Effects of a specific endothelin-1A antagonist on exercise-induced pulmonary haemorrhage (EIPH) in thoroughbred horses.

REASONS FOR PERFORMING STUDY: During high intensity exercise, the very high pulmonary artery pressure (Ppa) experienced by Thoroughbred horses is considered a major factor in the aetiology of exercise-induced pulmonary haemorrhage (EIPH). Recently, endothelin-1 (ET-1), a potent vasoconstrictive hormone, has been found to increase Ppa in horses at rest via binding to its ET-1A receptor subtype. In addition, plasma concentrations of ET-1 are increased in horses during and after high intensity exercise. HYPOTHESIS: If ET-1 increases Ppa during exercise in the horse, administration of a specific ET-1A antagonist would decrease Ppa and therefore EIPH. METHODS: Saline (CON) or an ET-1A receptor antagonist, TBC3214 (3 mg/kg bwt i.v.; ANTAG) was administered to horses 1 h prior to maximal incremental exercise on a high-speed treadmill. Gas exchange measurements were made breath-by-breath and blood samples collected during each 1 min stage to determine blood gases, acid-base status and cardiac output. EIPH was determined via bronchoalveolar lavage (BAL) approximately 30 min after exercise. RESULTS: The time to fatigue, gas exchange and cardiovascular responses were not different between groups (P>0.05). Resting and peak Ppa did not differ significantly between treatments. Most importantly, ANTAG did not decrease EIPH. CONCLUSIONS: These results do not support a deterministic role for ET-1 in the increased Ppa and therefore EIPH, during maximal exercise in the equine athlete. POTENTIAL RELEVANCE: Treatment with an ET-1A receptor antagonist does not appear to be a viable therapeutic intervention in the prevention of EIPH.

Exercise-induced pulmonary haemorrhage during submaximal exercise.

REASONS FOR PERFORMING STUDY: Maximally exercising horses achieve mean pulmonary artery pressures (Ppa(mean)) that exceed the minimum threshold (75 mmHg) estimated for pulmonary capillary rupture and exercise-induced pulmonary haemorrhage (EIPH). EIPH is not expected to occur during moderate submaximal exercise (i.e. 40-60% VO2max) since Ppa(mean) remains well below this threshold. HYPOTHESIS: Prolonged submaximal exercise (trotting) would precipitate locomotory respiratory uncoupling and cause EIPH. This would be present as a result of the most negative intrapleural pressures (as estimated by the minimum oesophageal pressure; Poes(min)) occurring simultaneously with the most positive Ppa (Ppa(peak)) to produce estimated maximal pulmonary artery transmural pressures (PATMPmax) that surpass the EIPH threshold. METHODS: Five Thoroughbred horses trotted to fatigue (approximately 25 min) at 5 m/sec on a 10% incline. Ventilation (V(E)), Poes, and Ppa were measured at 5 min intervals, and bronchoalveolar lavage (BAL) red blood cells (RBCs) were quantified 45 min post exercise. RESULTS: BAL revealed an increased EIPH (rest: 2.0 +/- 1 x 10(5), exercise: 17 +/- 10 x 10(5) RBCs/ml BALF; P<0.05), despite the highest Ppamean reaching only mean +/- s.e. 55 +/- 3 mmHg, while V(E), tidal volume and Poes(min) approached 70-80% of the values achieved at maximal running speeds (10% incline: 12-13 m/sec) by these same horses. The resulting PATMPmax was well above the level considered causative of EIPH. CONCLUSIONS: The finding of significant EIPH during submaximal exercise broadens the spectrum of performance horses susceptible to EIPH and supports studies that suggest that extravascular factors are of primary importance in the aetiology of EIPH. POTENTIAL RELEVANCE: Consideration of strategies such as the equine nasal strip for reducing negative extravascular pressures is warranted even for exercise at moderate intensities.

Inclined running increases pulmonary haemorrhage in the Thoroughbred horse.

