Effects of hydrocortisone and aminophylline on the aggregation of equine platelets in vitro

The purpose of this study was to evaluate in vitro the effects of hydrocortisone and aminophylline on adenosine diphosphate (ADP)-induced platelet aggregation in horses. Blood samples from 30 healthy Thoroughbred horses were collected by via jugular venipuncture to assess platelet aggregation. Platelet-rich and platelet-poor plasma were prepared from all samples by centrifugation and divided into three different aliquots. In the first aliquot, platelet aggregation was measured after platelet activation with 1 µM and 0.5 µM ADP (Group A). In the other two aliquots, the effect of a 10 min preincubation with hydrocortisone (Group B) or aminophylline (Group C) on ADP-induced aggregation at final ADP concentrations of 1 µM and 0.5 µM was observed. Platelet aggregation, recorded by an aggregometer, was evaluated by measuring the maximum degree of platelet aggregation and the initial velocities of platelet aggregation were obtained. Our results demonstrated the inhibitory effect of hydrocortisone and the induction effect of aminophylline on equine platelet responses in vitro.     

Effects of equine recombinant interleukin-1alpha and interleukin-1beta on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants

OBJECTIVES: To evaluate the effects of equine recombinant interleukin-1alpha (rEqIL-1alpha) and recombinant interleukin-1beta (rEqIL-1beta) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture. SAMPLE POPULATION: Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse. PROCEDURE: Expression constructs containing cDNA sequences encoding EqIL-1alpha and EqIL-1beta were generated, prokaryotically expressed, and the recombinant protein purified. Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse were separately randomized to receive rEqIL-1alpha or rEqIL-1beta treatments 10 to 500 ng/ml). Proteoglycan release was evaluated by 1,9-dimethylmethylene blue spectrophotometric analysis of explant media glycosaminoglycan (GAG) concentration and release of 35S-sulfate-labeled GAG to explant media. Proteoglycan synthesis was assessed by quantification of 35S-sulfate incorporation into proteoglycan. Explant media PGE2 concentrations were evaluated using a PGE2-specific enzyme-linked immunoassay. Data were collected at 48-hour intervals and normalized by DNA content. RESULTS: Proteoglycan release was induced by rEqIL-1alpha and rEqIL-1beta at concentrations > or =0.1 ng/ml, with 38 to 76% and 88 to 98% of total GAG released by 4 and 6 days, respectively. Inhibition of proteoglycan synthesis (42 to 64%) was observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. Increased PGE2 concentrations were observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. CONCLUSIONS AND CLINICAL RELEVANCE: The rEqIL-1 induced potent concentration-dependent derangement of equine chondrocyte metabolism in vitro. These findings suggest this model may be suitable for the in vitro study of the pathogenesis and treatment of joint disease in horses.     

The effects of aspirin and paracetamol on the aggregation of equine blood platelets

The responses of equine blood platelets in citrated platelet-rich plasma to arachidonic acid, U44069 (prostaglandin endoperoxide analogue), adenosine 5'-diphosphate, platelet-activating factor or collagen were investigated by turbidimetric aggregometry. Pre-treatment of the platelets with aspirin (1 mmol/1) or Paracetamo1 d-3 mmol/1) abolished shape change and aggregation in response to arachidonic acid; decreased the rate of aggregation in response to collagen, with no separate effect on shape change; had no marked effect on aggregation caused by the other agonists; but in no case transformed irreversible aggregation to reversible aggregation. We conclude that thromboxane A₂ generation is of minor importance in the aggregation of equine platelets, and in particular that thromboxane A₂ is not a significant mediator of irreversible aggregation.     

