and maintained a larger lumen diameter than those performed adjacent to the teniae.     

In vitro investigation of the effects of cyclooxygenase-2 inhibitors on contractile activity of the equine dorsal and ventral colon

OBJECTIVE: To evaluate the effect of 2 cyclooxygenase (COX)-2 inhibitors on contractile activity of the circular smooth muscle layer of the equine dorsal and ventral colon. SAMPLE POPULATION: Samples of the dorsal and ventral colon obtained from 10 healthy horses. PROCEDURE: Full-thickness tissue samples were collected from the dorsal colon in the area of the diaphragmatic flexure and the ventral colon in the area of the sternal flexure. Samples were cut into strips oriented along the fibers of the circular muscle layer and mounted in a tissue bath system for determination of contractile strength. Incremental amounts of etodolac, nabumetone, and indomethacin were added, and contractile activity was recorded. RESULTS: Response of the dorsal and ventral colon to nonsteroidal anti-inflammatory drugs (NSAIDs) was variable. Indomethacin induced the greatest reduction in contractile activity, followed by nabumetone. For etodolac, the difference from baseline values was only significantly reduced at the highest concentration used (1 X 10(5)M) for the ventral colon. CONCLUSIONS AND CLINICAL RELEVANCE: The NSAIDs that are designed to target the COX-2 isoform appeared to have variable effects on the contractile activity of the equine dorsal and ventral colon. Etodolac appeared to have the least effect on contractile activity, compared with the effects attributable to nabumetone, and would potentially have the fewest adverse effects relative to motility of the dorsal and ventral colon.     

Expression of the ether-a-go-go (ERG) potassium channel in smooth muscle of the equine gastrointestinal tract and influence on activity of jejunal smooth muscle

OBJECTIVE: To determine whether ether-a-go-go (ERG) potassium channels are expressed in equine gastrointestinal smooth muscle, whether ERG channel antagonists affect jejunal muscle contraction in vitro, and whether plasma cisapride concentrations in horses administered treatment for postoperative ileus (POI) are consistent with ERG channels as drug targets. SAMPLE POPULATION: Samples of intestinal smooth muscle obtained from 8 horses free of gastrointestinal tract disease and plasma samples obtained from 3 horses administered cisapride for treatment of POI. PROCEDURE: Membranes were prepared from the seromuscular layer of the duodenum, jejunum, ileum, cecum, large colon, and small colon. Immunoblotting was used to identify the ERG channel protein. Isolated jejunal muscle strips were used for isometric stress response to ERG channel blockers that included E-4031, MK-499, clofilium, and cisapride. Plasma concentrations of cisapride were determined in 3 horses administered cisapride for treatment of POI after small intestinal surgery. RESULTS: Immunoblotting identified ERG protein in all analyzed segments of the intestinal tract in all horses. The selective ERG antagonist E-4031 caused a concentration-dependent increase in jejunal contraction. Clofilium, MK-499, and cisapride also increased jejunal contraction at concentrations consistent with ERG channel block; effects of E-4031 and cisapride were not additive. Peak plasma cisapride concentrations in treated horses were consistent with ERG block as a mechanism of drug action. CONCLUSIONS AND CLINICAL RELEVANCE: The ERG potassium channels modulate motility of intestinal muscles in horses and may be a target for drugs. This finding may influence development of new prokinetic agents and impact treatment of horses with POI.     

A Botanical-Based Equine Nutraceutical Reduces Gastric Smooth Muscle Contractile Force In Vitro

The objective of this study was to evaluate the effect of a botanical-based equine nutraceutical on contractility of gastric smooth muscle in vitro. Gastric ulcers are prevalent in performance horses and negatively impact horse welfare. Gastric hypermotility has been positively associated with the development of gastric ulceration in nonequine species, and reduction of hypermotility may be protective against their development. Stomachs from 12 pigs processed for food at a provincially inspected abattoir were collected within 1 hour of slaughter. Explants of nonglandular gastric tissue were prepared and suspended in a tissue bath, attached to a force transducer, in the presence or absence of a simulated digest extract of the nutraceutical. Tissue was stimulated to contract using increasing doses of acetylcholine. Peak and mean contractile force over 1 and 2 minutes after exposure to acetylcholine were measured. Exposure of gastric smooth muscle to the nutraceutical significantly reduced contractility of the tissue. These data provide support for the use of this nutraceutical to reduce contractility of nonglandular gastric smooth muscle and may indicate a protective effect of this nutraceutical in horses with mechanically induced gastric ulcers. Future studies are needed to clarify the role of gastric hypermotility on development of equine gastric ulcers and to determine the effect of this nutraceutical on equine gastric contractility and ulcerogenesis in vivo.     