REASONS FOR PERFORMING STUDY: Capillary stress failure-induced (exercise-induced) pulmonary haemorrhage (EIPH) during intense running in horses is thought to involve both intravascular (i.e. mean pulmonary arterial pressure [Ppa] > 100 mmHg) and extravascular (e.g. negative inspiratory pressure swings) mechanisms. HYPOTHESIS: That inclined running would reduce breathing frequency (coupled to stride frequency) and increase tidal volume thus increasing lung volume changes and intrapleural pressure swings resulting in more pronounced EIPH. METHODS: Six Thoroughbred horses were run to volitional fatigue (incremental step test) on a level (L) and inclined (I; 10%) treadmill in random order. Pulmonary minute ventilation, arterial blood gases and mean Ppa were obtained during each run while EIPH severity was quantified via bronchoalveolar lavage (BAL) 30 mins post run. RESULTS: Time to fatigue did not differ between trials (P > 0.05). At end-exercise, breathing frequency was reduced (L, 127.8 +/- 3.0; I, 122.6 +/- 2.1 breaths/min; P < 0.05) and tidal volume increased (L, 11.5 +/- 0.6; I, 13.1 +/- 0.5 L; P < 0.05) during inclined running. No differences existed in end-exercise plasma [lactate] between trials (L, 24.5 +/- 2.9; I, 26.2 +/- 3.4 mmol/l, P > 0.05); however, the mean peak Ppa was reduced during the inclined run (L, 105+5; I, 96 +/- 4 mmHg, P < 0.05). In the face of reduced Ppa, EIPH severity was increased significantly (P < 0.05) during the inclined vs. level run (L, 37.0 +/- 11.7; I, 49.6 +/- 17.0 x 10(6) red blood cells/ml BAL fluid). CONCLUSIONS: Although inclined running lowered peak Ppa, EIPH severity was increased. It is likely that this effect resulted, in part, from an altered ventilatory pattern (i.e. increased tidal volumes and associated intrapleural pressure changes). POTENTIAL RELEVANCE: This conclusion supports an important role for extravascular factors in the aetiology of EIPH.

Movements of thoracic and abdominal compartments during ventilation at rest and during exercise.

The present investigation utilised simultaneous measurements of chest (Ch) and abdominal (Ab) circumferences and respiratory airflow to test the hypothesis that Ch circumferential expansion contributes proportionally little to tidal volume in the running Thoroughbred. During exercise, there were only small changes in Ch and Ab circumference and no increase with increasing tidal volume. At rest, walk and trot, the flow, Ch and Ab signals were in phase. However, during canter and gallop, the Ch and Ab changes were 180 degrees out of phase with each other and both were out of phase with airflow. In contrast to exercise, increase in ventilation at rest achieved by administration of lobeline resulted in a 4-6-fold increase in tidal volume; large excursions of the chest were always in phase with airflow. Furthermore, 3 horses showed an increase in chest circumference, demonstrating that chest stiffness per se does not preclude chest circumferential expansion. In conclusion, in the absence of significant increases in either Ch or Ab expansion during running, elongation of the thoracoabdominal segment may be the main determinant of tidal volume.

Structural and oxidative enzyme characteristics of the diaphragm.

During exercise, the horse can achieve oxygen uptakes and ventilations in excess of 200 ml/kg/min and 1800 l/min, respectively. Whether the diaphragm has the capacity to contribute substantially to inspiratory effort in the exercising horse is not known. To investigate the potential for the horse diaphragm to generate tension, lung displacement and sustain ventilatory function, we measured diaphragm thickness, muscle length and oxidative enzyme activity (citrate synthase) within the ventral, medial and dorsal costal and crural diaphragm. In the diaphragms of 6 mature horses (5 Thoroughbreds, one Quarter Horse; body mass (mean +/- s.e.) 475 +/- 14 kg, age 4 +/- 1 years), the mass of the freshly-excised diaphragm was 4.54 +/- 0.19 kg of which 79% was the costal diaphragm, 17% the crural diaphragm and 4% the central tendon. The medial costal region (2.1 +/- 0.1 cm) was significantly thicker (P<0.05) than either the ventral (1.4 +/- 0.1 cm) or dorsal (1.2 +/- 0.2 cm) costal regions and the crural diaphragm was significantly thicker (>3.2 +/- 0.3 cm, P<0.05) than any costal diaphragm region. With respect to the costal diaphragm, excised muscle length was greatest (P<0.05) in the medial costal (17.2 +/- 1.0 cm) than either the ventral costal (<12.6 +/- 1.5 cm) or dorsal costal (<13.9 +/- 1.8 cm) regions and therefore the medial region would be expected to exhibit the greatest absolute length change on inspiration. Citrate synthase activity was high throughout the diaphragm (40.8 +/- 113 to 55.3 +/- 9.7 micromol/g/min), but was not significantly different among regions. These structural characteristics and the oxidative potential of the horse diaphragm are consistent with the diaphragm providing a significant and substantial contribution to the inspiratory effort during exercise in the horse. Consequently, clinical and physiological investigations of exercise performance should not ignore the potentially crucial importance of the diaphragm.

Effect of body incline on cardiac performance.