The effect of a low molecular weight heparin on coagulation parameters in healthy cats

The low molecular weight heparin (LMWH), dalteparin sodium, was administered subcutaneously (100 IU/kg) to 8 healthy cats twice daily for 13 doses. Anti-activated factor X (anti-Xa) activity was measured prior to administration (time 0), and 4, 6, 8, and 12 h after the 1st dose, 4 h after administration of the 3rd dose, and at 4, 6, 8, and 12 h after the last dose. Four cats developed measurable anti-Xa activity 4 h following a single dose, returning to baseline by 6 h. Anti-Xa activity was not detected at any time point in 4 cats. Prothrombin time (PT), activated partial thromboplastin time (APTT), and antithrombin (AT) concentrations were unaffected by LMWH administration. Dalteparin, at 100 IU/kg SC, did not achieve anti-Xa activity in 4 out of 8 cats and failed to maintain anti-Xa activity beyond 4 h in the other 4 healthy cats.     

Effects of polysulfated glycosaminoglycan and hyaluronan on prostaglandin E2 production by cultured equine synoviocytes

OBJECTIVE: To investigate effects of the anti-arthritic agents hyaluronan and polysulfated glycosaminoglycan (PSGAG) on inflammatory metabolism in cultured equine synoviocytes. SAMPLE POPULATION: Synoviocytes cultured from samples obtained from the metacarpophalangeal joints of 4 horses. PROCEDURE: Equine synoviocytes were grown in monolayer culture. Synoviocytes were stimulated with lipopolysaccharide (LPS) and simultaneously treated with various concentrations of hyaluronan or PSGAG for 48 hours. Three hyaluronan preparations were compared. Prostaglandin E2 (PGE2) concentrations in culture medium were measured, using radioimmunoassay. RESULTS: The highest concentrations of hyaluronan and PSGAG tested inhibited PGE2 production. CONCLUSIONS AND CLINICAL RELEVANCE: Clinically achievable concentrations of hyaluronan and PSGAG inhibited PGE2 synthesis by cultured equine synoviocytes. This anti-inflammatory action may be a mechanism through which these agents exert anti-arthritic effects. The effect was obtained at concentrations that can be achieved by use of intra-articular, but not systemic, administration of hyaluronan or PSGAG.     

Effect of dietary corn oil supplementation on equine gastric fluid acid, sodium, and prostaglandin E2 content before and during pentagastrin infusion

The effect of corn oil (approximately 60% [wt/vol] linoleic acid) dietary supplementation on various components of equine gastric secretion was studied by use of a repeated-measures experimental design. Four healthy adult ponies were surgically fitted with gastric cannulas. The ponies were then fed a free-choice hay diet for 5 weeks, which was followed by 5 weeks of the same diet supplemented with 45 mL of corn oil daily. Gastric contents were analyzed under basal and pentagastrin-stimulated conditions once weekly during the latter 2 weeks on each diet. Gastric contents were collected at 30-minute intervals, and volume, hydrogen ion concentration, sodium content, and prostaglandin E2 (PGE2) content were measured. Data were analyzed by a linear fixed-effect modeling procedure. During the diet supplemented with corn oil, the ponies had, under basal and pentagastrin-stimulated conditions, significantly decreased acid output and significantly increased PGE2 and sodium outputs compared to those measured before corn oil supplementation. We conclude that corn oil supplementation may be an effective and inexpensive way to increase the protective properties of equine glandular gastric mucosa. This could be particularly helpful in reducing the chances of ulceration associated with nonsteroidal anti-inflammatory drug (NSAID) administration.     

Effects of mosapride citrate, metoclopramide hydrochloride, lidocaine hydrochloride, and cisapride citrate on equine gastric emptying, small intestinal and caecal motility

Objective

Although extensive work has been done to elucidate the beneficial and unfavorable effects of gastrointestinal prokinetic agents in humans, little is known on the effects of these agents in horses. In this study, we compared the effects of mosapride, metoclopramide, cisapride, and lidocaine on equine gastric emptying, jejunal and caecal motility and evaluated these agents’ adverse drug reactions (ADRs).

Animals

Seven healthy adult Thoroughbreds.

