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CVJ / VOL 50 / AUGUST 2009 821
Article
Effect of furosemide and furosemide–carbazochrome combination on
exercise-induced pulmonary hemorrhage in Standardbred racehorses
Cecilia I. Perez-Moreno, Laurent L. Couëtil, Suzanne M. Pratt, Hugo G. Ochoa-Acuña,
Rose E. Raskin, Mark A. Russell
Abstract — The objective was to quantify the effect of furosemide and carbazochrome on exercise-induced pul-
monary hemorrhage (EIPH) in Standardbred horses using red blood cell count and hemoglobin concentration in
bronchoalveolar lavage (BAL) fluid. Six healthy Standardbred horses with prior evidence of EIPH performed a
standardized treadmill test 4 h after administration of placebo, furosemide, or furosemide–carbazochrome com-
bination. Red blood cell (RBC) counts and hemoglobin concentrations were determined on the BAL fluid. The
RBC count in BAL ranges were (2903–26 025 cells/mL), (45–24 060 cells/mL), and (905–3045 cells/mL) for
placebo, furosemide, and furosemide–carbazochrome, respectively. Hemoglobin concentration ranges were
(0.03–0.59 mg/mL), (0.01–0.55 mg/mL), and (0.007–0.16 mg/mL) for placebo, furosemide, and furosemide–
carbazochrome groups, respectively. No significant differences were detected among treatments. However, there
was great variability among horses, suggesting that a larger sample size or better selection of horses was needed.
Résumé — Effet du furosémide et d’une combinaison de furosémide–carbazochrome sur l’hémorragie
pulmonaire induite par l’exercice chez les chevaux de course Standardbred. L’objectif était de quantifier l’effet
du furosémide et du carbazochrome sur l’hémorragie pulmonaire induite par l’exercice (HPIE) chez les chevaux
Standardbred en utilisant la numération des globules rouges et la concentration d’hémoglobine dans le liquide de
lavage broncho-alvéolaire (LBA). Six chevaux Standardbred en santé avec des signes antérieurs d’HPIE ont effectué
un test normalisé sur tapis roulant 4 heures après l’administration du placebo, du furosémide ou de la combinaison
furosémide–carbazochrome. Les numérations des globules rouges (GR) et les concentrations d’hémoglobine ont
été déterminées à partir du liquide du LBA. Les intervalles de numération des GR dans le LBA était de
[2903–26 025 cellules/mL], [445–24 060] et [905–3045] pour les groupes placebo, furosémide et furosémide–
carbazochrome, respectivement. Les intervalles de concentration de l’hémoglobine étaient de [0,03–0,59 mg/mL],
[0,01–0,55] et de [0,007–0,16] pour les groupes placebo, furosémide et furosémide — carbazochrome,
respectivement. Aucune différence significative n’a été détectée parmi les traitements. Cependant, la variabilité
entre les chevaux était très grande, suggérant qu’une taille d’échantillon plus importante ou une meilleure sélection
des chevaux était requise. (Traduit par Isabelle Vallières)
Can Vet J 2009;50:821–827
Introduction
Prevalence of exercise-induced pulmonary hemorrhage
(EIPH) can be as high as 80% to 90% in Standardbred
racehorses (1,2), and it has a significant adverse effect on per-
formance of racehorses (3,4). Several mechanisms have been
implicated in the etiology of EIPH. The predominant hypothesis
is that very high pulmonary vascular pressures occur during
strenuous exercise, resulting in stress-failure of the pulmonary
capillaries and consequent extravasation of blood into the
alveolar space (5,6). Another hypothesis is that EIPH is due to
impaired coagulation, but results from studies assessing coagu-
lation and fibrinolysis during exercise in horses have not been
conclusive (7–10).
Pulmonary hemorrhage and presence of blood at the nostrils
is sanctioned by all racing authorities. If a racehorse develops
epistaxis after exercise, it cannot race for the subsequent 7–25 d
depending on state regulations. Subsequent epistaxis episodes
Animal Science (Perez-Moreno, Russell), Veterinary Clinical Sciences (Couëtil), Comparative Pathobiology (Pratt, Ochua-Acuña,
Raskin), Purdue University, West Lafayette, Indiana 47906, USA.
