Temporal regulation of cytokine mRNA expression in equine recurrent airway obstruction.

Acute and chronic inflammation of the airway remains an important health problem for equids. "Heaves" or recurrent airway obstruction (RAO) remains one of the most commonly diagnosed conditions affecting the lung of older horses in Europe and the United States. The typical clinical signs of RAO include non-productive coughing, serous nasal discharge, labored expiratory effort, and flaring of the nostrils. Auscultation of the lungs of the affected horse often reveals abnormal respiratory sounds, described as crackles and wheezes, throughout the area of the lung field. These clinical signs occur secondary to an inflammatory response that results in bronchospasm, excessive mucus production and airway obstruction. This inflammatory response is characterized by the presence of excessive mucus and inflammatory cells, primarily neutrophils, in the small airways. Most evidence suggests that RAO is the result of a pulmonary hypersensitivity to inhaled antigens. Exposure of affected horses to hay dust, pollens, and mold spores leads to neutrophil accumulation in the lung and bronchospasm. The identification of allergen-specific IgE in bronchoalveolar lavage (BAL) fluid and sera of affected horses supports the involvement of a late phase, IgE-mediated, hypersensitivity reaction in the pathogenesis of equine RAO. The production of IgE antibodies is regulated by the cytokines IL-4 and IL-13. Using a quantitative PCR method we have reported that horses with RAO exhibit a modified Type 2 cytokine response characterized by the production of IL-4 and IL-13 mRNA, but not IL-5 mRNA in BAL cells. Interferon-gamma mRNA was also elevated, suggesting a mixed response. While these results are consistent with equine RAO being the result of an aberrant Type 2 cytokine response to inhaled allergens, others have failed to find any evidence of elevated Type 2 cytokine mRNA in BAL from horses with "heaves". It is likely that these disparate results could be the result of differences in the clinical stage of the affected animals or the timing of sample collection. Here, we report a diverse pattern of cytokine gene expression when sampling a group of affected horses over a period of time.

Characterization of a density-corrected ultrasonic pneumotachometer for horses.

A density-corrected ultrasonic pneumotachometer designed specifically for horses (UF202) was evaluated and characterized with the aid of a custom-built apparatus. UF202 provided voltage outputs for airflow through and gas density within the flowhead. Baseline stability for flow channel output (VUF202) was < 0.35% full scale (FS), and VUF202 was linear over a range of +/- 50 l/s (R2 > or = 0.9976). Under optimal conditions, VUF202 accuracy was determined to be +/- 1.00% FS and repeatability was +/- 0.78% FS. VUF202 resolution was 24 ml/s. The rise time for VUF202 was 18 ms, and the -3-dB point was 18 Hz; digital compensation provided a flat frequency response to 32 Hz. VUF202 gain appeared to be stable over a period of 8 days. Density channel output (PUF202) was linear over a range of 1.11-1.23 g/l (R2 > or = 0.9645). PUF202 accuracy was calculated to be +/- 0.50% FS, and PUF202 repeatability was determined to be +/- 0.30% FS. Resistance of the flowhead was determined to be 0.013 cmH2O.l-1.s at a flow of 40 l/s. On the basis of this study, we conclude that UF202 should facilitate accurate measurement of breath-by-breath airflow of exercising horses.

Ventilatory and blood gas dynamics at onset and offset of exercise in the pony.

The purpose of these experiments was to examine the temporal pattern of arterial carbon dioxide tension (PaCO2) to assess the relationship between alveolar ventilation (VA) and CO2 return to the lung at the onset and offset of submaximal treadmill exercise. Five healthy ponies exercised for 8 min at two work rates: 50 m/min 6% grade and 70 m/min 12% grade. PaCO2 decreased (P less than 0.05) below resting values within 1 min after commencement of exercise at both work rates and reached a nadir at 90 s. PaCO2 decreased maximally by 2.5 and 3.5 Torr at the low and moderate rate, respectively. After the nadir, PaCO2 increased across time during both work rates and reached values that were not significantly different (P greater than 0.05) from rest at minute 4 of exercise. Partial pressure of O2 in arterial blood and arterial pH reflected hyperventilation during the first 3 min of exercise. At the termination of exercise PaCO2 increased (1.5 Torr) above rest (P less than 0.05), reaching a zenith at 2-3 min of recovery. These data suggest that VA and CO2 flow to the lung are not tightly matched at the onset and offset of exercise in the pony and thus challenges the traditional concept of blood gas homeostasis during muscular exercise.