Procedure

Mosapride 1.0 mg/kg and 2.0 mg/kg, metoclopramide 0.2 mg/kg, and cisapride 1.0 mg/kg were dissolved in 100 mL distilled water for oral administration. Lidocaine 1.3 mg/kg was mixed with 500 mL saline for a 30-min intravenous infusion. Oral administration of 100 mL distilled water was used as control. Gastric emptying was evaluated using ¹³CO₂ breath test, and jejunal and caecal motility was assessed by electrointestinography.

Results

The present study demonstrates that mosapride at doses of 1.0 mg/kg and 2.0 mg/kg facilitates gastric emptying in horses. Improved jejunal motility was observed following administration of mosapride (1.0 mg/kg and 2.0 mg/kg), metoclopramide (0.2 mg/kg), and cisapride (1.0 mg/kg). Similarly, improved caecal motility was observed following administration of mosapride (2.0 mg/kg).

Conclusions and clinical relevance

This study shows that among the prokinetic agents studied here, only mosapride (2.0 mg/kg) promotes jejunal and caecal motility in horses. Considering mosapride ADRs profile, it is believed that this compound is useful in the treatment of diseases associated with decreased GI motility, including postoperative ileus.     

Effects of intravenous sodium bicarbonate and sodium acetate on equine acid-base status

Changes in blood gases, pH, and plasma electrolyte concentrations in response to intravenously infused sodium bicarbonate (NaHCO₃) and sodium acetate (NaCH₃CO₂) solutions (1.34 mEq/mL) in 5 light breed mares were investigated. Jugular venous blood samples were collected before and after completion of the infusions in 20-minute intervals for 200 minutes. Infusion of sodium bicarbonate and sodium acetate caused significant (P < .00l) increases in blood pH and bicarbonate ion concentration that persisted throughout the collection period. The elevation in blood pH and bicarbonate ion concentrations was greater (P < .01) for sodium bicarbonate than for sodium acetate immediately after the completion of the infusions but was not different (P > .05) thereafter. There were significant reductions (P < .01) in plasma-ionized calcium and potassium after infusion of both sodium bicarbonate and sodium acetate. This study found that significant metabolic alkalosis in horses and corresponding shifts in electrolyte concentrations can be induced by intravenous infusion of solutions of either sodium bicarbonate or sodium acetate, and they persist for at least 3 hours. These data show that the short-term elevation in pH and bicarbonate ion concentration is momentarily higher after infusion of sodium bicarbonate. This is likely due to the direct infusion of bicarbonate ions in the sodium bicarbonate treatment, such that further metabolism is not required to be effective. However, the longer-term alkalosis did not differ between isomolar solutions of sodium bicarbonate and sodium acetate.     

Evaluation of the influence of prostaglandin E2 on recombinant equine interleukin-1beta-stimulated matrix metalloproteinases 1, 3, and 13 and tissue inhibitor of matrix metalloproteinase 1 expression in equine chondrocyte cultures

OBJECTIVE: To determine the effects of prostaglandin E2 (PGE2) on recombinant equine interleukin (IL)-1beta-stimulated expression of matrix metalloproteinases (MMP 1, MMP 3, MMP 13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP 1) in vitro. SAMPLE POPULATION: Cultured equine chondrocytes. PROCEDURE: Stationary monolayers of first-passage chondrocytes were exposed to graduated concentrations of PGE2 with or without a subsaturating dose (50 pg/ml) of recombinant equine IL-1beta (reIL-1beta) to induce expression of MMP 1, MMP 3, MMP 13, and TIMP 1, followed by RNA isolation and northern blotting. In subsequent experiments, gene expression was similarly quantified from mRNA isolated from cultures pretreated with phenylbutazone to quench endogenous PGE2 synthesis, followed by exposure to reIL-1beta and exogenous PGE2 (5 mg/ml) with appropriate controls. RESULTS: Exogenous PGE2 (10 mg/ml) significantly reduced reIL-1beta-induced expression of MMP 1, MMP 3, MMP 13, and TIMP 1. Abrogation of cytokine induction with this dose of PGE2 was comparable to that for dexamethasone (10(-5) M) control. Similarly, pretreatment with phenylbutazone, followed by exposure to relL-1beta and PGE2 (5 mg/ml), was associated with a reduced expression of the genes of interest, an effect that was significant for MMP 1, MMP 13, and TIMP 1. CONCLUSIONS AND CLINICAL RELEVANCE: The MMP and TIMP 1 are important mediators in the pathophysiologic events in osteoarthritis. The potential for physiologically relevant regulation of expression of these genes by PGE2 is a consideration in the use of drugs that inhibit prostanoid synthesis in the treatment of equine arthropathies.     