Address all correspondence to Dr. Laurent L. Couëtil; e-mail: couetill@purdue.edu
This work was supported by the State of Indiana and the Purdue University School of Veterinary Medicine Research account;
funded by the total wager tax.
Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA
office (hbroughton@cvma-acmv.org) for additional copies or permission to use this material elsewhere.
822 CVJ / VOL 50 / AUGUST 2009
AR T I C L E
result in significantly longer interdiction of racing. Based on
the purported pathogenesis of EIPH, capillary wall stress could
be reduced by lowering pulmonary blood pressure. Furosemide
(such as Lasix) is a potent and rapidly acting loop diuretic com-
monly used to attenuate and manage EIPH (11,12). Furosemide
administration on race day as a prophylactic of EIPH is permit-
ted in almost all Thoroughbred, Standardbred, and Quarter
horse racing jurisdictions within the United States.
Several studies, however, have shown that despite the use of
furosemide, many horses continue to have blood in the trachea
(13,14). For this reason, adjunct bleeder medications such as
carbazochrome are currently used in some racing jurisdictions.
Carbazochrome salicylate, also known as Kentucky Red, is a
stable oxyepinephrine derivate classified among hemostatic drugs
as a capillary stabilizer (15). It is used clinically for the treat-
ment of hemorrhage due to capillary fragility in humans. The
mechanism of action of carbazochrome is unknown; however,
recent studies suggest that it reverses thrombin, tryptase, and
bradykinin-induced endothelial cell permeability by reducing
intracellular actin stress fiber formation and restoring inter-
cellular tight junctions (15,16). This drug is not approved
for use as a race-day medication by the Racing Medication
and Testing Consortium (RMTC) but the states of Louisiana,
Kentucky, Maryland, and Virginia do permit its use on race
day. Unfortunately, there is currently no scientific proof of the
efficacy of carbazochrome on pulmonary bleeding.
Previous studies have shown that horses with EIPH alone
or in combination with other respiratory diseases become
significantly more hypoxemic than control horses during stan-
dardized treadmill exercise tests (17–19). However, the effect
on gas exchange of drugs used to mitigate EIPH has not been
reported.
The objective of this study was to quantify the effect of furo-
semide and carbazochrome on EIPH in exercising Standardbred
racehorses using red blood cell count, hemoglobin, and protein
concentrations in bronchoalveolar lavage fluid as primary
outcome variables. The effect of treatment on hemostasis, gas
exchange, and performance during treadmill tests was also
evaluated.
Materials and methods
Horses
Six Standardbred horses (3 fillies and 3 geldings) between
3- and 9-years-old, with a mean weight of 452.5 6 9.7 kg were
used. Criteria for selection were prior evidence of epistaxis or
hemorrhage in the trachea confirmed by a regulatory veterinar-
ian after exercise; at least 6 mo on the bleeder’s list, no history
of upper airway obstruction, and no evidence of lameness or
dynamic airway obstruction during a high-speed treadmill test
conducted before the start of training. Horses were housed in
stalls, fed alfalfa grass hay and a grain mixture, and had free
access to water and salt. Horses were familiarized with laboratory
surroundings and were acclimatized to running on the high-
speed equine treadmill (SATO I; Equine Dynamics, Lexington,
Kentucky, USA). All horses were conditioned 5 d per wk for
6 wk to establish uniform fitness before study initiation, and all
the horses were able to complete the exercise protocol without
difficulty. Bronchoalveolar lavage fluid was obtained from all
horses 1 wk before undergoing strenuous exercise testing. Care
of the animals and all procedures performed and were approved
by Purdue University Animal Care and Use Committee.
Experimental design
The clinical trial was designed as a 3-way-cross-over, placebo-
controlled, Latin square design. Each horse completed 3 iden-
tical treadmill tests at 1-week intervals. Horses completed a
standardized treadmill test 4 h after receiving each of the 3 intra-
venous treatments in randomized order: placebo (20 mL; 0.9%
sterile saline solution), furosemide (250 mg) and furosemide
(250 mg) –carbazochrome salicylate (Wedgewood, Swedesboro,
New Jersey, USA) (100 mg) combination. Saline solution was
added to the drugs as needed to obtain a final volume of 20 mL
for each of the 3 treatments, and investigators were blinded to
the treatments.
