Effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation

OBJECTIVE: To investigate the effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation. SAMPLE POPULATION: Blood samples from 6 Thoroughbreds. PROCEDURE: The degree of fluorescence associated with binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITC-annexin V in unactivated and adenosine diphosphate (ADP)-, platelet activating factor (PAF)-, and A23187-activated platelet samples in unfixed and 0.5, 1.0, and 2.0% formaldehyde-fixed samples was assessed by use of flow cytometry. RESULTS: In samples incubated with FITC-anti-human fibrinogen antibody prior to fixation, addition of 2.0% formaldehyde resulted in a 30% increase in total fluorescence in ADP- and PAF-activated samples and a 60% increase in A23187-activated samples. Fixation for 24 hours prior to addition of antibody resulted in reduced fluorescence of samples containing antihuman fibrinogen antibody for all 3 concentrations of formaldehyde in PAF-activated samples. The addition of all 3 concentrations of formaldehyde after incubation with FITC-annexin V resulted in significant increases in fluorescence in unactivated and activated platelet samples. As length of fixation time increased, there was a gradual increase in fluorescence that was significant at 24 hours. CONCLUSIONS: Because fixation with 2.0% formaldehyde results in significant changes in fluorescence in activated platelet samples containing anti-fibrinogen antibody, lower concentrations of formaldehyde should be used to fix equine platelet samples. Formaldehyde-fixed platelet samples should be analyzed within 12 hours of fixation to avoid artifactual increases in fluorescence. Fixation of samples containing FITC-annexin V should be avoided because of significant increases in fluorescence that may interfere with interpretation of results.     

Dynamics in serum of the inflammatory markers serum amyloid A (SAA), haptoglobin,fibrinogen and alpha2-globulins during induced noninfectious arthritis in the horse

Despite the importance of noninfectious joint diseases in equine medicine, little is known about the acute phase response which may be elicited if the local inflammatory process of noninfectious arthritis is sufficiently strong, Therefore the aim of this study was to monitor the systemic inflammatory response during experimentally-induced noninfectious arthritis by studying the dynamics in serum of the acute phase proteins serum amyloid A (SAA), haptoglobin, fibrinogen and alpha2-globulins. Twenty-four Standardbred horses, age 3-7 years, found healthy on thorough clinical, radiological, haematological and serum biochemical examination, were injected aseptically into the right midcarpal joint with amphotericin B. Blood samples were drawn before induction of arthritis (0 h), and at 8, 16, 24, 36 and 48 h postinduction and then on Days 3, 4, 5 and 15 postinduction. All horses developed lameness with joint effusion and joint heat as well as increased respiratory rate, heart rate and body temperature. The lameness started to decline after 24-36 h and, in most animals, systemic signs disappeared on Day 2 postinjection. The concentration of the acute phase proteins increased following induction of arthritis. The SAA concentrations were higher than baseline concentrations from 16 h postinduction and were maximal at 36-48 h (227 times baseline concentration). The haptoglobin concentrations were higher than baseline concentrations from 24 h and were maximal at 48-96 h (1.14 times baseline concentration). The maximal concentrations of fibrinogen were seen between 36-72 h postinjection and increased on average 0.87 times from baseline concentrations. The fibrinogen concentrations were higher than baseline concentrations from 24 h postinjection. Alpha2-globulins concentrations showed a minor increase and increased 0.55 times from baseline concentrations. The markers had returned to baseline concentrations by Day 15. Our results demonstrate that amphotericin B-induced arthritis in a single joint gives rise to a systemic acute phase response measurable as increased concentrations in serum SAA, haptoglobin, fibrinogen and alpha2-globulins during the first 2 weeks of the condition and, thereby, that such an increase need not be indicative of infectious arthritis. Further research should be aimed at determining whether chronic noninfectious arthritis in the horse gives rise to increased acute phase protein concentrations in serum.     

Moment arms about the carpal and metacarpophalangeal joints for flexor and extensor muscles in equine forelimbs

OBJECTIVE: To determine whether muscle moment arms at the carpal and metacarpophalangeal joints can be modeled as fixed-radius pulleys for the range of motion associated with the stance phase of the gait in equine forelimbs. SAMPLE POPULATION: 4 cadaveric forelimbs from 2 healthy Thoroughbreds. PROCEDURE: Thin wire cables were sutured at the musculotendinous junction of 9 forelimb muscles. The cables passed through eyelets at each muscle's origin, wrapped around single-turn potentiometers, and were loaded. Tendon excursions, measured as the changes in lengths of the cables, were recorded during manual rotation of the carpal (180 degrees to 70 degrees) and metacarpophalangeal (220 degrees to 110 degrees) joints. Extension of the metacarpophalangeal joint (180 degrees and 220 degrees) was forced with an independent loading frame. Joint angle was monitored with a calibrated potentiometer. Moment arms were calculated from the slopes of the muscle length versus joint angle curves. RESULTS: At the metacarpophalangeal joint, digital flexor muscle moment arms changed in magnitude by < or = 38% during metacarpophalangeal joint extension. Extensor muscle moment arms at the carpal and metacarpophalangeal joints also varied (< or = 41% at the carpus) over the range of joint motion associated with the stance phase of the gait. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings suggest that, apart from the carpal flexor muscles, muscle moment arms in equine forelimbs cannot be modeled as fixed-radius pulleys. Assuming that muscle moment arms at the carpal and metacarpophalangeal joints have constant magnitudes may lead to erroneous estimates of muscle forces in equine forelimbs.     