Effects of heparin, citrate, and EDTA on plasma biochemistry of sheep: comparison with serum

This study was carried out to evaluate the features of neurological dysfunction in experimentally-induced bovine spongiform encephalopathy (BSE)-infected cattle using brainstem auditory evoked potentials (BAEP). The progressive prolongation of peak latency of waves III and V was observed right-and-left bilaterally at the onset of neurological symptoms. The peak latency of wave V and the I-V interpeak latency (IPL) in BSE cattle 22 and 24 months after intracerebral inoculation were significantly (P < 0.05) prolonged compared with the control cattle. In addition, the amplitude of the BAEP waves of the BSE cattle were low compared with the control cattle. Hearing loss occurred in the BSE cattle that showed advanced neurological symptoms such as tremor. It is thought that this BAEP data reflects a functional disorder in the central auditory nerve pathways characteristic of experimentally-induced BSE.     

Effects of heparin, citrate, and EDTA on plasma biochemistry of sheep: Comparison with serum

The effects of various types of anticoagulants on plasma biochemistry were studied in man and various animals, but limited information is existing for sheep plasma biochemistry. Ten clinically healthy Baloochi breed of sheep were blood sampled in different tubes containing each anticoagulants and plain tube for harvesting plasma and serum. The concentrations of glucose, cholesterol, total bilirubin, urea, creatinine, total protein, albumin, calcium, inorganic phosphorus, and magnesium and the activity of aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine kinase (CK) and gamma glutamyl transferase (GGT) were measured. Except for the amounts of GGT, bilirubin and inorganic phosphorus, other measured parameters were significantly lower in citrated plasma than that of serum. For corrected citrated plasma significant differences were seen for the concentrations of glucose, creatinine, calcium and the activity of ALP.Most parameters did not show any difference, but significant increase was seen for albumin concentration when heparin was used as an anticoagulant. Using EDTA as anticoagulant caused a significant difference for the concentrations of some of the measured parameters in plasma except glucose, GGT, cholesterol, albumin, bilirubin, CK, and inorganic phosphorus comparing with serum.     

Measurement of the activation of equine platelets by use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody

OBJECTIVE: To investigate the potential use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody for detection of the activation of equine platelets by use of flow cytometry. SAMPLE POPULATION: Platelets obtained from 6 Thoroughbreds. PROCEDURE: Flow cytometry was used to assess platelet activation as indicated by detection of binding of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-thrombospondin antibody to unactivated and ADP-, collagen-, platelet activating factor (PAF)-, and A23187-activated equine platelets. Human platelets were used as control samples. Determination of 14C-serotonin uptake and release was used to assess the extent of platelet secretion. RESULTS: Anti-human thrombospondin antibody failed to bind to equine platelets. Annexin V bound to platelets activated with PAF or A23187 when platelets had undergone secretion. Anti-human fibrinogen antibody bound to ADP-, PAF-, and A23817-activated platelets, but binding was not dependent on platelet secretion. The extent of binding of anti-fibrinogen antibody was less in equine platelets, compared with that for human platelets, despite maximal stimulation. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of equine platelets can be detected by use of fluorescent-labeled annexin V and anti-human fibrinogen antibody but not by use of anti-human thrombospondin antibody. These flow cytometric techniques have the potential for detection of in vivo platelet activation in horses at risk of developing thrombotic disorders.     