Standardized treadmill test (STT) protocol
All treadmill tests were performed in a climate-controlled
building (20°C to 22°C). On the day of each trial, the horse
was weighed and treatment was given. Water and feed were
withheld at the time of drug administration until completion of
the treadmill test. Horses were weighed again after test comple-
tion. A catheter was placed in the transverse facial artery for
blood sampling. Each horse wore a safety harness and was fitted
with a heart-rate monitor (Polar Equine S-610; Polar Electro,
New York, New York, USA) to ensure continuous recording of
heart rate throughout the exercise test.
The STT was preceded by warmup at zero slope (4-min walk;
2 m/s) and a 4-min trot (4 m/s), and consisted of 90-s incre-
mental speed steps (5,8,10,11, and 12 m/s) all at a 5% incline.
The incremental exercise testing was stopped when the horse
exhibited signs of fatigue and was unable to maintain its position
at the front of the treadmill or after 1 ½ min at 12 m/s. Run
time and distance covered were automatically recorded using a
computer program.
Blood sampling
All samples for clotting assays were evaluated in duplicate.
Baseline blood samples were collected while the horse was
at rest before the treadmill test. Blood was drawn by direct
venipuncture using Vacutainer into 2 pre-warmed (37°C)
tubes containing silaceous earth (diatomaceous earth) to esti-
mate activated clotting time (ACT) using a 2-tube technique
following the manufacturer’s instructions (BD Vacutainer
#366522; Franklin Lakes, New Jersey, USA). Blood samples for
evaluation of prothrombin time (PT)/partial thromboplastin
time (PTT) and D-dimer concentration were collected and
placed into 2 citrated (3.2%) tubes (BD Vacutainer Systems.
The PT and PTT were determined using a STA Compact
analyzer (Diagnostica Stago, Parsippany, New Jersey, USA).
The PT and PTT measurements are part of the routine coagu-
lation profile used at Purdue University Clinical Pathology
Laboratory and a reference range has been previously estab-
lished for horses. Determination of D-dimer concentration was
performed with 2 latex agglutination immunoassays validated
CVJ / VOL 50 / AUGUST 2009 823
AR T I C L E
for horses, the D-Di Test (Diagnostica Stago), and the Amax
Accuclot D-Dimer (Sigma Diagnostics, St Louis, Missouri,
USA) following the manufacturers’ instructions.
During the STT, blood was drawn after every 1st minute at
each speed step (5, 8, 10, 11, and 12 m/s). Blood was placed
into 1 chilled heparinized syringe for measurement of blood
gases (Rapidlab 800; Bayer, Tarrytown, New York, USA) and
in 2 chilled citrated, evacuated glass tubes for determination of
PT, PTT, and D-dimer concentrations. Additionally, aliquots
of blood were placed in 2 pre-warmed tubes for determina-
tion of ACT at the 3rd speed step (10 m/s) during the exercise
treadmill test.
Immediately at the end of the treadmill test, blood was drawn
by direct venipuncture using Vacutainer into 2 pre-warmed tubes
(37°C) for determination of ACT and into 2 citrated tubes for
PT/PTT and D-dimer concentration and immediately placed
in crushed ice. All ACT and D-dimer concentrations were
determined by the same investigator (CPM).
Post-exercise evaluation and bronchoalveolar
lavage (BAL)
One hour after the STT, horses were sedated with xylazine
hydrochloride (0.5 mg/kg, IV) and positioned in stocks. A
flexible video-endoscope (200-cm long, 9-mm diameter) was
advanced into the trachea and findings were recorded for later
analysis and grading of the amount of blood visible in the tra-
cheobronchial tree by a clinician unaware of the horse’s treat-
ment. A commonly used 4-point grading system was employed
(20). Visible tracheal mucus accumulation was also scored
using a 0–5 scale (21) where 0 = no visible mucus; 1 = singular
small aggregates; 2 = multiple aggregates only partly conflu-
ent; 3 = mucus ventrally confluent; 4 = large ventral pool; and
5 = profuse amounts of mucus occupying more than 25% of tra-
cheal lumen. The endoscope was then advanced further into the
main stem bronchus until it was wedged into the caudo-dorsal
airways of the right lung. Coughing was prevented by spray-
ing the airways with a 0.2% lidocaine solution (40–60 mL).