A novel model for equine recurrent airway obstruction.

Equine recurrent airway obstruction (RAO; a term combining both chronic obstructive pulmonary disease (COPD) and summer pasture associated obstructive pulmonary disease (SPAOPD)) is one of the most common equine respiratory diseases with up to 50% of horses affected worldwide. The etiopathogenesis of RAO is unknown although pulmonary hypersensitivity to inhaled mold antigens may be involved. Recent work in our laboratory demonstrating elevated levels of IL-4 and IL-13 mRNA in the airways and peripheral blood of horses with RAO is consistent with an atopic component to RAO. Little is known regarding the earliest phases of RAO in horses. Here we describe the development of a novel airway model for equine RAO that utilizes ovalbumin-coated polystyrene beads for airway sensitization and challenge. Aerosol challenge of sensitized ponies with OVA-coated microbeads resulted in decreased airway compliance, increased percentage of lymphocytes and neutrophils in the bronchoalveolar lavage fluid, and evidence of a Th2 cytokine response in the bronchoalveolar cells. These results suggest that this approach may be useful in describing the initial stages of RAO development in the horse.

The correlation of running ability and physiological variables in thoroughbred racehorses.

The running abilities of 25 Thoroughbred racehorses were measured at distances of 1200, 1600 and 20000 m. Various physiological variables were measured subsequently on the treadmill and correlated with running speed. There was a negative correlation for running speed with the velocity (VLa4) and work rate (WLa4) at which blood lactate reaches a steady-state concentration of 4 mmol/litre and a positive correlation with peak plasma lactate, suggesting that plasma lactate concentrations of faster horses rise more rapidly and to higher levels than do those of slower horses. The correlation between running speeds and heart rates (HR) was stronger for unfit than fit horses, suggesting that cardiovascular effects of training are more beneficial to slower horses. The significant correlation between running speeds and V200 suggests that the HR of faster horses increases more rapidly than in slower horses performing similar exercise. The correlation of running speeds and VO2max suggests that the HR of faster horses increases more rapidly than in slower horses performing similar exercise. The correlation of running speeds and VO2max suggests that faster horses utilise more oxygen during maximal intensity exercise. The correlation of running speeds with minimum pH and minimum HCO3- suggests that faster horses maintain speed at higher hydrogen ion (H+) concentrations. Correlations between running speeds and the measured variables were consistently stronger for the longer distance runs. Because VLa4 and WLa4 were obtained during sub-maximal exercise, these variables were determined to be the best correlates of running performance.

Interleukin-4 and interferon-gamma gene expression in summer pasture-associated obstructive pulmonary disease affected horses.

We hypothesised that horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) react to an allergen or allergens in their summer environment that is either absent or present at lower levels in their winter environment; and that such allergens stimulate SPAOPD-affected horses to produce a different T helper lymphocyte cytokine profile from that of control horses. The primary objective of this study was to determine the cytokine mRNA profile of T helper lymphocytes obtained from summer pasture-associated obstructive pulmonary disease (SPAOPD) affected horses when 1) the horses were showing signs of disease (summer) and 2) they were in clinical remission (winter). A further objective was to determine the differences between cytokine mRNA T helper lymphocyte profiles of control and affected horses in the summer and winter seasons. Interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression levels were increased in bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cell (PBMC) samples of affected horses during disease expression. No significant amounts of IL-5 mRNA were detected in any of the samples. These results suggest that there is an allergic component to SPAOPD of horses and that appropriate manipulation of the immune system could offer hope for treatment and prevention of the disease in the future. Further research studies will be needed to determine the most appropriate treatments to use to alter the antigen-stimulated cytokine profile being expressed by SPAOPD-affected horses or to alter the effects that these cytokines produce.

Pharmacokinetics of small doses of 3-methylindole given to horses.

The pharmacokinetics of 3-methylindole (3MI) given orally in 2 doses (10 mg/kg and 20 mg/kg) to horses were determined. The pharmacokinetic plasma-concentration profiles for 3MI (10- and 20-mg/kg dosages) in horses were represented by a 2-compartment open model with first-order absorption, as determined by nonlinear least-squares regression analysis. Absorption of 3MI at both dosages was rapid. Comparisons of the peak plasma concentrations, the postdistribution half lives, total clearances, and areas under the curve of the plasma-concentration profiles between the 10- and the 20-mg/kg dosages may indicate the presence of nonlinear or dose-dependent kinetics for 3MI in horses.