Effects of etamsylate on equine platelets: in vitro and in vivo studies

The aim of this study was to investigate whether etamsylate produces equine platelet activation. In vitro and in vivo studies were designed in which seven and eight adult healthy horses were included, respectively. In the in vitro study, citrated blood was incubated with different concentrations of etamsylate, and P-selectin expression and annexin V binding were determined by flow cytometry. In the in vivo study, blood was collected before and 1 and 2h after IV administration of etamsylate, and P-selectin expression was evaluated. In the in vitro study, a significant increase in P-selectin expression, leukocyte-platelet aggregate formation and annexin V binding were observed. In the in vivo study, a marked increase in P-selectin expression and heterotypic aggregate formation was seen in two and five horses, respectively, although no significant differences were detected when analyzing results from all the animals together. The results of the in vitro study indicate that etamsylate produces a pre-activation state in equine platelets, but this fact could be confirmed by the in vivo study.     

In vitro effects of prostaglandin E1, prostaglandin E2, indomethacin, histamine, and tuftsin on chemiluminescence response of bovine polymorphonuclear leukocytes

The in vitro effects of prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), indomethacin, histamine, and tuftsin on the chemiluminescence response of bovine polymorphonuclear cells (PMN) were determined. Addition of PGE1, PGE2, indomethacin, and histamine in vitro significantly suppressed the chemiluminescence response of bovine PMN's, whereas tuftsin had no effect. Suppression was dependent upon the continued presence of PGE1, PGE2, and histamine in the culture media. However, indomethacin's suppressive effect remained even after it was removed from the culture media. Hydrogen peroxide generated chemiluminescence was suppressed by high concentrations of indomethacin and histamine. Results of this study suggest possible pharmacologic or regulatory mechanisms for certain of these immune modulators in the control of the oxidative burst reaction of bovine PMN's.     

Amidolytic heparin activity and values for several hemostatic variables after repeated subcutaneous administration of high doses of a low molecular weight heparin in healthy dogs

OBJECTIVE: To determine effects of SC administration of repeated doses of a low molecular weight heparin (LMWH) in dogs. ANIMALS: 5 healthy dogs. PROCEDURE: Each dog received 6 injections (each injection, 150 U of anti-factor-Xa [anti-FXal/kg of body weight, SC) at 8-hour intervals. Blood samples were collected before and 2 hours after the first, second, third, and sixth injections to measure heparin activity, thrombin time, activated partial thromboplastin time (APTT), antithrombin activity, Hct, and platelet count. RESULTS: Heparin activity varied between 0.36+/-0.10 and 0.77+/-0.08 U of anti-FXa/ml (before and 2 hours after the third injection) and between 0.46+/-0.11 and 0.82+/-0.15 U of anti-FXa/ml (before and 2 hours after the sixth injection). Thrombin time and APTT were influenced only slightly. Platelet count, Hct, and antithrombin activity started to decrease significantly 2 hours after the second LMWH injection. Because of the increased consumption of antithrombin, antithrombin activity continuously decreased from 102.1+/-6.3% before the study to 91.0+/-3.0% at the end of the study. CONCLUSIONS AND CLINICAL RELEVANCE: Heparin plasma activity was only slightly higher than that recommended for LMWH treatment of humans, and none of the dogs had signs of increased bleeding. Thus, administration of heparin in accordance with this dosing regimen can be recommended for use in clinical studies. The screening tests investigated were not suitable for use in monitoring LMWH treatment of dogs. Assays that use chromogenic substrates are necessary to reliably monitor LMWH plasma concentrations in dogs.     

The effect of ticlopidine on canine platelets, fibrinogen, and antithrombin III activity

The effect of ticlopidine on platelet function, platelet number, mean platelet volume, antithrombin III activity, and fibrinogen was evaluated in 10 laboratory beagles. Ticlopidine (62 mg/kg) significantly inhibited ADP- and collagen-induced platelet aggregation within 2 days of the beginning of oral administration. Collagen-induced platelet 14C-serotonin release was not inhibited by day 9 of medication but was inhibited by day 20 in two of three beagles given medication for 32 days. Significant increases in mean platelet number were observed on days 2 and 5. The trend toward increased platelet number continued until day 16, at which time platelet number began to decrease toward baseline in three of three dogs treated for 32 days. Mean platelet volume (MPV) was significantly decreased compared to baseline on days 5 and 9. In three dogs treated for 32 days, the lowest MPV was observed on day 9 in two dogs and on day 12 in one dog. Significant changes were not observed in antithrombin III activity or fibrinogen with ticlopidine treatment.     