The BAL was then performed by infusing sterile saline solu-
tion (250 mL, 20°C) via a sterile polyethylene catheter passed
through the endoscope biopsy channel. Immediately after, fluid
was gently aspirated via a suction pump and immediately placed
on ice. All fluid samples were processed within 20 min of col-
lection. All BAL fluid collections were performed by the same
investigator (LLC).
Red blood cell and leukocyte counts were determined using an
automated cell counter (CELL-DYN 3700, Abbott Laboratories,
Abbott Park, Illinois, USA). Erythrocytes were also counted
using a Neubauer hemocytometer. Slides were made by centrifu-
gation in a Cytospin (Shandon Scientific, Cheshire, England)
and stained with modified Wright’s stain. Cytological evaluation
included a 200 differential cell count, in which the number of
macrophages, hemosiderophages, lymphocytes, neutrophils,
eosinophils, and mast cells were reported as an absolute number,
and as a percentage of total nucleated cells. Hemosiderophages
were also reported as a percentage of macrophages.
The remaining BAL fluid samples were stored at -80°C and
batch analysis of protein and hemoglobin concentration was
performed. Protein concentration in BAL fluid was assessed
by the bicinchoninic acid (BCA) method (Pierce Chemical
Company, Rockford, Illinois, USA). Hemoglobin was measured
in BAL fluid using a modified benzidine assay. Equine hemoglo-
bin (Sigma Chemical Company, St. Louis, Missouri, USA) was
used as standard for the assay. This sensitive and rapid assay was
adapted for microtiter plate format from Sigma Colorimetric
Assay, Procedure #527.
Drug safety
Safety of the medications was assessed during the study by daily
physical examination. In addition, blood analyses for complete
blood (cell) count (CBC) and serum biochemistry profile were
performed on each horse 24 h after drug administration.
Statistical analysis
The effects of carry-over, period of administration, sequence of
treatment, and horse on the measured variables and the inter-
action between treatment and horse were tested for statistical
significance using a Grizzle Model with the GLM Procedure in
SAS (SAS Institute, Cary, North Carolina, USA). This model
looks into the possibility of carryover effects when switching
treatments. These analyses were followed by Dunnet’s test for
multiple comparisons when a significant difference was found.
Spearman’s test was used for analysis of correlations between
variables. The t-test was used for comparison between subgroups.
The significance level was placed at a P-value of 0.05.
Results
The speed achieved for a heart rate of 200 bpm (V200) signifi-
cantly improved with training (P , 0.01). Mean test V200 ranged
from 9.36 to 10.24 m/s in all horses except one that tended to
push against the front strap, with a V200 74% to 82% lower
compared with the other horses (P . 0.05). All horses remained
sound and healthy during the study period.
Weight loss over the 4-hour period after treatment admin-
istration was 9.4 (1.1) kg, 14.8 (1.8) kg, and 13.8 (1.1) kg
for the placebo, furosemide, and furosemide–carbazochrome
groups, respectively (P = 0.07). No significant difference was
observed for distance covered (km) and time required to cover
this distance between treatment groups. Two animals could not
complete the entire test due to fatigue. Both horses reached the
last speed step (12 m/s) but could not sustain the pace for the
required 90 s.
All horses exercised at speeds eliciting maximum heart rate.
Heart rate increased linearly with exercise intensity until a pla-
teau was attained at a maximum of 220–223 bpm in all treat-
ments (P , 0.0001). There was great variability among horses
and no significant difference was detected between treatments
(P , 0.05).
The median EIPH endoscopy scores were 1.56 [range (0–3)],
1.12 [range (0–2)], and 1.0 [range (0–1)] for placebo, furo-
semide, and furosemide–carbazochrome treatments, respectively.