Maximal cardiac performance is improved in man during upright compared to supine exercise. Whether cardiac performance in quadrupeds is dependent upon body position is unknown. Therefore, we undertook the present investigation to determine if peak cardiac output (Qpeak) would be influenced by body inclination in the Thoroughbred horse. To test the hypothesis, four Thoroughbred horses performed an incremental exercise protocol (speed increased by 1 m/s/min to fatigue) on both a level (L) and inclined (I: 6 degrees) treadmill. Specifically, we hypothesised that Qpeak would be increased on the incline, as this represents a progression towards upright exercise. Cardiac output was determined using the Fick relationship from continuous measurements of pulmonary VO2 and paired arterial (carotid artery or transverse facial) and mixed venous (pulmonary artery) samples. Qpeak was significantly increased on the incline (L: 279 +/- 20; I: 336 +/- 17 l/min; P<0.05), while CaO2 was not significantly different (L: 25.5 +/- 1.1; I: 25.4 +/- 1.9 ml/100 ml), and therefore, whole body O2 delivery (QO2) was significantly increased (L: 70.7 +/- 4.9; I: 84.4 +/- 3.1 l/min; P<0.05). In conclusion, within the scope of this investigation, these data suggest that cardiac performance, as judged by increased Qpeak and QO2, is enhanced in the inclined body position. Furthermore, these findings provide preliminary information that level and incline treadmill exercise tests may yield significantly different results in the Thoroughbred horse and consequently this factor should be considered when interpreting exercise testing and performance data.

NO inhalation reduces pulmonary arterial pressure but not hemorrhage in maximally exercising horses.

In horses, the exercise-induced elevation of pulmonary arterial pressure (Ppa) is thought to play a deterministic role in exercise-induced pulmonary hemorrhage (EIPH), and thus treatment designed to lower Ppa might reasonably be expected to reduce EIPH. Five Thoroughbred horses were run on a treadmill to volitional fatigue (incremental step test) under nitric oxide (NO; inhaled 80 ppm) and control (N(2), same flow rate as per NO run) conditions (2 wk between trials; order randomized) to test the hypothesis that NO inhalation would reduce maximal Ppa but that this reduction may not necessarily reduce EIPH. Before each investigation, a microtipped pressure transducer was placed in the pulmonary artery 8 cm past the pulmonic valve to monitor Ppa. EIPH severity was assessed via bronchoalveolar lavage (BAL) 30 min postrun. Exercise time did not differ between the two trials (P > 0.05). NO administration resulted in a small but consistent and significant reduction in peak Ppa (N(2), 102.3 +/- 4.4; NO, 98.6 +/- 4.3 mmHg, P < 0.05). In the face of lowered Ppa, EIPH severity was significantly higher in the NO trial (N(2), 22.4 +/- 6.8; NO, 42.6 +/- 15.4 x 10(6) red blood cells/ml BAL fluid, P < 0.05). These findings support the notion that extremely high Ppa may reflect, in part, an arteriolar vasoconstriction that serves to protect the capillary bed from the extraordinarily high Ppa evoked during maximal exercise in the Thoroughbred horse. Furthermore, these data suggest that exogenous NO treatment during exercise in horses may not only be poor prophylaxis but may actually exacerbate the severity of EIPH.

Efficacy of nasal strip and furosemide in mitigating EIPH in Thoroughbred horses.

The purpose of this investigation was to study the effects of an equine nasal strip (NS), furosemide (Fur), and a combination of both (NS + Fur) on exercise-induced pulmonary hemorrhage (EIPH) at speeds corresponding to near-maximal effort. Five Thoroughbreds (526 +/- 25 kg) were run on a flat treadmill from 7 to 14 m/s in 1 m x s(-1) x min(-)1 increments every 2 wk (treatment order randomized) under control (Con), Fur (1 mg/kg iv 4 h prior), NS, or NS + Fur conditions. During each run, pulmonary arterial (Ppa) and esophageal (Pes) pressures were measured. Severity of EIPH was quantified via bronchoalveolar lavage (BAL) 30 min postrun. Furosemide (Fur and NS + Fur trials) reduced peak Ppa approximately 7 mmHg compared with Con (P < 0.05) whereas NS had no effect (P > 0.05). Maximal Pes swings were not different among groups (P > 0.05). NS significantly diminished EIPH compared with the Con trial [Con, 55.0 +/- 36.2; NS, 30.8 +/- 21.8 x 10(6) red blood cells (RBC)/ml BAL fluid; P < 0.05]. Fur reduced EIPH to a greater extent than NS (5.2 +/- 3.0 x 10(6) RBC/ml BAL; P < 0.05 vs. Con and NS) with no additional benefit from NS + Fur (8.5 +/- 4.2 x 10(6) RBC/ml BAL; P > 0.05 vs. Fur, P < 0.05 vs. Con and NS). In conclusion, although both modalities (NS and Fur) were successful in mitigating EIPH, neither abolished EIPH fully as evaluated via BAL. Fur was more effective than NS in constraining the severity of EIPH. The simultaneous use of both interventions appears to offer no further gain with respect to reducing EIPH.