The correlation between plasma anti-factor Xa activity and haemostatic tests in healthy dogs, following the administration of a low molecular weight heparin

The aim of the study was to examine how activated partial thromboplastin time (APTT, two different reagents), thrombin time (TT, thrombin activity in the reagent: 3 or 6 IU ml(-1)) and reaction time of the resonance thrombogram (RTG-r) in healthy dogs are influenced by low molecular weight heparin (LMWH). Three different LMWH doses were given subcutaneously or intravenously to groups, each of five healthy dogs. Mean plasma anti-FXa activities of 0.43, 0.88 and 1.86 anti-FXa IU ml(-1)were measured 2 min after intravenous injection of 25, 50 or 100 anti-FXa IU kg(-1). At this time, a dose-dependent increase of the coagulation times, above the baseline values (P < 0.05), was observed for all haemostatic tests. The significant prolongation of coagulation time lasted 10 minutes to 3 hours, and it was dependent on the test employed and LMWH dose. After subcutaneous LMWH injection of 50, 100 and 200 anti-FXa IU kg(-1), significant changes of the coagulation time above initial values were limited to the period around the time when maximum anti-FXa activities (0.23, 0.43 or 0.90 anti-FXa IU ml(-1)) were observed. For the tests which were less affected by the LMWH (APTT, TT([6 IU ml)(-1)(])), only small increases (< 4 seconds) were observed even after the highest subcutaneous LMWH dose. The correlation between plasma heparin activity and the relative alteration compared to the initial value (ratio), of the different coagulation tests was only moderate and considerably lower for RTG-r (r(s)= 0.526) than for the TT (r(s)= 0.711([6 IU ml(-1)]), r(s)= 0.780([3 IU ml(-1)])) and APTT (r(s)= 0.667([reagent 1]), r(s)= 0.727([reagent 2])). The low degree of prolongation, which was found particularly for the group tests APTT and TT([6 IU ml)(-1)]), reflects the low anti-thrombin activity of LMWH. The results indicate that measurement of anti-FXa activity with chromogenic substrates is the method of choice to control LMWH therapy in dogs, as is the case in humans. Copyright2000 Harcourt Publishers Ltd Copyright 2000 Harcourt Publishers Ltd.     

能量对低出生体重仔猪死亡率、断奶重和初乳摄入量的影响

文章探讨了能量补充对低出生体重仔猪的死亡率、断奶体重、日增重及初乳摄入量的影响。处理组选择22头母猪所产的306头活仔猪,其中极低出生体重(<1 kg)72头,低出生体重(1~1.2 kg)77头,分别于出生时和出生后8~12 h口服能量补充剂。对照组选择24头母猪所产的340头活仔猪,其中极低出生体重81头,低出生体重74头。结果显示:能量补充较对照组显著降低了极低出生重仔猪哺乳3 d的死亡率(P <0.05),有降低低出生重仔猪死亡率的趋势(P=0.07)。总的来说,能量补充剂有降低仔猪哺乳3 d死亡率的趋势(P=0.06)。能量补充较对照组对极低出生重仔猪哺乳7和21 d存活优势率显著提高了4.04和3.59倍(P <0.05)。对照组较处理组显著提高了仔猪的断奶重(P <0.05)。对照组与处理组对仔猪的日增重、初乳摄入量及出生产量的影响均无显著差异(P> 0.05)。处理组母猪初乳产量在数值上比对照组低(P> 0.05),但处理组母猪初乳产量均匀性在数值上较对照组好。综上所述,能量补充通过为极低出生体重仔猪直接提供能量而不是通过提高初乳的摄入量来降低其断奶前的死亡率。