Slow fiber cluster pattern in pig longissimus thoracis muscle: implications for myogenesis

Recent evidence implicates fiber type proportions as playing a role in meat eating quality, and in pigs it has been suggested that the slow oxidative fibers contribute to both juiciness and tenderness. The fiber distribution in pigs is different from that found in most other species, in which the various types of skeletal muscle fiber are distributed in a "checkerboard" pattern, because in pigs the slow oxidative fibers have a clustered distribution. The initial processes leading to fiber clustering are likely to occur during myogenesis, but the precise mechanistic aetiology of this patterning and whether the slow oxidative fiber clusters occur in a random or ordered fashion is unknown. In the present study longissimus thoracis muscle from Large White crossbred pigs was sampled at the 10th rib, 48 h postmortem. Transverse cryo-sections were cut and histochemically stained to allow the identification of the main muscle fiber types: slow oxidative, fast glycolytic, and fast oxidative glycolytic. Images of the sections were captured and analyzed using point processes and Voronoi Tesselations to examine the randomness and spatial distribution of the clusters of slow oxidative fibers found in pig longissimus thoracis muscle. The results showed that an assumption of complete spatial randomness can be rejected and that a mathematical model incorporating a minimum distance of 1.7 to 2.0 microm between cluster centers produced fiber patterns similar to those observed in the original transverse sections of the muscle. In addition, if it assumed that the central fiber in each cluster is derived from primary myoblast progenitors, these results suggest that there may be some degree of repulsion between the primary fibers during the initial stages of cluster formation. The mechanistic basis of such repulsion is not clear, but it is speculated that secreted factors, such as sonic hedgehog or myostatin may play a role.     

Beta2-agonists and cAMP inhibit protein degradation in isolated chick (Gallus domesticus) skeletal muscle

1. The role of beta2-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours. 2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective hbetaagonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP. 3. The decrease in the rate of total protein degradation induced by 10(-5)M clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10(-5)M ICI 118.551, a selective fbeta2antagonist. 4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10(-4)M dibutyryl-cAMP or isobutylmethylxanthine. 5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by fbeta2adrenoceptors, with the participation of a cAMP-dependent pathway.     

Validation and clinical utility of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone in the horse

REASONS FOR PERFORMING STUDY: Parathyroid hormone (PTH) plays a critical role in the regulation of mineral metabolism in mammals. Until recently, the standard method for PTH measurement has been the 2nd generation intact-PTH (I-PTH) assay. Current evidence indicates that the I-PTH assay binds to the PTH molecule and to an inactive N-terminally truncated PTH fragment that tends to accumulate in the blood of uraemic patients. Therefore, a new 3rd generation PTH assay that detects only the whole PTH molecule (W-PTH; cyclase-activating PTH [CAP]) has been developed. OBJECTIVES: To validate this more specific W-PTH assay for measurement of equine PTH and evaluate its clinical utility. METHODS: W-PTH and I-PTH were measured in plasma samples from normal horses (adults and foals) and horses with nutritional secondary hyperparathyroidism (N2HPT) and with chronic renal failure (CRF). Replicate measurements and dilutional paralellism were used for assay validation. Changes in blood ionized calcium were induced by EDTA and CaCl2 administration. RESULTS: Performance of the W-PTH assay (accuracy, sensitivity, specificity and ability to detect changes in PTH in response to changes in calcium) was similar to that of the I-PTH assay. Surprisingly, the relative W-PTH concentration in normal horses and foals was higher than the relative I-PTH concentration. W-PTH values remained higher than I-PTH during acute hypo- and hypercalcaemia. An increase in both W-PTH and I-PTH concentrations was found in horses with N2HPT. In horses with CRF, W-PTH and I-PTH values were very low and no increase in I-PTH was observed. CONCLUSIONS: The W-PTH assay can be used for measurement of equine PTH. POTENTIAL RELEVANCE: The use of W-PTH assay is likely to improve the diagnosis of mineral metabolism in horses.     

