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Abstract

Objective—To determine effect of leukocyte depletion on hematologic, morphologic, and metabolic variables of equine jejunum after induction of arterial lowflow ischemia and reperfusion by use of an extracorporeal circuit.

Animals—14 healthy adult horses.

Procedure—A segment of jejunum was surgically removed and maintained in an isolated circuit for 3 hours (control group), arterial flow was reduced to 20% of baseline for 40 minutes followed by 1 hour of reperfusion (low-flow group), or leukocyte depletion was filter-induced, and low-flow ischemia and reperfusion were conducted as in the low-flow control group (filter-treated group). Various metabolic, hemodynamic, and histomorphologic variables were evaluated, including effects of electrical field stimulation and L- N-nitro-arginine-methyl-ester (L-NAME) on contractile activity.

Results—The extracorporeal circuit appeared to maintain the jejunum within physiologic limits for an extended period. Low-flow ischemia with reperfusion induced significant differences in various measurements, compared with control specimens. Significant differences were not detected between the low-flow and filter-treated groups. Myeloperoxidase activity was greater in the low-flow group than the control group, whereas a difference was not detected between control and filter-treated groups.

Conclusions and Clinical Relevance—The extracorporeal circuit maintained intestine for 3 hours in a physiologic state and may be used for simulation of tissue injury. Leukocyte depletion generally did not attenuate the effects of low-flow ischemia and reperfusion on equine small intestine. ( Am J Vet Res 2001;62:87–96)

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in American Journal of Veterinary Research

Abstract

Objective—To determine efficacy of an extracorporeal circuit to maintain a segment of equine large colon for 3.5 hours and to evaluate the effect of low arterial flow on histologic and metabolic variables.

Sample Population—Segments of large colon from 15 healthy adult horses.

Procedure—The pelvic flexure was surgically removed and maintained in an isolated circuit. In the control group, tissue was evaluated for 3.5 hours, whereas in the low-flow group, arterial flow was reduced to 20% of baseline for 40 minutes followed by 2 hours of reperfusion. Various metabolic and hemodynamic variables were evaluated at 30-minute intervals. Effects of nitric oxide (NO) and L-N-nitro-arginine- methyl-ester (L-NAME) on contractile activity were determined, and histomorphologic evaluation was performed at the completion of the study.

Results—Low-flow ischemia with reperfusion caused significant histomorphologic differences, compared with the control group. In the low-flow group, significant differences included reduction in PaCO2, reduction in bicarbonate concentrations, increase in PaO2, and an increase in base deficit in arterial and venous blood samples. Other significant differences included increases in PCV, protein concentration, total WBC count, and albumin clearance for the low-flow group. Differences were not detected in inhibitory activity of the low-flow group relative to the control tissue with or without addition of NO and L-NAME.

Conclusion—The extracorporeal circuit maintained a segment of equine intestine for 3.5 hours and can be used to simulate ischemic injury. The extracorporeal circuit provides the potential to investigate pharmaceutic agents that can minimize intestinal injury. (Am J Vet Res 2000;61:1042–1051)

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in American Journal of Veterinary Research

Abstract

Objective—To determine expression of cyclooxygenase (COX) genes 1 and 2 (also called prostaglandin-endoperoxide synthases 1 and 2) and stability of housekeeping gene expression during low-flow ischemia and reperfusion in the jejunum of horses.

Animals—5 healthy adult horses.

Procedures—Horses were anesthetized, and two 30-cm segments of jejunum were surgically exteriorized. Blood flow was maintained at baseline (untreated) values in 1 (control) segment and was decreased to 20% of baseline (low-flow ischemia) for 75 minutes, followed by 75 minutes of reperfusion, in the other (experimental) segment. Biopsy samples were collected from experimental segments at baseline (T0), after 75 minutes of ischemia (T1), and after 75 minutes of reperfusion (T2); samples were collected from control segments at T0 and T2. Horses were euthanized 24 hours after induction of ischemia (T3), and additional samples were collected. Samples were evaluated histologically. Total RNA was extracted; expression of COX genes and stability of 8 housekeeping genes were determined via quantitative real-time PCR assays.

Results—COX-1 and COX-2 genes were constitutively expressed in baseline samples. Low-flow ischemia resulted in significant upregulation of COX-2 gene expression at each subsequent time point, compared with baseline values. The most stably expressed reference genes were β-actin and hypoxanthine phosphoribosyltransferase, whereas glyceraldehyde 3-phosphate dehydrogenase and β-2 microglobulin were the least stably expressed.