Mucus score medians were 2 [range (0–3)] for all groups. A
significant correlation was observed between EIPH endoscopy
score and the mucus score (r = 0.61; P = 0.0065). However,
there was no effect of treatment on EIPH endoscopy and
824 CVJ / VOL 50 / AUGUST 2009
AR T I C L E
mucus scores. No significant correlation was found between
endoscopy score and manual RBC numbers in BAL fluid
(r = 0.39; P . 0.1) or between mucus score and RBC in BAL
fluid (r = -0.24; P . 0.1). Moreover, neutrophil proportion in
BALF of the horses was not significantly correlated with mucus
score (r = -0.12, P . 0.5). Average BAL fluid recovery ranged
from 59% to 70%, with no significant differences between
treatments.
Automatic RBC count failed to enumerate erythrocytes
in BAL fluid under 30 000 cells and consequently, were
not included in the analysis. Manual RBC counts from the
1st week of testing were not available, therefore 6 observa-
tions were missing. The RBC counts in BAL ranges were
(2903–26 025 cells/mL), (445–24 060 cells/mL), and
[905–3045 cells/mL) for placebo, furosemide, and furose-
mide–carbazochrome, respectively (n = 4 per group). There was
a significant correlation between manual RBC count and hemo-
globin concentration in BAL fluid (r = 0.97; P , 0.0001). The
average protein concentration ranged from 483 to 661 mg/mL
(Figure 1). Variability between horses was present (P , 0.05).
There was a highly significant correlation between protein
concentration in BAL fluid and manual RBC count (r = 0.9;
P , 0.0001). Baseline hemoglobin concentration ranged from
0.004 to 0.026 mg/mL. Mean concentration of hemoglobin
after exercise testing ranged from 0.06 to 0.20 mg/mL
(Figure 2). There were no significant differences between
treatments for RBC, protein and hemoglobin concentrations,
probably because of the variability among horses (P , 0.05).
An attempt to compare individual horses was based on
hemoglobin concentration in BAL fluid in the placebo group.
Animals were grouped into 2 categories of 3 horses each and
were compared between more severe bleeders and less severe
bleeders. A significant difference was found (P , 0.05) between
these 2 groups (Figure 3). Moreover, 2 horses did not follow
the expected trend in response to treatment administration;
therefore, a new comparison between treatments was performed
after their exclusion. A significant treatment effect was found for
protein concentration in BALF (P = 0.0006), and a trend was
found for hemoglobin concentration (P = 0.054). There was no
significant correlation between hemoglobin concentration and
endoscopy score (r = 0.33; P . 0.1).
Total nucleated cell numbers are presented in Table 1.
Absolute numbers and percentages of cell types showed no sig-
nificant effect of treatment; however, there was large variability
between horses (P , 0.05). Percentage and absolute numbers of
hemosiderophages showed no statistically significant difference
between treatments; however, animals in all treatment groups
had hemosiderin present in the macrophages. There was no
significant correlation between manual RBC count and hemo-
siderophage numbers in BAL fluid (r = 0.39; P . 0.1). There
was no effect of treatment on blood chemistry and hematology
parameters. There were; however, significant differences among
horses (P , 0.05).
The ACT, PT, and PTT values are shown in Table 2.
Compared with baseline values, exercise produced a significant
decrease in mean ACT of about 58% to 61% in all groups
(P , 0.0001). Post-exercise ACT was 34% to 43% lower
than the pre-exercise values in the 3 groups (P , 0.0001).
Prothrombin time showed a significant decrease during the last
2 speed steps of the treadmill test compared with baseline values
(P , 0.01). Partial thromboplastin time values only showed
a shortened time compared with baseline values at 11 m/s
(P , 0.05). Prothrombin time and PTT returned to baseline
values after exercise testing (P . 0.05). However, no treatment
Figure 1. Protein concentrations (mg/mL) in BAL fluid after a
standardized treadmill test in horses (P = 0.16). PLA — placebo,
FUR — furosemide; FU R 1 CBZ — furosemide-carbazochrome
combination. Data from individual horses are displayed.
PLA FUR FUR 1 CBZ
Protein concentration (mg/mL)
3
2.5
2
1.5
1
0.5
0
Figure 2. Hemoglobin concentrations (mg/mL) in BAL fluid after
a standardized treadmill test in horses (P = 0.35). Data from
individual horses is displayed.
