BACKGROUND: Opioids and nitric oxide (NO) are functionally linked in the regulation of intestinal motility. OBJECTIVES: To investigate the role of NO in the opium induced bowel dysfunction in mice. METHODS: Sixty-six male mice received incrementally doses of the following treatments in six groups for 5 consecutive days: 1) Opium (0.2, 0.3, 0.4, 0.5 and 0.6mg/30g/day), 2) N-nitro-L-arginine methyl ester (L-NAME, 5,7.5,10,15 and 20mg/kg/day), 3) L-arginine (5-20mg/kg/day), 4) Opium+L-NAME, 5) Opium+L-arginine and 6) distilled water. At the end of the treatment, the abdomen was opened; some pieces of duodenal and proximal colon were taken to determine NO synthase (NOS) expression and nitrite levels, and some isolated rings from those parts of small and large intestine were prepared and transferred to the organ bath system to study intestinal motility. RT-PCR was used to determine the NOS gene expression. To determine the small intestinal transit, 30 mice in six groups, were used for oral administration of charcoal+gum in vivo. RESULTS: Opium decreased amplitude of the duodenum and ileum contractions, but increased frequency of duodenal and mid colon contractions (P<0.05). While the gene expression of inducible, neuronal and endothelial NOS was increased in colon (P<0.05), a reduced neuronal and endothelial NOS gene expression was shown in duodenum. The charcoal+gum transit was decreased in opium-treated animals compared to the control group (19.9%). However, L-arginine increased this transit while L-NAME decreased it. CONCLUSIONS: Opium induced intestinal smooth muscle spasms, which result in the decreased intestinal movements. The alterations in NOS gene expression may be a compensation mechanism against opium-induced intestinal dysfunction.

This study determined the presence of nitric oxide synthesis isoforms (nNOS, iNOS, and eNOS) in thoracic spinal cord segments and nodose ganglia of rats with gamma-irradiated livers. Male rats (n = 32) were divided into equal groups A, B, C, and D. In group A, the controls, no radiation was applied, while groups B, C, and D received 10 Gy of ionising gamma radiation. The rats of group B were euthanized at the end of the first day (d1), those of group C on the second day (d2), and those of group D on the third day (d3). The liver, spinal cord segments, and nodose ganglion tissues were dissected and fixed, and the liver sections were examined histopathologically. The other tissues were observed through a light microscope. Regeneration occurred at the end of d3 in hepatocytes which were radiation-damaged at the end of d1 and d2. On d1, some nNOS-positive staining was found in the neuronal cells of laminae I–III of the spinal cord and in neurons of the nodose ganglion, and on d3, some staining was observed in lamina X of the spinal cord, while none of note was in the nodose ganglion. Dense iNOS-positive staining was seen on d1 in the ependymal cells of the spinal cord and in the glial cells of the nodose ganglion, and on d3, there was still considerable iNOS staining in both tissues. There was clear eNOS-positive staining in the capillary endothelial cells of the spinal cord and light diffuse cytoplasmic staining in the neurons of the nodose ganglion on d1, and on d3, intense eNOS-positive staining was visible in several endothelial cells of the spinal cord, while light nuclear staining was recognised in the neurons of the nodose ganglion. The nNOS, iNOS, and eNOS isoforms are activated in the spinal cord and nodose ganglion of rats after ionising radiation insult to the liver.

To study the effect of follicle aspiration on the follicular, luteal, and uterine dynamics and hemodynamics associated with estradiol and nitric oxide changes, five aged mares >18 years were granted from Police department due to aging were subjected to transvaginal dominant follicle aspiration (TVA) on Day 12 after ovulation (Day 0). Before TVA, three complete estrous cycles were studied. The three large follicles were tracked after spontaneous ovulation and follicle aspiration starting from the day of aspiration (Day 0) till Day 12 and Day 15 of the first and second estrous cycles following aspiration. Follicle number, diameter, antral diameter, area, antral area, color area, color area %, granulosa area, and granulosa color area % in relation to the diameter of the dominant and subordinate follicles were determined during the spontaneous estrous cycles. Follicle diameter, antral diameter, area, antral area, color area, color area %, granulosa area, granulosa color area %, CL color area % and uterine horns color area % in relation to days before or after follicle aspiration were determined during the spontaneous estrous cycles. Blood samples collected on the same day of ultrasound and Doppler assessment were used to assay estradiol and nitric oxide concentrations. Days after ovulation influenced Uterine horn area (P<0.05) and color area (P<0.05), CL color area (P< 0.05) and CL color area % (P<0.05), Estradiol (E2, P<0.0001), and Nitric oxide (NO, P<0.0001). Days after aspiration influenced follicle antrum (P<0.01), follicle circulatory (P <0.0001), CL diameter (P<0.05), CL area (P<0.05), and E2 (P=0.007). Aspiration influenced the follicle circulatory (P<0.0001) and CL color area (P<0.05) and tended to influence E2 (P>0.05). In conclusion, aspiration of the dominant follicle simulates spontaneous ovulation in aged mares. Day 12 after ovulation could be selected for aspirating the dominant follicle without disturbing the follicular dynamics and hemodynamics. Aspirated dominant follicle become a corpus luteum with 2-3 days after aspiration.

