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.     

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.     

Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: Relationship with microvascular density during the estrous cycle

Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P₄) (5 ng/ml or 1 μg/ml) or estradiol 17β (E₂β) (50 pg/ml or 1 μg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and inducible forms of NOS in the uterus was assessed by Western blot and immunocytochemistry. Vascular density in endometrial tissue was determined on histologic sections. In the luteal phase, compared to the follicular phase, endometrial NO production increased without exogenous hormones and with exogenous E₂β (1 μg/ml). Although immunocytochemistry revealed iNOS and eNOS expression in the endometrium, no positive signal for iNOS was detected by Western blot. Endothelial NOS was observed in endometrial glands, endothelial cells, fibroblasts, blood and lymphatic vessels. Endometrial eNOS expression was the highest in the follicular and mid-luteal phases while it was found to be the lowest in the early luteal phase. In the follicular phase, hyperplasia of endometrial tissue with respect to myometrium was detected. No difference in vascular density was present between phases. All together, NO may play some roles in both proliferative and secretory phases of endometrial development in the mare.     

Evaluation of urinalysis as an aid in the diagnosis of equine grass sickness

To determine whether urinalysis can aid the diagnosis of equine grass sickness, samples of urine from 15 horses with acute grass sickness, eight horses with subacute grass sickness, 17 co-grazing horses and 17 stabled control horses were analysed. The samples from all of the horses with grass sickness had a significantly higher specific gravity, higher protein and creatinine concentrations and a significantly lower pH; the samples from the horses with acute grass sickness also had significantly higher glucose concentrations. These differences may support a diagnosis of grass sickness but they are not pathognomonic for the disease.     

Endotoxin induction of nitric oxide synthase and cyclooxygenase-2 in equine alveolar macrophages

OBJECTIVE: To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS). Sample Population-Alveolar macrophages obtained from 12 horses. PROCEDURE: Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 microg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde 3-phosphate dehydrogenase. In a second study, cells were incubated with LPS or vehicle for 24 hours. Culture medium was assayed for COX-2 and iNOS activity by determining prostaglandin E2 (PGE2) and total nitrite concentrations, respectively. RESULTS: Lipopolysaccharide induces COX-2 and iNOS mRNA in equine alveolar macrophages. Sequencing revealed that PCR products for COX-2 and iNOS had a high degree of nucleotide homology with the human sequences (91% COX-2, 93% iNOS). Production of mRNA for COX-2 and iNOS was accompanied by induction of enzyme activity. Comparing PCR fragment production, expression of mRNA for iNOS appeared to be less than that for COX-2. Induction of COX-2, but not iNOS, was LPS-concentration dependent. Conclusion-Lipopolysaccharide induces COX-2 and iNOS in equine macrophages. CLINICAL RELEVANCE: The induction of iNOS and COX-2 by LPS in equine macrophages suggests these enzymes may be important in the pathophysiology of sepsis. Pharmacologic modulation of iNOS and COX-2 activity may represent a novel therapeutic target in the management of endotoxemia in horses.     

Radiographic identification of the equine ventral conchal bulla

Involvement of the ventral conchal sinus (VCS) is an important diagnostic and prognostic feature in cases of the equine sinus disease. The authors aimed to ascertain if the caudo-dorsal extension of the VCS, the ventral conchal bulla (VCB) is identifiable on plain radiographs of cadaver skulls without sinus disease. Bilateral frontonasal sinus flaps were made in 10 equine cadaver skulls. Plain lateral, lateral oblique and dorso-ventral radiographs were then obtained followed by the same views taken with stainless steel wire outlining the caudal border of the VCB. Plain radiographs were randomised and blindly evaluated by two observers who marked where they believed the VCB to be positioned. This was then correlated with the true position of the VCB using radiographs with wires in place. The ease of identification of the VCB was classified as 'easy' or 'difficult'. The VCB was correctly identified in 70 per cent of lateral radiographs, but only 45 per cent of lateral oblique radiographs and 17 per cent of dorso-ventral radiographs. If a clinician was confident that he or she could identify the VCB, they were usually correct. Conversely if the clinician judged VCB identification as 'difficult', they usually identified it incorrectly. In the authors' clinical experience, the VCB of horses with sinusitis involving this compartment is more radiologically evident than in clinically normal horses. Knowledge of the normal radiographic anatomy of this structure should aid clinicians in identifying horses with sinusitis affecting the VCS.     

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.     

Vascular anatomy of the equine small colon

The vasculature of 22 small colons from dead adult ponies was perfused with latex or barium sulphate solution. The vascular anatomy was studied by use of dissection and alkali digestion of the latex specimens and microangiography of the barium sulphate-perfused specimens. The small colon is supplied by the caudal mesenteric artery. The left colic artery arises from the caudal mesenteric artery, which then becomes the cranial rectal artery. Branches from the left colic and cranial rectal arteries form anastomosing arcades that become narrower distally along the length of the small colon. From these arcades arise terminal arteries, which enter the small colon wall and give rise to a subserosal, an intermuscular, and a large submucosal plexus, with frequent anastomoses between them. The venous drainage closely parallels the arterial supply, except near to its origin from the portal vein, when the left colic vein and caudal mesenteric vein are separate from the corresponding arteries.     