PLA FUR FUR 1 CBZ
Hemoglobin concentration
(mg/mL)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Figure 3. Individual hemoglobin concentrations (mg/mL) in BAL
fluid from racehorses in the placebo group after a standardized
treadmill test. More severe bleeders (BR, CH, WH) compared
with less severe bleeders (DT, JO, TT). (P , 0.05), n = 6.
BR CH WH DT JO TT
Individual hemoglobin
concentration (mg/mL)
Animal
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
CVJ / VOL 50 / AUGUST 2009 825
AR T I C L E
effect was observed in ACT, PT, or PTT before, during, or
after exercise.
D-dimer concentration measured by the D-Di Test was
, 500 ng/mL in 5 of the 6 horses. Amax Accuclot revealed con-
centrations of , 250 ng/mL in 3 horses for all treatments, with
the other 3 having concentrations of no more than 500 ng/mL,
even during exercise. Only D-dimer concentrations using Amax
Accuclot showed a significant increase as exercise progressed
(P , 0.001) and showed also an increase after exercise compared
to baseline values (P , 0.05). Differences between horses were
detected (P , 0.05). No differences between treatment groups
were observed at any point of the testing with either D-Di Test
or Amax Accuclot.
Blood pH decreased significantly for the 3 treatments in all
animals as exercise progressed (P , 0.001). A nonsignificant
increase in arterial partial pressure of carbon dioxide (PaCO2)
was observed in all horses (P . 0.05); however, a significant
decrease in arterial partial pressure of oxygen (PaO2) was found
as the treadmill speed increased (P , 0.0001) (Figure 4). No
significant differences were observed between treatments for
pH, PaCO2, or PaO2.
Discussion
The main finding of this study was that pre-exercise administra-
tion of furosemide or furosemide-carbazochrome combination
did not affect the severity of pulmonary bleeding or the perfor-
mance of horses that had a history of EIPH. Training resulted
in a significant improvement in cardiovascular capacity (V200) of
the horses and all animals in the study were on the same level of
fitness before the beginning of the trial. Peak heart rates reached
by the horses during the treadmill tests showed that a level of
strenuous exercise was attained for each treatment run. These
findings are in agreement with other studies for Standardbred
horses that were heavily exercised on a track and a treadmill (22),
suggesting that although we cannot entirely imitate the environ-
ment of the horses running on a track, the exercise intensity
required to reproduce EIPH was attained.
All horses had some degree of mucus in the trachea during
post-exercise endoscopy consistent with previous reports (23).
However, our findings suggest that the amount of blood or
mucus present in the trachea 1 h post-exercise does not indi-
cate the degree of inflammation or bleeding that these horses
may have in the deep lung sampled during BAL. Neutrophil
proportion in BALF in this study was within the reference
range reported for healthy horses (24–26). Total nucleated cell
numbers and differential cell counts were similar between treat-
ments and horses and no specific inflammatory changes were
present in BAL fluid.
Five of the 18 endoscopies performed in the study showed
no evidence of blood at the trachea, although all of the animals
had measurable concentrations of hemoglobin in BAL fluid after
exercise. Indeed, our study provides evidence that the severity of
the hemorrhage present in the lungs is underestimated by endos-
copy. Presence of hemosiderophages in the BALF has also been
used as a method to estimate the degree of bleeding (27,28).