5-Hydroxytryptamine mediated contractions in isolated preparations of equine ileum and pelvic flexure: pharmacological characterization of a new 5-HT(4) agonist

The effects of 5-hydroxytryptamine (5-HT), HTF 919, a new 5-HT(4) agonist, and the antagonists SB 203-186 (5-HT(4)) and tropisetron (5-HT(3)) on intestinal motility were tested in vitro on isolated preparations of horse ileum and pelvic flexure. Concentration-response curves were created by cumulative application of the agonists with or without preincubation of the antagonists. The 5-HT preparation induced a concentration-dependent contraction in equine ileum and pelvic flexure. The results indicate that 5-HT receptors are present in all parts of equine intestine investigated in this study. Tropisetron was found to act as a noncompetitive antagonist in all locations of the equine intestine. SB 203-106 was confirmed as an antagonist to 5-HT in the equine ileum circular muscle, in pelvic flexure circular and longitudinal muscle. Nevertheless, a discernible increase of smooth muscle contractions caused by HTF 919 could only be observed in pelvic flexure. In accordance with an earlier study in the guinea pig, in the equine gut HTF 919 acted as a partial agonist for the 5-HT(4) receptor with an affinity constant in the nanomolar range. It is concluded that 5-HT receptors, and especially their subtypes, may represent a promising target for the treatment and prevention of gastrointestinal (GI) motility disorders in horses.     

In vitro effects of tachykinins on the smooth musculature of horse gut

The contractile effects of the tachykinins eledoisin, substance P and neurokinin A and B were investigated in vitro on circular and longitudinal muscle strips from horse duodenum, ileum and colon. Circular smooth muscle of the small intestine was highly responsive, large intestine circular smooth muscle less so, while longitudinal muscle from all gut segments was much less sensitive. pD₂ values and intrinsic activities on small intestine circular muscle indicated differences in receptor distribution between the duodenum and ileum: NK₃ and a smaller number of NK₂ receptors being present in the duodenum, and NK₂ receptors predominating in the ileum. Notwithstanding this, eledoisin and neurokinin B were the most active substances on duodenum and ileum, respectively. These findings suggest that tachykinins may play a role in equine gastrointestinal pathophysiology.     

5-Hydroxytryptamine-4 receptor messenger ribonucleic acid levels and densities in gastrointestinal muscle layers from healthy dairy cows

Serotonin (5-hydroxytryptamine, 5-HT) is involved in gastrointestinal tract (GIT) motor functions through binding to specific receptors located in the GIT walls. The objectives of the current study were to compare mRNA levels and binding sites of 5-HT(4) receptors (5-HTR(4)) in smooth muscle layers from the fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows, and to verify whether mRNA and protein expression were correlated. Smooth muscle samples were prepared by scraping the mucosa and submucosa from full-thickness intestinal wall samples. The mRNA levels of 5-HTR(4) were measured by real-time PCR and expressed relative to those of the housekeeping gene glyceraldehyde phosphate dehydrogenase. Binding studies were performed using the 5-HTR(4) antagonist [(3)H]GR113808. The mRNA levels of 5-HTR(4) were affected (P < 0.05) by location along the GIT. The mRNA levels of 5-HTR(4) in the ELSC and the ileum were greater than in the PLAC (P = 0.05 and P = 0.07, respectively) but similar to those of all other locations. The competitive binding of [(3)H]GR113808 to suspended membranes from the fundus abomasi, pylorus, cecum, and ELSC was best fit by a 2-site receptor model, whereas it was best fit by a 1-site receptor model in the ileum and PLAC. The mRNA levels and numbers of 5-HTR(4) were not correlated (r = 0.14; P = 0.71). In conclusion, mRNA and binding sites for 5-HTR(4) are present in the smooth muscle layer of the entire GIT of dairy cows and may play a role with respect to motility. The effects of activation of this receptor subtype may be different among GIT locations due to differences in the amount of high- relative to low-affinity binding sites.     

