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Abstract

Objective—To evaluate the activation status of neutrophils in blood samples obtained from horses with naturally occurring colic associated with strangulating obstruction, nonstrangulating obstruction, or inflammatory bowel disease.

Animals—30 horses with naturally occurring colic and 30 healthy control horses.

Procedure—Activation status of neutrophils was determined by assessing the number of neutrophils that could pass through filters with 5-µm pores, cellsurface CD11-CD18 expression, and alterations in size and granularity of neutrophils.

Results—Horses with impaction or gas colic did not have evidence of activated neutrophils. Horses with inflammatory bowel disease consistently had evidence of activated neutrophils, including decreased leukocyte deformability, increased CD11-CD18 expression, increased neutrophil size, and decreased neutrophil granularity. Horses with strangulating colic had variable results. Of horses with strangulating colic, 7 of 14 had marked changes in filtration pressures, 5 of 14 had increased CD11-CD18 expression, 6 of 14 had changes in neutrophil size, and 5 of 14 had changes in neutrophil granularity. Among horses with strangulating colic, changes in deformability, size, and granularity of neutrophils correlated with an adverse outcome.

Conclusions and Clinical Relevance—Activated neutrophils were detected in all horses with inflammatory bowel disease and a few horses with strangulating colic. Correlation of activated neutrophils with horses that had strangulating colic that died or were euthanatized indicates that activated neutrophils are a negative prognostic indicator. Additional studies are needed to determine whether activated neutrophils contribute directly to the adverse outcome in horses with strangulating colic. (Am J Vet Res 2003;64:1364–1368)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To investigate the effects of acupuncture on neural activity detected by use of manganeseenhanced functional magnetic resonance imaging (fMRI) and elucidate the relationship between somatic acupoint stimulation and brain activation.

Animals—40 New Zealand White rabbits.

Procedure—Manganese-enhanced fMRI was performed in anesthetized rabbits manipulated with electroacupuncture (EA) on Zusanli (ST-36) and Yanglingquan (GB-34) acupoints. Image acquisition was performed on a 1.5T superconductive clinical scanner with a circular polarized extremity coil. T1- weighted images were acquired sequentially as follows: baseline, after mannitol injection, after manganese infusion, and 5 and 20 minutes after initiation of EA.

Results—Changes in focal neural activity were detected by use of manganese-enhanced fMRI. Stimulation on Zusanli (ST-36) for 5 minutes resulted in activation of the hippocampus, whereas stimulation on Yanglingquan (GB-34) resulted in activation of the hypothalamus, insula, and motor cortex. Activation became less specific after 20 minutes of EA. Furthermore, stimulation on ipsilateral acupoints led to bilateral brain activation.

Conclusions and Clinical Relevance—Each acupoint has a corresponding cerebral linkage, and stimulation on these points resulted in time-dependent neural activation. Understanding the linkage between peripheral acupoint stimulation and central neural pathways may provide a useful guide for clinical applications of acupuncture. (Am J Vet Res 2001;62:178–182)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine whether platelets and neutrophils become activated in dogs during short-distance sled-pulling activity.

Animals—18 physically fit adult Siberian Huskies.

Procedure—Dogs were allocated into 2 teams (9 dogs/team). Each team ran a course of approximately 6.4 km while pulling a sled that contained 2 people. Blood samples were collected immediately before and within 10 minutes after completion of sled-pulling activity. Blood was aspirated into sterile syringes and immediately transferred to evacuated tubes containing EDTA solution. Platelet activation status was evaluated by determining cell-surface P-selection expression, number of platelet aggregates and platelet microparticles, mean platelet-component (MPC) concentration, and mean platelet-component distribution width (MPCDW) concentration. Neutrophil activation status was evaluated by determining cell-surface CD11/CD18 expression, neutrophil size, and neutrophil granularity.

Results—Short-duration strenuous sled-pulling activity was associated with lower MPC concentration, higher MPCDW concentration, and higher cell-surface P-selectin expression after activation with phorbol myristate acetate. An increase in neutrophil CD11/CD18 expression and a decrease in neutrophil granularity were also observed after exercise.