Effect of L-NAME on oxygen uptake kinetics during heavy-intensity exercise in the horse.

There is evidence that oxidative enzyme inertia plays a major role in limiting/setting the O(2) uptake (VO(2)) response at the transition to higher metabolic rates and also that nitric oxide (NO) competitively inhibits VO(2) within the electron transport chain. To investigate whether NO is important in setting the dynamic response of VO(2) at the onset of high-intensity (heavy-domain) running in horses, five geldings were run on a treadmill across speed transitions from 3 m/s to speeds corresponding to 80% of peak VO(2) with and without nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor (20 mg/kg; order randomized). L-NAME did not alter (both P > 0.05) baseline (3 m/s, 15.4 +/- 0.3 and 16.2 +/- 0.5 l/min for control and L-NAME, respectively) or end-exercise VO(2) (56.9 +/- 5.1 and 55.2 +/- 5.8 l/min for control and L-NAME, respectively). However, in the L-NAME trial, the primary on-kinetic response was significantly (P < 0.05) faster (i.e., reduced time constant, 27.0 +/- 2.7 and 18.7 +/- 3.0 s for control and L-NAME, respectively), despite no change in the gain of VO(2) (P > 0.05). The faster on-kinetic response was confirmed independent of modeling by reduced time to 50, 63, and 75% of overall VO(2) response (all P < 0.05). In addition, onset of the VO(2) slow component occurred earlier (124.6 +/- 11.2 and 65.0 +/- 6.6 s for control and L-NAME, respectively), and the magnitude of the O(2) deficit was attenuated (both P < 0.05) in the L-NAME compared with the control trial. Acceleration of the VO(2) kinetics by L-NAME suggests that NO inhibition of mitochondrial VO(2) may contribute, in part, to the intrinsic metabolic inertia evidenced at the transition to higher metabolic rates in the horse.

Relationship of pulmonary arterial pressure to pulmonary haemorrhage in exercising horses.

Exercise-induced pulmonary haemorrhage (EIPH) is characterised by blood in the airways after strenuous exercise and results from stress failure of the pulmonary capillaries. The purpose of this experiment was to establish a threshold value of transmural pulmonary arterial pressure at which haemorrhage occurs in the exercising horse. Five geldings, age 4-14 years, were run in random order once every 2 weeks at 1 of 4 speeds (9, 11, 13, 15 m/s); one day with no run was used as a control. Heart rate, pulmonary arterial pressure and oesophageal pressure were recorded for the duration of the run. Transmural pulmonary arterial pressure was estimated by electronic subtraction of the oesophageal pressure from the intravascular pulmonary arterial pressure. Within 1 h of the run, bronchoalveolar lavage was performed and the red and white blood cells in the fluid were quantified. Red cell counts in the lavage fluid from horses running at 9, 11 and 13 m/s were not significantly different from the control value, but after runs at 15 m/s, red cell counts were significantly (P<0.05) higher. White cell counts were not different from control values at any speed. Analysis of red cell count vs. transmural pulmonary arterial pressure indicated that haemorrhage occurs at approximately 95 mmHg. Red cell lysis in the lavage fluid was also apparent at transmural pulmonary arterial pressures above 90 mmHg. We conclude that, in the exercising horse, a pulmonary arterial pressure threshold exists above which haemorrhage occurs, and that pressure is often exceeded during high speed sprint exercise.

Analysis of the right ventricular function in the exercising horse: use of the Fourier Transform.

The objective of this study was to develop and test a technique to allow dynamic cardiac function to be studied during exercise in the horse. Blood pressure waveforms in the exercising horse are difficult to interpret because of the large influence of stride and respiration. A method has been devised to study dynamic right ventricular variables during high-speed exercise in the horse. A Fast Fourier Transform was performed on the digitised pressure waveforms and the frequency components associated with stride and respiration were removed. An inverse Fourier Transform was then performed to generate a time-domain pressure signal. Several dynamic right ventricular variables were calculated using the derived signal. Various parameters associated with removing frequencies from the frequency-domain pressure signal were changed to determine their influence on the variables. Most of the variables were not sensitive to these parameters. When compared during separate exercise bouts, some variables differed among runs, while others were not significantly different. Using the signal separation technique described here, right ventricular function of an exercising horse can be critically analysed.