The weak correlation between the number of hemosiderophages
Table 1. Total nucleated cell numbers (cells/mL) and
200 differential cell count with percentage and absolute numbers
in racehorses after a standardized treadmill test. Values are
mean (SD)
Treatment
Furosemide-
Placebo Furosemide Carbazochrome
[mean 6 (s)] [mean 6 (s)] [mean 6 (s)]
WBC (cells/mL) 305 (207.7) 311 (220) 306 (147.6)
Macrophages (% of total) 56.5 (11.7) 59.16 (9.66) 58.33 (10.03)
Hemosiderophages 13.66 (20.6) 15.5 (18.6) 11.33 (15.8)
(% of macrophages)
Lymphocytes (% of total) 39 (10.8) 34.33 (9.52) 36.33 (8.5)
Neutrophils (% of total) 2.66 (0.8) 3.5 (1.64) 3.66 (1.97)
Mast cells (% of total) 1.83 (1.5) 3 (2.6) 1.66 (1.2)
Eosinophils (% of total) 0 ( 0 ( 0 (
Macrophages (cells) 113 (23.4) 118.33 (19.3) 116.66 (20)
Hemosiderophages (cells) 17.27 (29.6) 19.8 (26.6) 12.45 (15.7)
Lymphocytes (cells) 78 (21.7) 68.66 (19) 72.66 (17)
Neutrophils (cells) 5.33 (1.6) 7 (3.3) 7.33 (4)
Mast cells (cells) 3.66 (2.9) 6 (5.2) 3.33 (2.4)
Eosinophils (cells) 0 ( 0 ( 0 (
Figure 4. Arterial partial pressure of oxygen (PaO2) (mmHg)
in racehorses during a standardized treadmill test. Values are
mean 6 standard deviation (s).
PLA FUR FUR 1 CBZ
PaO2 (mmHg)
110
105
100
95
90
85
80
75
70
65
60
5 8 10 11 12
Speed (m/s)
Table 2. Activated clotting time (ACT), prothrombin time (PT), and
partial thromboplastin time (PTT) in racehorses before, during, and
after a standardized treadmill test
Treatment
Furosemide-
Time Placebo Furosemide Carbazochrome
(s) [mean 6 (s)] [mean 6 (s)] [mean 6 (s)]
Pre ACT 103.2 (0.33)a 113.4 (0.31)a 100.2 (0.29)a
ACT (10 m/s) 39.6 (0.25)ab 42.6 (0.27)ab 42 (0.26)ab
Post ACT 58.2 (0.21)ab 64.2 (0.16)ab 65.4 (0.21)ab
Pre PT 11.3 (0.35)a 11.06 (0.48)a 11.06 (0.31)a
PT (5 m/s) 11.3 (0.34) 11.5 (1.13) 11.1 (0.33)
PT (8 m/s) 11.2 (0.26) 11.1 (0.55) 11.1 (0.31)
PT (10 m/s) 11.2 (0.23) 11.1 (0.47) 10.9 (0.32)
PT (11 m/s) 11.2 (0.24)a 10.9 (0.52)a 10.9 (0.04)a
PT (12 m/s) 11.3 (0.25)ab 10.9 (0.4)ab 11.0 (0.3)ab
Post PT 11.4 (0.43)b 10.9 (0.47)b 11.2 (0.2)b
Pre PTT 45.1 (2.34)a 44.8 (2.18)a 45.4 (2.45)a
PTT (5 m/s) 45.65 (1.84)bc 44.6 (2.16)bc 45.4 (2.64)bc
PTT (8 m/s) 44.5 (1.93) 45.0 (3.22) 43.9 (2.25)
PTT (10 m/s) 44.4 (2.1)c 44.25 (1.96)c 43.46 (2.54)c
PTT (11 m/s) 44 (2.16)ab 43.9 (2.03)ab 43.5 (2.32)ab
PTT (12 m/s) 44.1 (2.62)c 44.3 (1.96)c 43.3 (1.9)c
Post PTT 44.5 (2.62) 44.6 (2.3) 44.2 (2.37)
a,b,c Data with identical superscripts are significantly different from each other
within the same treatment group (P , 0.05), n = 6.
826 CVJ / VOL 50 / AUGUST 2009
AR T I C L E
present in BAL fluid and hemoglobin concentration (r = 0.38;
P = 0.11), however, suggests that the quantification of this type
of cell in BAL fluid may be an inaccurate method of measuring
acute EIPH, as only past hemorrhage is indicated (3,28–30).
Although we have no information on the type of protein pres-
ent in the recovered fluid, the strong correlation between RBC
numbers and protein concentration in BAL fluid suggests
that protein concentration could be an indicator of severity of
hemorrhage. However, the smaller effect of treatment on BALF
protein concentration implies that BALF protein may not be
as good an indicator of severity of hemorrhage in the lungs as
hemoglobin. This suggests that the amount of protein leaking
from the pulmonary vasculature into the airway lumen was
small, compared with protein levels present in the epithelial
lining fluid.