Cardiopulmonary effects of positive end-expiratory pressure in anesthetized horses.

The cardiopulmonary effects of 0, 5, 10, and 15 cm of H2O positive end-expiratory pressures (PEEP) were determined in anesthetized, spontaneously breathing horses, using a 4 by 4 Latin-square design with one repetition. Cardiac output, alveolar-arterial oxygen tension difference, alveolar ventilation, dead space/tidal volume ratio, and carbon dioxide elimination were not significantly altered by the procedure. As PEEP was increased, alveolar and arterial oxygen tensions, respiratory exchange ratio, and pH decreased, whereas arterial carbon dioxide tension and oxygen consumption increased. These results indicate PEEP is contraindicated in laterally recumbent spontaneously ventilating anesthetized horses breathing air, because it causes alveolar hypoventilation and does not improve pulmonary gas exchange.

Cross-sectional area of the aditus laryngis and rima glottidis before and after transection of the left recurrent laryngeal nerve in the horse.

The ventral-to-dorsal height of the rima glottidis was measured from lateral pharyngeal radiographs after correction for magnification. The rima glottidis height was used to enlarge accurately endoscopic photographs of 5 horses taken before and after transection of the left recurrent laryngeal nerve. Areas of the rima glottidis and aditus laryngis were measured, using a computerized digitizer. Mean area of the aditus laryngis was 1,908 mm2 before neurectomy and 1,346 mm2 after neurectomy (P = 0.025). Mean area of the rima glottidis was 1,198 mm2 before neurectomy and 805 mm2 after neurectomy (P = 0.025). Mean width of the rima glottidis was 31 mm before neurectomy and 20 mm after neurectomy (P = 0.001). Significant differences were not found between the pre- and postneurectomy heights of the rima glottidis.

Quantification of antigen-specific antibody concentrations in tracheal lavage fluid of horses with summer pasture-associated obstructive pulmonary disease.

OBJECTIVE: To determine whether horses with summer pasture-associated obstructive pulmonary disease (SPAOPD) have increased concentrations of antigen-specific IgG and IgE in tracheal lavage fluid, compared with values in clinically normal horses. ANIMALS: 8 horses (6 females, 2 geldings; 6 Quarter Horses, 2 Appaloosas), 14 to 23 years old and with previous diagnosis of SPAOPD, served as the principal group; 8 horses (2 females, 6 geldings; 1 Quarter Horse, 7 Thoroughbreds), 6 to 9 years old, with no evidence of respiratory tract disease, served as the control group. PROCEDURE: Data were collected twice during a 1-year period: when all SPAOPD-affected horses were manifesting clinical signs of disease (July), and when all SPAOPD-affected horses appeared clinically normal (February). On each occasion, clinical evaluations were performed and blood and tracheal lavage fluid samples were collected. Transtracheal lavage supernatant was evaluated for mold antigen-specific IgG and IgE concentrations. RESULTS: Median IgE relative antibody unit (RAU) values were significantly higher in control, compared with principal, horses. The SPAOPD-affected horses had increased concentrations of specific IgG for only 1 antigen, during winter sample collection. CONCLUSION: Antigen-specific IgG and IgE RAU values were not increased in SPAOPD-affected horses when these horses were manifesting clinical signs of disease.

Correlation of clinical score, intrapleural pressure, cytologic findings of bronchoalveolar fluid, and histopathologic lesions of pulmonary tissue in horses with summer pasture-associated obstructive pulmonary disease.