Cardiorespiratory impact of the nitric oxide synthase inhibitor L-NAME in the exercising horse.

To investigate the role of nitric oxide, NO, in facilitating cardiorespiratory function during exercise, five horses ran on a treadmill at speeds that yielded 50, 80 and 100% of peak pulmonary oxygen uptake (V(O(2)) peak) as determined on a maximal incremental test. Each horse underwent one control (C) and one (NO-synthase inhibitor; N(G)-L-nitro-arginine methyl ester (L-NAME), 20 mg/kg) trial in randomized order. Pulmonary gas exchange (open flow system), arterial and mixed-venous blood gases, cardiac output (Fick Principle), and pulmonary and systemic conductances were determined. L-NAME reduced exercise tolerance, as well as cardiac output (C, 291+/-34; L-NAME, 246+/-38 L/min), body O(2) delivery (C, 74.4+/-5. 5; L-NAME, 62.1+/-5.6 L/min), and both pulmonary (C, 3.07+/-0.26; L-NAME, 2.84+/-0.35 L/min per mmHg) and systemic (C, 1.55+/-0.24; L-NAME, 1.17+/-0.16 L/min per mmHg) effective vascular conductances at peak running speeds (all P<0.05). On the 50 and 80% trials, L-NAME increased O(2) extraction, which compensated for the reduced body O(2) delivery and prevented a fall in V(O(2)). However, at peak running speed in the L-NAME trial, an elevated O(2) extraction (P<0. 05) was not sufficient to prevent V(O(2)) from falling consequent to the reduced O(2) delivery. At the 50 and 80% running speeds (as for peak), L-NAME reduced pulmonary and systemic effective conductances. These data demonstrate that the NO synthase inhibitor, L-NAME, induces a profound hemodynamic impairment at submaximal and peak running speeds in the horse thereby unveiling a potentially crucial role for NO in mediating endothelial function during exercise.

Effect of furosemide on pulmonary blood flow distribution in resting and exercising horses.

We determined the spatial distribution of pulmonary blood flow (PBF) with 15-micron fluorescent-labeled microspheres during rest and exercise in five Thoroughbred horses before and 4 h after furosemide administration (0.5 mg/kg iv). The primary finding of this study was that PBF redistribution occurred from rest to exercise, both with and without furosemide. However, there was less blood flow to the dorsal portion of the lung during exercise postfurosemide compared with prefurosemide. Furosemide did alter the resting perfusion distribution by increasing the flow to the ventral regions of the lung; however, that increase in flow was abated with exercise. Other findings included 1) unchanged gas exchange and cardiac output during rest and exercise after vs. before furosemide, 2) a decrease in pulmonary arterial pressure after furosemide, 3) an increase in the slope of the relationship of PBF vs. vertical height up the lung during exercise, both with and without furosemide, and 4) a decrease in blood flow to the dorsal region of the lung at rest after furosemide. Pulmonary perfusion variability within the lung may be a function of the anatomy of the pulmonary vessels that results in a predominantly fixed spatial pattern of flow distribution.

Influence of frusemide on dynamic cardiac variables during exercise.

Exercising horses have extremely high right and left atrial pressures. Limitation in ventricular function (i.e. relaxation) may play a role in these high pressures. We studied relaxation characteristics of the right ventricular myocardium and the impact of frusemide (2.0 mg/kg bwt i.v.) on these characteristics in horses exercising at 8, 10, 12 and 14 m/s. Exercise tests were performed 4 h after administration of frusemide. Right ventricular (RV) pressure was analysed using Fast Fourier Transform techniques to remove non cardiac components of the pressure signal. Mean right atrial (RA) pressure increased with exercise and was significantly attenuated at all speeds by frusemide. RV maximum and minimum rates of pressure change with respect to time (RV + dP/dtmax, RV-dP/dtmax) increased with exercise and RV relaxation time constant (RV tau) and time of RV relaxation from 65-20% of the difference between maximum and minimum ventricular pressure (delta 65-20) decreased with exercise. Frusemide produced no significant differences in +dP/dtmax, -dP/dtmax, RV tau or delta 65-20 except at 12 m/s where RV tau was longer after frusemide (23.4 ms for frusemide vs. 19.7 ms for control). Significant reductions in stroke volume were seen at 8, 10 and 14 m/s after frusemide. These results suggest that the reduction of atrial pressure by frusemide is not due to changes in ventricular relaxation rate.