Evaluation of the gene for inducible nitric oxide synthase as a radiosensitizer under hypoxic and oxic conditions

The radiosensitizing effect of inducible nitric oxide synthase (iNOS) was evaluated, in vitro, in a feline vaccine‐associated sarcoma (VAS) cell line and a canine osteosarcoma cell line (D17). The gene encoding the human iNOS was cloned into an expression plasmid under the control of a cytomegalovirus immediate early promoter. Transient transfections were performed in feline VAS cells and D17 cells. Nitric oxide was measured in the supernatant media 48 h later as an indirect measurement of iNOS expression. Cells were irradiated using cobalt‐60 under hypoxic or oxic conditions, and clonogenic assays were used to evaluate the effects of gene transfer on the sensitivity of cells to radiation. The results demonstrated that iNOS had no significant effect on improving the radiosensitivity of cells under oxic conditions. However, under hypoxic conditions, iNOS gene transfer significantly improved radiosensitization in osteosarcoma cells. These results demonstrate the feasibility of improving the outcome of radiotherapy in dogs with large bulky tumours using iNOS gene therapy.     

The role of prostanoids and nitric oxide in endotoxin-induced hyporesponsiveness of equine digital blood vessels.

Endotoxin has been implicated in the pathophysiology of acute laminitis. The aim of this study was to examine the direct effects of endotoxin on isolated equine digital blood vessels. Equine digital veins (EDV), incubated in Krebs-Henseleit solution containing lipopolysaccharide (LPS) (1 microg/ml) became hyporesponsive to 5-HT after 16 h. Cycloheximide and ibuprofen blocked this effect of LPS and increased the maximum response obtained to 5-HT when compared to control vessels. L-nitroarginine methyl ester (L-NAME) reversed the hyporesponsiveness caused by LPS. Vessels maintained in culture medium containing LPS also became hyporesponsive to 5-HT, an effect which was completely prevented by ibuprofen but only partially reversed by L-NAME. Measurements were made of 6-keto PGF1alpha and nitrite production by segments of equine digital artery and vein in culture medium alone or co-cultured with peripheral blood leucocytes. LPS did not stimulate nitrite production from vessel segments but increased nitrite release from leucocytes, an effect which was inhibited by cycloheximide and L-NAME. Lipopolysaccharide increased 6-keto PGF1alpha production by blood vessels, an effect which was inhibited by cycloheximide and ibuprofen but not L-NAME. No synergistic effect on release of nitrite or 6-keto PGF1alpha was noted in co-cultures of blood vessels and leucocytes. These data suggest that induction of cyclo-oxygenase by LPS was a major cause of hyporesponsiveness of digital blood vessels to 5-HT. Release of nitric oxide was not detectable in LPS-stimulated blood vessels maintained in culture even in the presence of activated leucocytes yet L-NAME did protect against LPS-induced hyporesponsiveness indicating nitric oxide synthase induction may play some role in the effect of LPS. These findings are important in furthering our understanding of the pathophysiological mechanisms underlying the vascular changes which occur in acute laminitis.     

Neuronal nitric oxide synthase is heterogeneously distributed in equine myofibers and highly expressed in endurance trained horses

Mammalian skeletal muscle expresses splice variants of neuronal nitric oxide synthase (nNOS). Skeletal muscles have a metabolically heterogeneous population of myofibers, and fiber composition in equine skeletal muscle is correlated with athletic ability in endurance events. In this study, we investigated whether nNOS expression in equine skeletal muscle is related to fiber type and endurance training. Biopsy samples obtained from the gluteus medius of sedentary- (SH) and endurance-trained (TH) horses were examined for the electrophoretic mobility of myosin heavy chain (MHC) and NOS activity. Serial tissue cross-sections were stained for myosin ATPase and nicotinamide adenine dinucleotide (NADH) reductase, and also immunostained for nNOS. The gluteus medius of TH had higher levels of nNOS expression and activity when compared to muscle from SH. In SH, nNOS was restricted to the subsarcolemmal area while in TH nNOS was also present at cytoplasmic sites. A splice variant of nNOS was heterogeneously distributed among the different myofibers, its expression being higher in fast-oxidative-glycolytic type IIA fibers than in fast-glycolytic type IIX fibers and absent in slow-twitch type I fibers. Trained horses had a significantly higher relative content of type IIA fibers, a greater oxidative capacity, and a lower percentage of type IIX fibers when compared with SH. The differences in muscle fiber typing between the 2 groups of horses reflected alterations that probably resulted from the endurance-training program. Overall, these results show that nNOS is differentially expressed and localized in the gluteus medius according to the fiber type and the athletic conditioning of the horses.     

Getah virus as an equine pathogen.