Recombinant equine growth hormone administration: effects on synovial fluid biomarkers and cartilage metabolism in horses

REASONS FOR PERFORMING STUDY: Recombinant equine growth hormone (reGH) has recently been evaluated for effects on body condition and wound healing. It has the potential to influence articular cartilage via stimulation of IGF-1. OBJECTIVES: To investigate effects of administration on synovial joint metabolism. METHODS: Six mature horses were given 20 microg/kg bwt reGH daily for 8 weeks by i.m. injection. Three control horses were injected with sterile water. Serum and synovial fluid samples were collected at 6, 8, 11 and 16 weeks for GH and IGF-1 assays. Articular cartilage harvested at week 16 was evaluated by Western analysis using monoclonal antibodies BC-13, BC-4, 8-A-4 and CH-3. RESULTS: Concentrations of IGF-1 in serum and synovial fluid were significantly elevated (P < 0.05) at 6 and 8 weeks in the reGH group. Glycosaminoglycan concentrations in synovial fluid were significantly less than controls at these time points, suggesting that reGH may modulate proteoglycan metabolism in articular cartilage. In the reGH group, there were not any alterations in synovial fluid content of 3B3(-) epitope or aggrecan metabolite, or in aggrecan or link protein catabolites retained within cartilage, that might be expected with development of osteoarthritis. CONCLUSIONS: Intramuscular administration of reGH may be a more efficient means of delivery of IGF-1 to joints for cartilage resurfacing initiatives. POTENTIAL RELEVANCE: We found no alterations in cartilage metabolism indicative of development of osteoarthritis.     

Effect of photoperiod on hepatic growth hormone receptor 1A expression in steer calves

Photoperiod manipulation, specifically a long-day photoperiod (LDPP), increases milk production in lactating cattle. We have previously reported that the galactopoietic effect of LDPP is associated with an increase in circulating IGF-I, which seems to occur independently of changes in concentrations of GH, IGFBP-2, and IGFBP-3. This study tested the hypothesis that LDPP increases the expression of GH receptor (GHR) 1A messenger RNA (mRNA) in the liver. Two groups of Holstein steer calves (98 +/- 4 d old) were maintained indoors and exposed to LDPP (16-h light: 8-h dark; n = 6) or short-day photoperiod (SDPP; 8-h light: 16-h dark; n = 6) for 60 d. Calves were individually fed a grain- and alfalfa-based diet. Jugular blood samples were collected weekly and via cannula at 15-min intervals for a 4-h period on d 1, 26, and 55 of the study to monitor pulsatile hormone secretion. Serum was harvested and assayed for IGF-I, prolactin (PRL), and GH using RIA. Liver biopsies were obtained at 3-wk intervals to quantify changes in hepatic IGF-I and GHR 1A mRNA using real-time PCR. Steer BW increased during the study but did not differ between treatments. No differences in ADG or total DMI were observed. Relative to SDPP, calves on LDPP had higher (P < 0.05) serum IGF-I concentrations. Concentrations of PRL increased (P < 0.01) in calves exposed to LDPP compared with calves exposed to SDPP. Differences (P < 0.05) in pulsatile GH secretion were also detected. Hepatic IGF-I and GHR 1A mRNA were positively correlated with circulating IGF-I concentrations, and although both increased with time, they were not affected by photoperiod treatment. These results confirm that LDPP increases circulating concentrations of IGF-I, but this occurs independently of changes in IGF-I synthesis and GHR 1A mRNA expression in the liver. Therefore, our hypothesis that LDPP increases the expression of GHR 1A mRNA in the bovine liver is rejected.     

Evaluation of herd sampling for Salmonella isolation on midwest and northeast US dairy farms

Epidemiologic investigations of Salmonella infections in dairy cattle often rely on testing fecal samples from individual animals or samples from other farm sources to determine herd infection status. The objectives of this project were to evaluate the effect of sampling frequency on Salmonella isolation and to compare Salmonella isolation and serogroup classification among sample sources on 12 US dairy farms sampled weekly for 7-8 weeks. Three herds per state were enrolled from Michigan, Minnesota, New York and Wisconsin based upon predefined herd-size criteria. Weekly samples were obtained from cattle, bulk tank milk, milk filters, water and feed sources and environmental sites. Samples were submitted to a central laboratory for isolation of Salmonella using standard laboratory procedures. The herd average number of cattle fecal samples collected ranged from 26 to 58 per week. Salmonella was isolated from 9.3% of 4049 fecal samples collected from cattle and 12.9% of 811 samples from other sources. Serogroup C1 was found in more than half of the samples and multiple serogroups were identified among isolates from the same samples and farms. The percentage of herd visits with at least one Salmonella isolate from cattle fecal samples increased with overall herd prevalence of fecal shedding. Only the three herds with an average fecal shedding prevalence of more than 15% had over 85% of weekly visits with at least one positive fecal sample. The prevalence of fecal shedding from different groups of cattle varied widely among herds showing that herds with infected cattle may be classified incorrectly if only one age group is tested. Testing environmental sample sources was more efficient for identifying infected premises than using individual cattle fecal samples.