Mean hemoglobin concentration in BAL was not statistically
different between groups; however, the standard deviations (s)
were comparatively large and may have masked small treatment
effects. In fact, a reduction of 25% and 70% in hemoglobin
concentration was observed in the furosemide and furosemide —
carbazochrome treatments, respectively, when compared with the
placebo group. Furthermore, the difference in bleeding sever-
ity detected between individual horses suggests that selection
of the horses is an important factor that should be taken into
consideration when designing a study. In this regard, Kindig
et al (3) observed a significant reduction in BAL RBC count
in 5 furosemide treated horses compared to placebo. However,
mean RBC count in the placebo group was 55.0 3 106 cells/mL
of BAL fluid, which is approximately 5 times higher than that
in our study. Similarly, Geor et al (31) reported a mean RBC
count approximately 6 times higher than that in our study.
Differences in characteristics of the study population, intensity
of exercise, technique, and BAL fluid volume used, and analysis
could also account for the variation in results between these
studies and our study.
Poor statistical power may also be an important consider-
ation when trying to explain the lack of significance in our
results. Due to the difficulty in recruiting the desired number
of 8 horses, the sample size originally sought, the statistical
power of the analyses performed on RBC and hemoglobin
concentration with 6 horses was 0.60, less than the desired
value of 0.80 when the alpha error was set at 0.05. Given the
variability (s) we observed in our horses, a sample size of more
than 8 animals would have been needed to achieve a power of
80%. Consequently, our probability of detecting a clinically
important difference, if it had existed, was reduced.
In humans, heavy exercise has been associated with activation
of both blood coagulation and fibrinolysis (32,33). The ACT
baseline values in our horses were in accordance with other
reports (34). McKeever et al (7) reported that clotting time
returned to pre-test levels 15 min after cessation of exercise. Our
findings suggest that enhancement of coagulation during exercise
may still be present for a short time after cessation of exercise.
Additionally, these results do not support the hypothesis of a
coagulation defect as a mechanism of EIPH. Previous studies
in horses could not detect differences in PT and PTT measure-
ments after exercise (8,35), although speed of exercise did not
exceed 10 m/s. No effect of furosemide-carbazochrome combi-
nation on coagulation parameters was detected herein, suggest-
ing that the beneficial effect of carbazochrome, if it attenuates
pulmonary hemorrhage, may be due to another mechanism.
Studies in humans showed that fibrinolysis is increased
after exercise (36,37). We did detect an effect of exercise on
fibrinolysis in our horses; however, the differences in D-dimer
concentrations at rest and during exercise suggest that D-dimer
concentration in horses is highly variable. Matsumoto et al (38)
found that carbazochrome had an inhibitory effect on tissue
plasminogen activator (t-PA) on endothelial cells in culture.
In the present study D-dimer concentration was not affected
by treatment, suggesting that carbazochrome may not act as
a hemostatic agent through modulation of endothelial cell
function.
The degree of exercise-induced arterial hypoxemia and hyper-
capnia in all animals was consistent with previous reports
(39–45). In our study, arterial oxygenation worsened as treadmill
speed increased in all horses, in agreement with prior studies in
horses with and without EIPH (17,18). In the present study,
PaO2 and PaCO2 were not affected by treatment, but because
treatment did not significantly affect the degree of EIPH
exhibited by the horses we cannot discount the possibility
that furosemide and carbazochrome would have improved gas
exchanges if indeed the drug combination would have reduced
pulmonary bleeding.
In conclusion, this study found that furosemide–
carbazochrome combination had no detectable effect on
the severity of exercise-induced pulmonary hemorrhage in
Standardbred horses compared with furosemide and placebo
treatments. Furthermore, none of the treatments abolished
pulmonary hemorrhage following intense exercise in these
horses. Horses, however, showed a 25% and 70% reduction in
hemoglobin concentration in BALF when receiving furosemide
and furosemide–carbazochrome combination, respectively, when
compared with the placebo group. Consequently, further studies
are warranted, but should take into account the shortcomings
described in this study in order to determine if horses with
EIPH may indeed benefit from carbazochrome therapy. CVJ
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