OBJECTIVE To determine the impact of mechanical ventilation (MV) and perfusion conditions on the efficacy of pulse-delivered inhaled nitric oxide (PiNO) in anesthetized horses. ANIMALS 27 healthy adult horses. PROCEDURES Anesthetized horses were allocated into 4 groups: spontaneous breathing (SB) with low (< 70 mm Hg) mean arterial blood pressure (MAP; group SB-L; n = 7), SB with physiologically normal (≥ 70 mm Hg) MAP (group SB-N; 8), MV with low MAP (group MV-L; 6), and MV with physiologically normal MAP (group MV-N; 6). Dobutamine was used to maintain MAP > 70 mm Hg. Data were collected after a 60-minute equilibration period and at 15 and 30 minutes during PiNO administration. Variables included Pao2, arterial oxygen saturation and content, oxygen delivery, and physiologic dead space-to-tidal volume ratio. Data were analyzed with Shapiro-Wilk, Mann-Whitney U, and Friedman ANOVA tests. RESULTS Pao2, arterial oxygen saturation, arterial oxygen content, and oxygen delivery increased significantly with PiNO in the SB-L, SB-N, and MV-N groups; were significantly lower in group MV-L than in group MV-N; and were lower in MV-N than in both SB groups during PiNO. Physiologic dead space-to-tidal volume ratio was highest in the MV-L group. CONCLUSIONS AND CLINICAL RELEVANCE Pulmonary perfusion impacted PiNO efficacy during MV but not during SB. Use of PiNO failed to increase oxygenation in the MV-L group, likely because of profound ventilation-perfusion mismatching. During SB, PiNO improved oxygenation irrespective of the magnitude of blood flow, but hypoventilation and hypercarbia persisted. Use of PiNO was most effective in horses with adequate perfusion.

Objective—To image the spatial distribution of pulmonary blood flow by means of scintigraphy, evaluate ventilation-perfusion (VA/Q) matching and pulmonary blood shunting (Qs/Qt) by means of the multiple inert gas elimination technique (MIGET), and measure arterial oxygenation and plasma endothelin-1 concentrations before, during, and after pulse-delivered inhaled nitric oxide (PiNO) administration to isoflurane-anesthetized horses in dorsal recumbency. Animals—3 healthy adult Standardbreds. Procedures—Nitric oxide was pulsed into the inspired gases in dorsally recumbent isoflurane-anesthetized horses. Assessment of VA/Q matching, Qs/Qt, and Pao2 content was performed by use of the MIGET, and spatial distribution of pulmonary blood flow was measured by perfusion scintigraphy following IV injection of technetium Tc 99m–labeled macroaggregated human albumin before, during, and 30 minutes after cessation of PiNO administration. Results—During PiNO administration, significant redistribution of blood flow from the dependent regions to the nondependent regions of the lungs was found and was reflected by improvements in VA/Q matching, decreases in Qs/Qt, and increases in Pao2 content, all of which reverted to baseline values at 30 minutes after PiNO administration. Conclusions and Clinical Relevance—Administration of PiNO in anesthetized dorsally recumbent horses resulted in redistribution of pulmonary blood flow from dependent atelectatic lung regions to nondependent aerated lung regions. Because hypoxemia is commonly the result of atelectasis in anesthetized dorsally recumbent horses, the addition of nitric oxide to inhaled gases could be used clinically to alleviate hypoxemia in horses during anesthesia.

Objective-To assess effects of in vitro meloxicam exposure on metabolism in articular chondrocytes from dogs with naturally occurring osteoarthritis Sample-Femoral head cartilage from 16 dogs undergoing total hip replacement Procedures-Articular cartilage samples were obtained. Tissue sulfated glycosaminoglycan (SGAG), collagen, and DNA concentrations were measured. Collagen, SGAG, chondroitin sulfate 846, NO, prostaglandin E2 (PGE2), and matrix metalloproteinase (MMP)-2, MMP-3, MMP-9, and MMP-13 concentrations in culture medium were analyzed. Aggrecan, collagen II, MMP-2, MMP-3, MMP-9, MMP-13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4, ADAMTS-5, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, interleukin-1β, tumor necrosis factor-α, cyclooxygenase-1, cyclooxygenase-2, and nducible nitric oxide synthase gene expression were evaluated. Comparisons between tissues cultured without (control) and with meloxicam at concentrations of 0.3, 3.0, and 30.0 μg/mL for up to 30 days were performed by means of repeated-measures analysis. Results-Meloxicam had no effect on chondrocyte SGAG, collagen, or DNA concentrations. Expression of ADAMTS-5 was significantly decreased in all groups on all days, compared with the day 0 value. On day 3, culture medium PGE2 concentrations were significantly lower in all meloxicam-treated groups, compared with values for controls, and values remained low. Culture medium MMP-3 concentrations were significantly lower on day 30 than on day 3 in all meloxicam-treated groups. Conclusions and Clinical Relevance-Results suggested that in vitro meloxicam treatment of osteoarthritic canine cartilage for up to 30 days did not induce matrix degradation or stimulate MMP production. Meloxicam lowered PGE2 release from this tissue, and effects on tissue chondrocyte content and matrix composition were neutral.