Conclusions and Clinical Relevance—Low-flow ischemia resulted in upregulation of COX-2 gene expression in the jejunum of horses. Housekeeping genes traditionally used as internal standards may not be stable in this tissue during arterial low-flow ischemia and reperfusion.

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in American Journal of Veterinary Research

Abstract

Objective—To determine the response to neostigmine of the contractile activity of the jejunum and pelvic flexure and the effects of a continuous rate infusion (CRI) of neostigmine in horses.

Animals—7 adult horses and tissue from 12 adult horses.

Procedures—A CRI of neostigmine (0.008 mg/kg/h) or placebo was administered to 6 horses in a crossover study design. Gastric emptying was evaluated by the acetaminophen test. The frequency of defecation and urination and the consistency and weight of feces were recorded throughout the experiment. The effect of neostigmine on smooth muscle contractile activity was evaluated in tissues from the jejunum and pelvic flexure. The effect of neostigmine and acetylcholine after incubation with muscarinic receptor antagonists (atropine and DAU 5884) and an acetylcholinesterase inhibitor (edrophonium) was also investigated in vitro.

Results—No difference was observed between neostigmine and placebo for time to reach peak plasma acetaminophen concentration and absorption rate constant. A CRI of neostigmine increased fecal production and frequency of urination. Neostigmine induced a dose-dependent increase of contractile amplitude in jejunum and pelvic flexure muscle strips. Incubation of muscle strips with atropine and DAU 5884 inhibited the response to acetylcholine and neostigmine. Incubation of smooth muscle strips from the jejunum with edrophonium increased the response to acetylcholine and had no effect on the response to neostigmine in vitro.

Conclusions and Clinical Relevance—A CRI of neostigmine increased fecal production and urination frequency in horses. A CRI of neostigmine did not decrease gastric emptying. Neostigmine stimulated contractile activity of jejunum and pelvic flexure smooth muscle strips in vitro.

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in American Journal of Veterinary Research

Abstract

Objective

To compare the strength of the sutured linea alba, in vitro, using 2 suture patterns.

Animals

12 clinically normal llamas.

Procedure

2 incisions in the linea alba of 12 llamas were closed with a simple continuous or inverted cruciate pattern, and tissue was harvested after 10 days. In 6 llamas, the simple continuous line was intact; the inverted cruciate specimens contained 6 sutures. In 6 llamas, 1 knot was excised in the simple continuous pattern to simulate a failed line; the cruciate pattern contained 5 knots. Tissue sections were taken from cranial, between, and caudal to the linea alba incisions to compare fascial thickness. The sutured specimens were mounted in a mechanical testing system and tested to failure. A mixed-model ANOVA was used to evaluate the effects of suture pattern and incisional position on mechanical properties.

Results

Significant differences were not found between suture patterns or between location for yield force, failure force, or yield strain, whereas failure strain was lower for the intact simple continuous pattern than the inverted cruciate pattern (P = 0.003). From histomorphometric analysis, the caudal tissue specimens were significantly thinner than the middle tissue specimen cranial to the umbilicus (P = 0.006).

Conclusion

There was no significant difference in monotonic breaking strength of the linea alba sutured with the simple continuous or inverted cruciate pattern.

Clinical Relevance

These results justify the use of the simple continuous pattern over the cruciate pattern for ventral midline closure in llamas because of the ease of placement and speed. (Am J Vet Res 1996;57:938–942)

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in American Journal of Veterinary Research

SUMMARY

The ultrastructural injury that develops sequentially in the ascending colon during experimentally induced ischemia was examined in 6 halothane-anesthetized horses. Colonic ischemia was created by 2 types of vascular occlusion 24 cm proximal and distal to the pelvic flexure.

In all horses, transmural vascular compression was created. The colonic venous circulation was obstructed in 3 horses, whereas in the other 3 horses, arterial and venous circulation was obstructed. Two additional horses were anesthetized as controls for determination of any morphologic alterations associated with the experimental protocol.