OBJECTIVE: To correlate clinical score, intrapleural pressure, cytologic findings of bronchoalveolar lavage fluid (BALF), and histologic lesions of pulmonary tissue in horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD). ANIMALS: 8 adult horses affected with SPAOPD and 6 adult horses without evidence of respiratory tract disease. PROCEDURE: Clinical score, change in intrapleural pressure (deltaPpl) during tidal breathing, results of cytologic examination and bacteriologic culture of BALF, and results of histologic examination of pulmonary parenchyma were evaluated. RESULTS: Clinical scores for SPAOPD-affected horses (median, 5.75; range, 4.0 to 7.5) were significantly greater, compared with clinically normal horses (median, 2.0; range, 2.0 to 3.0). Cytologic examination of BALF from SPAOPD-affected horses revealed predominantly nondegenerate neutrophils. Histologic lesions were identified throughout pulmonary tissue and included severe accumulation of mucus and neutrophils within the small airways, metaplasia of bronchiolar goblet cells, and mild peribronchial infiltrate. Histologic examination of specimens collected via percutaneous biopsy was predictive of disease and corresponded to findings at postmortem examination. Clinical score and deltaPpl were highly correlated with mucus accumulation in the airways of affected horses. Peribronchial inflammatory infiltrate correlated with percentage of neutrophils in BALF of affected horses. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical scoring and deltaPpl provided valid estimates of disease severity. Findings from cytologic examination of BALF of SPAOPD-affected horses varied, although, in most instances, it was diagnostically useful. Severe mucus accumulation in the airways was the most remarkable histopathologic finding in SPAOPD-affected horses. Examination of biopsy specimens collected from pulmonary parenchyma was consistently useful in diagnosing SPAOPD.

Comparative responses of bronchial rings to mediators of airway hyperreactivity in healthy horses and those affected with summer pasture-associated obstructive pulmonary disease.

OBJECTIVE: To compare responses of bronchial rings obtained from healthy horses and horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) to selected mediators of airway hyperreactivity in vitro. SAMPLE POPULATION: Bronchial rings from 6 healthy horses and 6 horses affected with SPAOPD. PROCEDURE: Bronchial rings obtained from each group of horses were mounted in organ baths and attached to force transducers interfaced with a polygraph. After applying 2g of tension, each ring was allowed to equilibrate for 45 minutes in Tyrode's solution at 37 C. Cumulative concentration-response relationships to graded concentrations of selected mediators (10(-8) to 10(-4) M) were determined and analyzed for significance at each concentration. RESULTS: Acetylcholine, histamine, 5-hydroxytryptamine, and leukotriene D4 induced concentration-dependent contractile responses in bronchial rings. Prostaglandin F2alpha induced weak and inconsistent contractile responses. The other 2 agents, norepinephrine and substance P, did not induce concentration-dependent responses. Considering the overall group-drug effect, acetylcholine, histamine, 5-hydroxytryptamine, and leukotriene D4 were effective in inducing consistent concentration-dependent contractile responses in both groups. Only 5-hydroxytryptamine and histamine induced significant responses in contractility between groups. The response of bronchial rings from horses with SPAOPD to 5-hydroxytryptamine was significantly greater than those from control horses, whereas the response to histamine was significantly lower. Significant responses were evident at concentrations ranging from 10(-6) to 10(-4) M for both drugs. CONCLUSIONS AND CLINICAL RELEVANCE: Because the airways of horses with SPAOPD had increased responsiveness to 5-hydroxytryptamine in vitro, treatment modalities using 5-hydroxytryptamine antagonists should be investigated to address this phenomenon.

Summer pasture-associated obstructive pulmonary disease in horses: 21 cases (1983-1991).

Medical records of 21 horses with summer pasture-associated obstructive pulmonary disease were reviewed, and history, signalment, clinical signs, radiographic signs, clinicopathologic data, and therapeutic response were determined. Most affected horses were used as pleasure horses, and for the most part, remained at pasture when not in use. The mean age (+/- SD) was 13.7 +/- 3.6 years. Clinical signs included intermittent nasal discharge, cough, tachypnea, labored expiratory effort, and crackles and wheezes on auscultation. Radiography frequently revealed interstitial patterns in the lung fields; in horses with chronic disease, pulmonary overinflation was evident. Hemogram was usually normal, and transtracheal wash fluid was characterized by nondegenerate neutrophils.

Chronic obstructive pulmonary disease in horses in Louisiana.