Conclusions and Clinical Relevance—Results of this study provide evidence of priming and activation of platelets and activation of neutrophils after strenuous short-duration sled-pulling activity. Additional studies will be needed to determine whether these changes have adverse effects on animal performance or induce tissue injury. (Am J Vet Res 2003;64:855–859)

Full access
in American Journal of Veterinary Research

Abstract

Objectives

To determine whether platelets become activated and form platelet-platelet or platelet-neutrophil aggregates, or both, when subjected to shear.

Sample Population

Blood obtained from 3 Thoroughbreds.

Procedures

Blood, with PCV adjusted to 32 (low hematocrit) or 60 (high hematocrit)%, was subjected to shear rates of 11.25, 22.5, 45, 90, 225, and 750/s for 3 minutes by use of a cone-plate viscometer. Flow cytometric techniques were used to identify activated platelets, platelet-platelet aggregates, and platelet-neutrophil aggregates.

Results

Shear resulted in decreased platelet count, increased mean platelet volume, platelet activation, and formation of platelet-platelet and platelet-neutrophil aggregates. These changes occurred at lower shear rates in blood with high hematocrit. Platelet-neutrophil aggregate formation was inhibited by blocking P-selectin, but not CD11/CD18 receptors.

Conclusions

Shear-induced platelet activation and aggregate formation occur at physiologic shear rates.

Clinical Relevance

Shear-induced platelet activation may explain the exercise-associated platelet-neutrophil aggregates observed in Thoroughbreds undergoing treadmill exercise. (Am J Vet Res 1998;59:1243-1246)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate a point-of-care coagulation analyzer (PCCA) in dogs with coagulopathies and healthy dogs.

Animals—27 healthy and 32 diseased dogs with and without evidence of bleeding.

Procedure—Prothrombin time (PT), activated partial thromboplastin time (aPTT), and activated clotting time (ACT) were determined, using a PCCA and standard methods.

Results—Using the PCCA, mean (± SD) PT of citrated whole blood (CWB) from healthy dogs was 14.5 ± 1.2 seconds, whereas PT of nonanticoagulated whole blood (NAWB) was 10.4 ± 0.5 seconds. Activated partial thromboplastin time using CWB was 86.4 ± 6.9 seconds, whereas aPTT was 71.2 ± 6.7 seconds using NAWB. Reference ranges for PT and aPTT using CWB were 12.2 to 16.8 seconds and 72.5 to 100.3 seconds, respectively. Activated clotting time in NAWB was 71 ± 11.8 seconds. Agreement with standard PT and aPTT methods using citrated plasma was good (overall agreement was 93% for PT and 87.5% for aPTT in CWB). Comparing CWB by the PCCA and conventional coagulation methods using citrated plasma, sensitivity and specificity were 85.7 and 95.5% for PT and 100 and 82.9% for aPTT, respectively. Overall agreement between the PCCA using NAWB and the clinical laboratory was 73% for PT and 88% for aPTT. Using NAWB for the PCCA and citrated plasma for conventional methods, sensitivity and specificity was 85.7 and 68.4% for PT and 86.7 and 88.9% for aPTT, respectively.

Conclusions and Clinical Relevance—The PCCA detected intrinsic, extrinsic, and common pathway abnormalities in a similar fashion to clinical laboratory tests. (Am J Vet Res 2001;62:1455–1460)

Full access
in American Journal of Veterinary Research

Abstract

Objectives

To determine whether platelets become activated and form platelet-neutrophil aggregates during near-maximal treadmill exercise in horses.

Animals

4 Thoroughbreds.

Procedure

Horses were subjected to 4 standardized exercise tests on a treadmill, and blood samples were collected before exercise, at treadmill speed of 12 m/s, and 5 minutes after exercise. Flow cytometric techniques were used to identify activated platelets, and flow cytometric and microscopic techniques were used to identify platelet-neutrophil aggregates.

Results

Platelet-neutrophil aggregates increased from 2.8 ± 0.4% at rest to 17.2 ± 1.1% and 14.7 ± 1.6% during and after exercise, respectively. Platelet activation was not detected during or after exercise.

Conclusions

Platelet-neutrophil aggregates consistently form during strenuous exercise in horses. (Am J Vet Res 1998;59:393–396)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To establish the existence of platelet-derived proteins in equine plasma, with the future goal of developing an assay for the detection of in vivo platelet activation.

Animals

5 mature healthy horses.