Full-thickness colonic biopsy specimens were obtained from the antimesenteric border of the pelvic flexure at 0, 0.25, 0.5, 1, 1.5, 1.75, 2, 2.25, 2.5, 3, 3.5, 4, 4.5, and 5 hours during occlusion, and were studied by light and transmission electron microscopy. Morphologic alterations did not develop in the colon of control horses. Mucosal congestion was observed by light microscopy in the colon of horses with experimentally induced ischemia, but congestion developed early in those with obstructed colonic venous circulation, compared with those having arterial and venous obstruction. Inter- and intracellular vacuolation and loss of staining initially resulted in groups of 3 to 5 superficial luminal epithelial cells. Alterations in the glandular epithelium lagged behind those in the superficial epithelium, but were observed in both groups by 2 hours of obstruction. These changes progressed to 100% sloughing of all epithelium by 4.5 to 5 hours.

The initial cellular alterations, which were observed by transmission electron microscopy, developed at 0.25 hour in horses with colonic venous obstruction and was characterized by inter- and intracellular edema. By 1 hour in horses with colonic venous obstruction, vacuoles were observed within the basal lamina and some vacuoles contained intracellular organelles. These cellular changes were followed by increases in the intercellular gap and breaks between degenerating and more normal-appearing superficial epithelium, which led to sloughing of the epithelium. Endocrine cells by 1 hour also had evidence of ischemic injury.

Injury to the vascular circulation, including congestion and platelet accumulations within the mucosal capillaries was apparent by 0.25 hour in horses with venous obstruction. By 1 to 1.5 hours in both groups of horses with experimentally induced ischemia, loss of vascular integrity and leukocyte migration frequently were observed. Platelets, proteinaceous material, and cellular debris continued to accumulate, and by 2.25 hour capillary plugging frequently was observed. These results indicated that the initial ultrastructural injury in the ischemic colon consisted of degenerative changes in epithelial cells, which led to sloughing of degenerating and necrotic cells. Although injury between the 2 types of vascular obstruction differed, end results were similar. Ischemic vascular injury may lead to further vascular thrombosis and necrosis, resulting in an irreversible injury or contribute to difficulty in medically managing horses with natural ischemia during the perioperative period.

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in American Journal of Veterinary Research

Abstract

Objective

To determine whether xanthine oxidase and dehydrogenase activities are altered during low flow ischemia and reperfusion of the small intestine of horses.

Animals

5 clinically normal horses without histories of abdominal problems.

Procedure

With the horse under general anesthesia, a laparotomy was performed and blood flow to a segment of the distal jejunum was reduced to 20% of baseline for 120 minutes and was then reperfused for 120 minutes. Biopsy specimens were obtained before, during, and after ischemia for determination of xanthine oxidase and dehydrogenase activities, and for histologic and morphometric analyses.

Results

Percentage of xanthine oxidase activity (as a percentage of xanthine oxidase and dehydrogenase activity) was not altered during ischemia and reperfusion. An inflammatory response developed and progressed during ischemia and reperfusion. Mucosal lesions increased in severity after ischemia and reperfusion. Mucosal surface area and volume decreased during ischemia and continued to decrease during reperfusion. Submucosal volume increased slightly during ischemia, and continued to increase during reperfusion.

Conclusions and Clinical Relevance

Evidence for conversion of xanthine dehydrogenase to xanthine oxidase during ischemia was not found. Factors other than production of reactive oxygen metabolites may be responsible for progressive epithelial loss, decrease in mucosal surface area and volume, and increase in submucosal volume observed in this study. Other methods of determining xanthine oxidase activity that detect the enzyme in sloughed epithelial cells should be used to better define the importance of this pathway in jejunal reperfusion injury in horses. (Am J Vet Res 1998;59:772-776)

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in American Journal of Veterinary Research

SUMMARY

Microvascular permeability of the jejunum of clinically normal equids and microvascular permeability associated with 60 minutes of ischemia (25% baseline blood flow) and subsequent reperfusion were investigated. Eight adult horses were randomly allotted to 2 equal groups: normal and ischemic/reperfusion injury. Lymphatic flow rates, mesenteric blood flow, and lymph and plasma protein concentrations were determined at 15-minute intervals throughout the study. Microvascular permeability was determined by estimates of the osmotic reflection coefficient, which was determined when the ratio of lymphatic protein to plasma protein concentration reached a constant minimal value as lymph flow rate increased (filtration-independent lymph flow rate), which occurred at venous pressure of 30 mm of Hg. Full-thickness jejunal biopsy specimens were obtained at the beginning and end of each experiment, and were prepared for light microscopy to estimate tissue volume (edema) and for transmission electron microscopy to evaluate capillary endothelial cell morphology.