OBJECTIVE: To evaluate chronic obstructive pulmonary disease of horses in Louisiana by assessing the signalment, history, environmental factors, clinical signs, and treatment of such horses. DESIGN: Epidemiologic mail survey. SAMPLE POPULATION: 83 of 240 veterinarians contacted by mail agreed to take part in the survey. Veterinarians contacted were listed as mixed-animal or equine practitioners in the 1991/1992 directory of the Louisiana Veterinary Medical Association or had submitted a specimen from a horse to the Louisiana Veterinary Medical Diagnostic Laboratory within the past 2 years. PROCEDURE: The survey contained 47 questions designed to elicit information from owners and veterinarians about horses reported to have chronic obstructive pulmonary disease (COPD). Questions were included to evaluate age, breed, sex, vaccination history, respiratory disease history, environment of primary activity, level of exercise, primary residence (pasture or stall), condition of pasture or barn, type and condition of feed, clinical signs, concurrent conditions, and treatment regimen prescribed. Information from the returned forms was analyzed by using a microcomputer program designed for epidemiologic data. RESULTS: Of the 83 veterinarians who agreed to participate, 31 returned 71 completed questionnaires for horses affected with COPD. Most affected horses were mature in age, kept on pasture, and had developed clinical signs during the summer months. The most consistent clinical signs were dry coughing, slight serous nasal discharge, labored expiratory effort, and flaring nostrils. CLINICAL IMPLICATIONS: Summer pasture-associated obstructive pulmonary disease appears to be precipitated by factors different than those associated with the traditionally diagnosed form of COPD and, thus, successful management measures may also vary.

Complications associated with immunotherapy of equine phycomycosis.

Five horses with pythiosis of the limbs were treated unsuccessfully by surgery or topical application of amphotericin B, or both. Follow-up immunotherapy resulted in 1 horse responding favorably. Three horses were cured of the fungal infection but developed osteitis or deep-seated laminitis, which necessitated their destruction. The remaining horse, which had severe anemia, died before the course of vaccination was completed.

Characterization of normal tidal breathing flow-volume loops for thoroughbred horses.

The purpose of this study was to characterize the normal equine tidal breathing flow-volume loop (TBFVL). The study was performed using 18 healthy Thoroughbred horses. TBFVLs constructed from data collected from resting horses had a typical biphasic inspiratory and expiratory phase. The interindividual variability of the indices used to describe TBFVLs was in the range 16-32%, which is comparable to the variability of other measures of equine pulmonary mechanics. The large variability of these data probably limits the value of resting TBFVL indices for detecting subclinical respiratory conditions in individual horses. Factor analysis of these data revealed that in excess of 90% of the variance of the initial response variables could be explained in terms of three common factors. Varimax rotation of these three common factors provided three subsequent factors that were readily identifiable as (1) a factor describing the time-volume relationships of TBFVLs, responsible for 81% of the total variance, (2) a factor explaining the expiratory portion of the TBFVL, explaining 12% of the variance, and (3) a factor describing the inspiratory portion of the loops, responsible for the remaining 7% of the variance. The analysis also provided standardized factor scoring coefficients for use in subsequent studies using similar experimental techniques.

The effects of three models of airway disease on tidal breathing flow-volume loops of thoroughbred horses.

The effects of histamine and methacholine aerosols and of a fixed inspiratory resistance on tidal breathing flow-volume loops (TBFVL) were investigated using 18 unsedated, standing, healthy thoroughbred horses. The data were first analysed using traditional flow-volume loop indices and then reduced using standardized factor scoring coefficients obtained in a previous study in this laboratory using similar experimental techniques. On the basis of resting TBFVL analysis, the degree of pulmonary dysfunction caused by inhalation of histamine and methacholine aerosols with concentrations of 10 and 2 mg/ml, respectively, was similar. The fixed resistance also caused significant changes in the resting spirogram and TBFVL indices, suggesting that this model may prove valuable for further studies involving upper respiratory tract (URT) conditions. Administration of histamine and methacholine aerosols resulted in significant changes in all factor scores, although most of the observed changes were due to the effects of these aerosols on the respiratory rate. These findings re-emphasize the importance of the effects of respiratory rate on pulmonary mechanics. Application of the resistance resulted in significant changes in factor score 3, the 'inspiratory' factor, which lends support to the validity of this model for URT conditions. The close agreement between the factor scores obtained under controlled conditions in this study and in a previous study in this laboratory confirms that the factor analysis used for both of these studies provides an adequate means of reducing TBFVL data obtained from thoroughbred horses. The large intra- and inter-individual variation observed both with the indices of TBFVL and with the factor scores limits the potential of these variables for detecting individual animals with obstructive airway disease. Re-evaluation of these indices under the stress of exercise may reduce the variability observed in these data and may increase the magnitude of differences between different animals, providing a means of detecting individual animals with subclinical obstructive airway conditions.

Responses of guinea-pig lung parenchymal strips to tracheobronchial lavage fluid from horses affected with summer pasture-associated obstructive pulmonary disease.