Objective-To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. Sample Population-Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. Procedure-The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interluekin-1, tumor necrosis factor-α, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP. Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). Results-All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and nonstimulated cultures. Conclusion and Clinical Relevance—Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP.

Objective--To test the hypothesis that endothelium-derived nitric oxide modulates vasomotor reactivity in equine digital arteries. Design--Digital arteries were isolated from adult horses, and their vasodilator properties were examined in an in vitro controlled environment. Animals--Five adult horses (1 gelding, 4 mares) without evidence of hoof or vascular disease were studied. Procedure--Arterial rings with or without endothelium were exposed to endothelium-dependent vasodilator drugs in the presence or absence of a pharmacologic inhibitor of the enzyme nitric oxide synthase. Results--Vasodilator effects of 3 endothelium-dependent vasorelaxant agents were significantly greater in endothelium-intact vessels than in endothelium-denuded vessels. Moreover a nitric oxide synthase inhibitor reduced vasodilator responses to endothelium-dependent vasodilators in endothelium-intact arteries, but had no discernable effects in endothelium-denuded arteries. Conclusions--These findings indicate the presence of endothelium-derived relaxing factor/nitric oxide in blood vessels of horses, and identify vascular endothelium as an endogenous modulator of vasomotor tone in the digital arteries of this species.

Achyranthes japonica Nakai (A. japonica) is a medicinal herb found widely distributed throughout Korea. The biological activities of A. japonica are well-documented and include anti-fungal, anti-inflammatory, and immunity enhancement. The objective of the present study was to investigate the immune-related activities of A. japonica extract in dogs. The extract was acquired by ethanol extraction and purified by filtration. To examine the effect of A. japonica extract on immune cell viability, human lymphocytes, such as Jurkat T-cells and Ramos B-cells, were exposed to the extract. After treatment with the extract, the number of Ramos B-cells was increased, whereas Jurkat T-cells remained unaffected. Griess assay revealed decreased nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated mouse macrophage Raw 264.7 cells after exposure to A. japonica extract. To evaluate the in-vivo effect in dogs, feed containing A. japonica extract was provided to 8 dogs for 2 months. Blood samples were collected before, during, and after consumption of the feed. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples and the number of T-cells and B-cells were assessed using flow cytometry with anti-dog fluorescein isothiocyanate (FITC)-conjugated CD3 and anti-dog phycoerythrin (PE)-conjugated CD21 antibodies, respectively. We observed a significant increase in the average number of B-cells in the PBMCs during ingestion of the feed containing A. japonica. In addition, enzyme-linked immunosorbent assay (ELISA) revealed a decrease in the levels of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, in 3 out of 8 dogs and increased levels of interleukin-10 (IL-10), an anti-inflammatory cytokine, in 4 out of 8 dogs. Taken together, we believe that these changes indicate that A. japonica extract is beneficial in improving the immunity of dogs by stimulating B-cells and inducing production of anti-inflammatory responses.

OBJECTIVE To assess changes in biochemical and biophysical properties of canine RBCs during cold (1° to 6°C) storage in a licensed RBC additive solution (the RBC preservation solution designated AS-1) supplemented with ascorbic acid. SAMPLE Blood samples from 7 neutered male Greyhounds; all dogs had negative results when tested for dog erythrocyte antigen 1.1. PROCEDURES Blood was collected into citrate-phosphate-dextrose and stored in AS-1. Stored RBCs were supplemented with 7.1mM ascorbic acid or with saline (0.9% NaCl) solution (control samples). Several biochemical and biophysical properties of RBCs were measured, including percentage hemolysis, oxygen-hemoglobin equilibrium, and the kinetic rate constants for O2 dissociation, carbon monoxide association, and nitric oxide dioxygenation. RESULTS Greyhound RBCs stored in AS-1 supplemented with ascorbic acid did not have significantly decreased hemolysis, compared with results for the control samples, during the storage period. CONCLUSIONS AND CLINICAL RELEVANCE In this study, ascorbic acid did not reduce hemolysis during storage. Several changes in stored canine RBCs were identified as part of the hypothermic storage lesion.