The histological observations on the large intestine of the goose (Anser anser) during the pre- and post-hatching periods

The development of the cecum and colon in the goose was investigated during the period from the 15th to 28th day of the incubation and from 1 to 30 days of age after hatching by light microscopy. By day 15 of the incubation, in the cecum and colon, the lumen was surrounded by pseudostratified epithelium. The previllous ridges appeared at 15th and 17th days of the incubation in the colon and ceca, respectively. At the base of previllous ridges, the epithelium changed into a simple prismathic epithelium at 15th and 17th days of the incubation in the colon and cecum, respectively. The villi appeared at the 21st days of the incubation. The crypts and goblet cells appeared on the first day after hatching. In the pre-hatching period, the lamina muscularis mucosa was present only in the colon. The submucosa consisted of loosely aggregated connective tissue in the pre-hatching period. In the post-hatching period, it consisted of a very thin layer of connective tissue. Its presence was only obvious where the cells of the submucosal nerve plexus or occasional large blood vessels considerably increased its thickness. The nerve plexus corresponding to the Auerbach's plexus of the mammalian intestine and submucosal nerve plexus appeared by 15th days of the incubation. From the 15th to 28th day of incubation, the tunica muscularis consisted of circular smooth muscle cells in the ceca. On the 28th day of the incubation a thinner longitudinal muscle layer added to the circular muscle layer. In the colon there was an outer longitudinal and a thicker circular muscle layer.     

Morphological changes in the small intestinal smooth muscle layers of horses suffering from small intestinal strangulation. Is there a basis for predisposition for reduced contractility?

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.     

Prostaglandin E2 and reactive oxygen metabolite damage in the cecum in a pony model of acute colitis

The objective of this project was to determine early tissue biochemical events associated with increased colonic secretion during the acute stage of castor-oil-induced colitis by measuring cecal mucosal and submucosal malondialdehyde (MDA) and prostaglandin E₂ (PGE₂), levels in ponies. Intestinal tissue (inflamed or healthy) samples were obtained from 4 age- and sex-matched Shetland ponies. Biochemical methods were used to determine MDA and PGE₂ levels in intestinal tissue samples from inflamed and healthy equine intestine. Inflamed tissue MDA and PGE₂ levels increased with time after castor oil challenge and correlated with granulocyte infiltration, as determined by myeloperoxidase levels in a companion study. Elevated intestinal tissue MDA levels suggest that lipid peroxidation could be attributed to reactive oxygen metabolites (ROM) released from stimulated, recruited, and resident granulocytes. Tissue levels of MDA and PGE₂ suggest a role for granulocyte-derived mediators of intestinal inflammation in the massive secretory response in cases of acute equine colitis. Tissue MDA and PGE₂ levels may be useful laboratory tools to quantify and characterize intestinal secretory inflammatory responses in acute inflammatory conditions in the equine colon.     

Neuromuscular and vascular hamartoma of the small intestine in an F344 rat

An intestinal mass was found in the border area of the jejunum and ileum of a 110-week-old male F344 rat. Histopathologically, the mass protruded into the lumen and was covered with intestinal epithelium, exhibiting a normal architecture. The lesion was located in the submucosa and consisted of loose connective tissue, smooth muscle, scattered ganglion cells, and blood vessels of various sizes. Although these components showed an irregular and disordered structure, no cellular atypia, increased proliferation activity, or invasive growth to adjacent tissues were detected. Immunohistochemical analyses revealed that smooth muscle, ganglion, and endothelial cells were positive for α-smooth muscle actin and vimentin, S-100, and CD34 and von Willebrand factor, respectively, indicating maturation of these cells. Thus, the mass was diagnosed as a neuromuscular and vascular hamartoma of the small intestine. To the best of our knowledge, this is the first report of this type of lesion in rodents.     