The response of parenchymal strips from guinea-pig lungs to tracheobronchial lavage fluid (TBLF) collected from 8 normal horses and from 8 affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) was determined. TBLF was collected during the summer (July) and winter (February) seasons. The serum/TBLF urea nitrogen ratio was used to standardize the mediator concentration in the TBLF. Four strips were used from each guinea-pig. The first strip did not receive any antagonist and served as the control. The second, third and fourth strips received antagonists of PGE2, LTD4 and PAF, respectively at 10(-6) mol/L for 30 min. The tissues were then precontracted with a dose of histamine (10(-5) mol/L) and their responses to 1 ml of TBLF were determined. The study showed that TBLF obtained in the summer from unaffected horses produced a significantly greater relaxation than that from the affected horses, whereas TBLF obtained in the winter from unaffected or affected horses did not cause a significantly different degree of relaxation. Among the antagonist-treated strips, only those exposed to the PGE2 blocker showed a significant reduction in the relaxation caused by TBLF obtained in the summer from SPAOPD horses. This suggests that PGE2 is an important mediator present in the summer in the TBLF from horses affected with SPAOPD.

Vasorelaxation responses to insulin in laminar vessel rings from healthy, lean horses.

Hyperinsulinemia causes laminitis experimentally and is a risk factor for naturally occurring laminitis. The aim of this study was to investigate the effects of insulin on laminar vascular relaxation and to induce insulin-associated vascular dysfunction in vitro. Relaxation responses of isolated laminar arterial and venous rings to acetylcholine and insulin were evaluated. To alter vascular function in response to insulin, all vessel rings were incubated with insulin or vehicle, submaximally contracted, administered insulin again and relaxation responses recorded. Laminar arteries were also incubated with the mitogen-activated protein kinase (MAPK) inhibitor, PD-98059. Relaxation in response to acetylcholine was not different between arteries and veins, but veins relaxed less in response to insulin than arteries. In arteries incubated with insulin, the subsequent relaxation response to insulin was blunted. Veins had minimal relaxation to insulin regardless of incubation. Arteries incubated with PD-98059 relaxed more in response to insulin than arteries not exposed to PD-98059, indicating that MAPK plays a role in maintenance of basal tone in laminar arteries. A differing response of laminar veins and arteries to insulin-induced relaxation may be important in understanding the link between hyperinsulinemia and laminitis. In vitro induction of vascular dysfunction in response to insulin in laminar arteries may be useful for testing therapeutic interventions and for understanding the pathophysiology of laminitis.

Insulin resistance in equine digital vessel rings: an in vitro model to study vascular dysfunction in equine laminitis.

REASONS FOR PERFORMING STUDY: One of the causes of equine laminitis is hyperinsulinaemia, which may be associated with endothelial dysfunction and insulin resistance of vessels. HYPOTHESIS AND OBJECTIVES: Insulin resistance can be induced in palmar digital vessels by continued exposure to insulin in vitro. The objective was to evaluate this in vitro model for future studies. METHODS: Palmar digital vessel segments were collected immediately after euthanasia from horses with normal insulin/glucose blood values. Four arterial and 4 venous rings (3 mm wide) were prepared and each ring mounted in a tissue bath, containing Tyrode's solution at 37°C, 2 g tension was applied and the rings allowed to equilibrate for 45 min. Of the 4 rings of each vessel type, one was used as a control. One each of the remaining 3 rings was used for incubation with insulin (to induce resistance), wortmannin (to block PI3-kinase) and PD-098059 (to block MAP-kinase), respectively, for 30 min. After the incubation period, the rings were contracted with phenylephrine. When the response reached a plateau, a single dose of insulin was added to the baths and the response of each ring monitored for 30 min. RESULTS: Insulin relaxed the control rings and those treated with PD 098059 but contracted those pretreated with insulin and wortmannin. Normal relaxation responses of the rings were converted to contractions by insulin resistance. Insulin resistance was confirmed by the qualitative response of insulin-incubated and wortmannin-incubated rings. CONCLUSIONS: This study demonstrated successful induction of insulin resistance in both arterial and venous rings. It also suggested that the MAP-kinase pathway plays a minor role in controlling vasomotor tone under normal physiological conditions. POTENTIAL RELEVANCE: The study suggests that the induction of insulin resistance in equine palmar digital vessel rings is reliable and provides a good in vitro model for studying the vascular insulin resistance which may occur in equine laminitis.