Getah virus is a member of the genus Alphavirus in the family Togaviridae and has been frequently isolated from mosquitoes. Seroepizootiologic studies indicate that the virus is mosquito-borne and widespread, ranging from Eurasia to southeast and far eastern Asia, the Pacific islands, and Australasia. The natural host animal of the virus was not known until the first recognized occurrence of Getah virus infection among racehorses in two training centers in Japan in 1978. Outbreaks of clinical disease due to Getah virus infection occur infrequently, and only one outbreak has been reported outside Japan; this was in India in 1990. Clinical signs of the disease are mild and nonlife-threatening and are characterized by pyrexia, edema of the hind limbs, swelling of the submandibular lymph nodes, and urticarial rash, as reported in the 1978 epizootic. The morbidity was 37.9% (722 of 1903 horses) in one training center, with 96% of 722 affected horses making a full clinical recovery within a week without any significant sequelae. Antibodies against Getah virus were detected in 61.2% (172 of 281) and 55.8% (254 of 455) of horses at two training centers, respectively. Virus isolation can be attempted in VERO, RK-13, BHK-21, and many other cell lines as well as in suckling mouse brain. Blood plasma collected from suspect cases of infection at the onset of pyrexia is the specimen of choice. A diagnosis of Getah virus infection can also be confirmed serologically based on testing acute and convalescent phase sera by using SN, CF, HI, and ELISA tests. An inactivated vaccine is available for the prevention and control of Getah virus infection in horses in Japan.     

Dynamic compressive strain inhibits nitric oxide synthesis by equine chondrocytes isolated from different areas of the cartilage surface

REASONS FOR PERFORMING STUDY: Chondrocytes within articular cartilage respond to the mechanical stresses associated with normal joint loading via a series of signalling pathways. Specific biomolecules, such as nitric oxide (NO), have been implicated in these mechanotransduction processes. It has been shown that the synthesis of NO can be inhibited by dynamic compressive strain of chondrocytes in vitro which, in turn, leads to an up-regulation of specific metabolic parameters. HYPOTHESIS: Chondrocytes isolated from different joint locations and seeded in agarose constructs respond in a distinct manner to the application of dynamic compression. METHODS: Chondrocytes were isolated separately from the equine patella groove and the femoral condyle, representing high loaded areas (HLA) and low loaded areas (LLA), respectively, of 6 specimens of different ages. The cells were seeded in agarose constructs and cultured either in an unstrained state or strained under dynamic loading at 1 Hz for 48 h. The synthesis of nitric oxide (NO), proteoglycan synthesis and chondrocyte proliferation were assessed. RESULTS: Equine chondrocytes were found to synthesise significant basal levels of NO, regardless of topographical origin or age of tissue. Marked differences in both proteoglycan synthesis and cell proliferation were, however, revealed between the 2 chondrocyte subpopulations. Dynamic compression inhibited NO synthesis but significant alterations in proteoglycan synthesis and cell proliferation were apparent in a minority of cases. CONCLUSIONS AND POTENTIAL RELEVANCE: The differential response of the subpopulations of chondrocytes derived from the HLA and LLA provides a potential mechanism which enables the biomechanical demands of differing joint regions to be maintained.     

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.     

Normal motility of the cecum and right ventral colon in ponies

To study the normal motility of the cecum and right ventral colon (RVC) in 3 mature Shetland ponies, a 6-part, indwelling, intraluminal catheter system was used to measure intraluminal pressure changes. Three catheters were placed in the cecum at 10, 25, and 40 cm from the cecocolic orifice, and 3 catheters were placed in the RVC at 10, 20, and 30 cm from the cecocolic orifice. Recordings were made during the interdigestive period beginning 2 weeks after surgical operation was done. Frequent, low-amplitude peaks (0.35 +/- 0.13 coordinated peaks/min) were seen involving the cecal body and caudal cecal base, which represented a haustra-to-haustra mixing pattern. Coordinated pressure peaks originated in the cecal body and progressed to the cranial cecal base (0.07 +/- 0.01/min) or originated in the cranial cecal base and progressed to the cecal body (0.07 +/- 0.04/min). Associated with a loud rush of ingesta heard on transabdominal auscultation and progression of liquid ingesta confirmed with barium contrast radiography, there was a series of coordinated, progressive pressure peaks which originated in the cecal body, sequentially involved the cecal base, traversed the cecocolic orifice, and extended into the RVC (0.36 +/- 0.05/min). It seemed that a pacemaker region existed in the cecal body and initiated the important aborally propagated progressive pattern responsible for the transit of ingesta from the cecum to the RVC. A separate mechanism for the transit of gas was not identified. In the RVC, infrequent, nondirectional, low-amplitude segmental pressure peaks (0.12 +/- 0.06/min), and aborally progressive coordinated pressure peaks originating at the beginning of the RVC (0.09 +/- 0.02/min), occurred.(ABSTRACT TRUNCATED AT 250 WORDS)