The osmotic reflection coefficient for normal equine jejunum was 0.19 ± 0.06, and increased significantly (P < 0.0001) to 0.48 ± 0.05 after the ische- mia/reperfusion period. Microscopic evaluation revealed a significant increase (P < 0.0001) in submucosal and serosal volume and capillary endothelial cell damage in horses that underwent ischemia/reperfusion injury. Results indicate that ischemia/re-perfusion of the equine jejunum caused a significant increase in microvascular permeability.

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in American Journal of Veterinary Research

Summary

Sixteen horses were allotted at random to 3 groups: vehicle only; low dosage (vehicle and 3 mg of U-74389G/kg of body weight); high dosage (vehicle and 10 mg of U-74389G/kg). These solutions were given prior to reperfusion. The ascending colon was subjected to 2 hours of ischemia followed by 2 hours of reperfusion. Before, during, and after ischemia, full-thickness colonic tissue biopsy specimens were obtained for measurement of malondealdehyde (mda) concentration and myeloperoxidase activity and for morphologic evaluation.

Although increases were not significant, mda concentration and myeloperoxidase activity increased during ischemia and reperfusion. Administration of U-74389G did not have significant effects on mda concentration and myeloperoxidase activity. However, the lower dosage tended (P = 0.08) to reduce myeloperoxidase activity at 30 and 60 minutes of reperfusion.

In horses of the vehicle-only group, ischemia induced a decrease in mucosal surface area that was continued into the reperfusion period (P ≤ 0.05). Administration of U-74389G at both dosages (3 and 10 mg/kg) prevented the reperfusion-induced reduction in mucosal surface area, which was significant at 60 minutes (high dosage; P = 0.05) and 90 minutes (low and high dosages; P = 0.02). After initial reduction in horses of all groups, mucosal volume increased for the initial 60 minutes of reperfusion.

Our results indicate that lipid peroxidation may be partially involved in continued cellular death after ischemia of the ascending colon of horses. The 21-aminosteroid, U-74389G, prevented further loss of mucosa and partially attenuated the induced increase in myeloperoxidase activity during reperfusion of the ascending colon.

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in American Journal of Veterinary Research

Abstract

Objective

To determine the effects of the 21-amino-steroid, U-74389G, on reperfusion of the equine jejunum, using total (TVO) and partial (PVO) vascular occlusion during the ischemic period.

Design

TVO: 16 healthy horses were randomly allotted to 3 groups—4 horses received the vehicle alone, 6 horses received a low dosage (3 mg/kg of body weight), and 6 horses a high dosage (10 mg/kg) of U-74389G. PVO: 10 healthy horses were randomly allotted to 2 groups—5 horses received the vehicle alone, and 5 horses received the low dosage (3 mg/kg) of U-74389G.

Procedure

TVO was induced for 1 hour followed by 2 hours of reperfusion. During PVO, blood flow was reduced to 20% of baseline for 2 hours, followed by 2 hours of reperfusion.

For both models, either the vehicle alone or the drug was given 15 minutes prior to reperfusion. Samples were obtained before, during, and after ischemia for determination of myeloperoxidase (MPO) activity, malondealdehyde (MDA) concentration, concentration of conjugated dienes (PVO experiment only), and morphometric analysis.

Results

TVO: tissue concentration of MDA and MPO activity were not altered in any group by ischemia or reperfusion. During ischemia, mucosal volume and surface area were reduced. After reperfusion, no further reduction occurred. After initial decrease in submucosal volume during ischemia, there was a significant increase after reperfusion in the vehicle-only group (P < 0.05). PVO: there were no alterations in the concentration of either MDA or conjugated dienes. There was a significant increase in the activity of MPO during ischemia and reperfusion (P< 0.05). These effects were similar for the vehicle-only and drug groups. During ischemia, there was a significant decrease in mucosal surface area and volume (P< 0.05), that was continued during reperfusion for the vehicle-only group (P< 0.05). Submucosal volume increased during reperfusion (P< 0.05). Serosal volume was increased during ischemia and reperfusion.

Conclusions and Clinical Relevance

Reduced blood flow during ischemia (PVO group) caused continued loss in mucosal volume and surface area during reperfusion. At the dosage given, the 21-aminosteroid, U-74389G, was not effective in preventing continued reduction in mucosal volume and surface area after restoration of blood supply in the horses subjected to reduced blood flow. (Am J Vet Res 1996; 57:762–770)

Free access
in American Journal of Veterinary Research