Evaluation of nitric oxide as an inhibitory neurotransmitter in the equine ventral colon

OBJECTIVE: To determine the role of nitric oxide and an apamin-sensitive nonadrenergic noncholingeric inhibitory transmitter on contractility of the ventral colon of horses. SAMPLE POPULATION: Strips of the circular and longitudinal muscle layers and taenia of the ventral colon from 14 horses. PROCEDURE: Muscle strips were suspended in tissue baths and attached to force transducers. Contractile activity of circular, longitudinal, and taenia muscle strips in response to electrical field stimulation was measured after addition of apamin and a nitric oxide inhibitor, N-nitro-L-arginine methyl ester (L-NAME). RESULTS: Electrical field stimulation reduced contractile activity in the circular muscle layer and taenia but not the longitudinal muscle layer. Addition of L-NAME significantly reduced inhibitory contractile activity at all frequencies for the circular muscle layer, whereas a significant effect was evident for the taenia only at the highest frequency. The combination of L-NAME and apamin resulted in a significant reduction in inhibition of the taenia at all frequencies but for circular muscle only at lower frequencies. CONCLUSIONS AND CLINICAL RELEVANCE: Nitric oxide and an apamin-sensitive neurotransmitter appear to mediate a component of inhibitory transmission in the circular muscle and taenia, but not the longitudinal muscle layer, of the equine ventral colon. Nitric oxide has a role in regulating contractile activity of the equine ventral colon, and nitric oxide synthase inhibitors may be useful in horses with ileus of the large colon.     

Pharmacological modulation of postprandial colonic motor activity in the pony

The contractile activity of the equine large intestine exhibited a biphasic response to feeding: enhancement of migrating complexes passing along the colon and an increase of 50% in cyclic variations in smooth muscle at intervals of 20 min on the left ventral colon for a period of 5 to 7 h postfeeding. The cholinergic agonist, bethanechol (50 micrograms/kg subcutaneously), induced both the migrating complexes and the cyclic variations at intervals of 10-15 min. In contrast, the intra-arterial infusion of PGF2 alpha (3 micrograms/kg/min) increased the contractile activity during infusion, but without inducing distinct patterns of activity. Atropine but not indomethacin or flunixin pre-treatment prevented the effects of postprandial, cholinergic and PGF2 alpha stimulation of colonic motility, suggesting that the gastrocolonic reflex involved mainly cholinergic stimulation of the caecum and replicated colon, including the prostaglandin F2 alpha excitatory effects.     

Evaluation of substance P as a neurotransmitter in equine jejunum

OBJECTIVE: To determine whether substance P (SP) functions as a neurotransmitter in equine jejunum. SAMPLE POPULATION: Samples of jejunum obtained from horses that did not have lesions in the gastrointestinal tract. PROCEDURE: Jejunal smooth muscle strips, oriented in the plane of the circular or longitudinal muscle, were suspended isometrically in muscle baths. Neurotransmitter release was induced by electrical field stimulation (EFS) delivered at 2 intensities (30 and 70 V) and various frequencies on muscle strips that were maintained at low tension or were under contraction. A neurokinin-1 receptor blocker (CP-96,345) was added to baths prior to EFS to interrupt SP neurotransmission. Additionally, direct effects of SP on muscle strips were evaluated, and SP-like immunoreactivity was localized in intestinal tissues, using indirect immunofluorescence testing. RESULTS: Substance P contracted circularly and longitudinally oriented muscle strips. Prior treatment with CP-96,345 altered muscle responses to SP and EFS, suggesting that SP was released from depolarized myenteric neurons. Depending on orientation of muscle strips and stimulation variables used, CP-96,345 increased or decreased the contractile response to EFS. Substance P-like immunoreactivity was detected in the myenteric plexus and circular muscle layers. CONCLUSIONS AND CLINICAL RELEVANCE: Substance P appears to function as a neurotransmitter in equine jejunum. It apparently modulates smooth muscle contractility, depending on preexisting conditions. Effects of SP may be altered in some forms of intestinal dysfunction. Altering SP neurotransmission in the jejunum may provide a therapeutic option for motility disorders of horses that are unresponsive to adrenergic and cholinergic drugs.     

Intestinal choristoma in the subcutis of a dog

A 2-year-old, spayed female, Labrador Retriever-cross presented with a subcutaneous mass of several weeks' duration in the right flank region. Surgical excision and histologic examination were performed. The 1.0-cm-diameter mass was circumscribed, unencapsulated, and cystic with a bilayer wall. The inner layer resembled intestinal mucosa, including a tall columnar lining epithelium, crypt-like glands containing scattered neuroendocrine cells that were strongly immunopositive for synaptophysin, and a supporting lamina propria-like fibrovascular tissue that contained lymphocytes and plasma cells. The outer layer was 1- to 2-mm thick and was composed of intersecting and blending bundles of smooth muscle and collagen. Given the presence of organized intestinal tissues in the subcutis, the lesion was consistent with intestinal choristoma.