Procedure

Platelet-rich plasma and platelet-poor plasma were prepared from anticoagulated blood. Platelets were separated from plasma proteins by gel filtration, then activated with 0.5 μM platelet-activating factor. Protease inhibitors were added, and the released platelet proteins were harvested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on the released platelet proteins and platelet-poor plasma, and the resultant silver-stained bands were compared. Immunoblot analysis was performed on released platelet proteins, using an antibody to human thrombospondin; human platelet-derived proteins served as the positive control for the antibody.

Results

Released platelet proteins in the presence of β-mercaptoethanol (reduced samples) contained several proteins that were not observed in plasma including (mean ± SEM) 194 ± 2, 159 ± 2, 151 ±2, 104 ± 2, and 95 ± 1 kd. Immunoblots of released platelet proteins had a prominent 180 ± 2-kd protein in reduced samples that was recognized by an antibody to human thrombospondin, and with prolonged color development, 2 additional less prominent proteins (166 ± 1 and 155 ± 1 kd) were observed.

Conclusions

Several proteins are released from activated equine platelets that are not detectable in normal equine plasma. Thrombospondin is one of the high molecular mass proteins released by activated equine platelets.

Clinical Relevance

An assay can be developed for detection of thrombospondin in equine plasma and may be useful for detection of in vivo platelet activation in horses. (Am J Vet Res 1997;58:954–960)

Free access
in American Journal of Veterinary Research

Summary

Acyloxyacyl hydrolase (aoah) is a lysosomal enzyme found in neutrophils and macrophages that acts to partially deacylate the lipid-A component of the endotoxin of gram-negative bacteria rendering it less toxic, yet maintaining much of its immunostimulatory potential. We have found that the activity of neutrophil aoah per cell increased during localized inflammation. The purpose of this study was to determine the mechanism(s) responsible for these increases in neutrophil aoah activity. Because changes in neutrophil maturity commonly are associated with inflammation, intravascular infusion of purified gram-negative bacterial lipopolysaccharide and sc injection of bovine recombinant granulocyte colony-stimulating factor was used to induce large numbers of circulating immature neutrophils. Immature neutrophils were found to have aoah activity equal to that of mature cells; however, when neutrophils were stimulated in vitro with known activators, aoah activity of activated cells was more than that of unstimulated cells. The increase in aoah activity was inversely related to prestimulation activity. Increases in aoah activity after neutrophil activation were not a result of de novo synthesis of the enzyme, because cycloheximide did not prevent activation-induced increases in activity.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine the influence of activated equine neutrophils on sulfated glycosaminoglycan metabolism of equine articular cartilage in vitro.

Animals

Articular cartilage explants harvested from the metacarpophalangeal joints of 7 horses.

Procedure

Proteoglycan degradation and synthesis were measured bv release of glycosaminoglycan from the explants, and incorporation of [35Slsulfate into newly synthesized glycosaminoglycan.

Results

Activated equine neutrophils significantly increased the release of glycosaminoglycan from explant matrix and the magnitude of that response was influenced by duration of exposure. This response varied significantly between horses, but was detected as early as 3 hours after co-cultures were initiated. In addition to enhancing degradation, incubation of explants with activated neutrophils for 3 days caused significant inhibition of glycosaminoglycan synthesis during a subsequent 3-hour pulse-labeling period. This response varied significantly between individual animals, but age was not a predictive factor.

Conclusion

Neutrophils may have a critical role in the process of cartilage degradation during equine inflammatory joint disease. (Am J Vet Res 1996;57:1738–1747)

Free access
in American Journal of Veterinary Research

Summary

Fibrin dots were induced in eyes of dogs by injection of autogenous citrated plasma into the anterior chamber. Twenty-four hours after dot formation, one 50-μl drop of tissue plasminogen activator at a concentration of 5 mg/ml (group 1, n = 7) was administered topically 9 times at 5-minute intervals, or a collagen shield that was hydrated with tissue plasminogen activator at a concentration of 5 mg/ml (group 2, n = 7) was applied. The contralateral eye served as a nontreated control. Serial photographs were taken of the fibrin dots after topical application of tissue plasminogen activator. Computerized morphometric analysis was then used to evaluate changes in cross-sectional surface area of the fibrin dot. There was no significant mean percentage decrease in dot surface area of treated eyes of group-1 dogs or in treated eyes of group-2 dogs. In addition, there was no significant difference in mean percentage decrease in dot surface area between treated eyes of group-1 and group-2 dogs.

Free access
in American Journal of Veterinary Research