Respiration
EFFECTS
Physiology
(1976) 27, 305-321;
OF SIMULATED
NATAL10
Department
have been studied
exercise at
exposure
marked
635 and
animals,
associated
outputs.
in P,,
[Hb]
transport
(617 to 1245 cap/mm’) about
30”,. Calculations
in the hypoxic
IN DOGS’
AHMAD ROSTAMI
qf Medicine,
Dmrer,
Cola. 80220.
moderate
animal
remained
chamber.
hyperventilation
significantly
At both levels of PB the dogs
and low Pa,,>‘s
(28 mm Hg at
ml:min kg) reflecting
O2 content.
Capillary
after 3 weeks at a Pa of 435, whereas indicate because
density
and hypoxic
in the hypoxic 0, content
in the sternothyroid
the average
dogs
difference
muscle doubled
muscle fiber diameter
decreased
of the muscle fiber is relatively
in tiber size and intercapillary
Altitude
higher
distances.
Hypoxia
Capillary Cardiac
P,, ‘s decreased arterial-venous
that the Paz in the ‘lethal corner’
of the decrease
the
after 3 weeks at 435 mm Hg with a 4 mm
and mixed venous
but arterial
unchanged.
treadmill
in Denver at a Pa of 635 mm Hg and after 3 weeks
\io>’ s were high (12.4 and 11.4
exercise. Arterial
U.S.A
muscle and on the cardiocirculatory
Q, HR. SV and systemic blood pressures were normal in both normoxic
(82 to 56 and 42 to 36 mm Hg. respectively) 0,
Amsterdam
TRANSPORT
on skeletal
with pulmonary
and Hct increased
at rest as well as during
and systemic
Company,
dogs (16-39 kg) at rest and during
were obtained
I7 mm Hg at 435 mm Hg). Resting
Hg decrease
School
to a Pa of 435 mm Hg using a hypobaric
panting,
high respiratory
ON 0,
altitude
conscious,
IS”; incline. Measurements
of continuous showed
qf Colorado
to simulated
on awake,
Publishing
MANUEL GIMENEZ, and SHEILA H. EBY
University
Abstract. The effects of exposure system
ALTITUDE
BANCHERO,
qf Physiology,
Norrh-Holland
Muscle
density output
0,
Hemoglobin
fiber size
transport
Ventilation
Acclimatization to chronic hypoxia involves several cardiopulmonary adjustments which contribute to supply 0, to the body tissues. Resting 0, consumption in hypoxia is the same as in normoxia but the partial pressure of 0, in the cells is assumed to be somewhat lower. It is during exercise in hypoxia that the maximal 0, consumption, ix, the maximal aerobic power is reduced in relation to normoxic standards (Balke et al., 1965: Buskirk et al., 1967; Klausen et al., 1966; Piiper et al., 1966). Buskirk (1969) suggested that the factors limiting aerobic capacity reside in skeletal muscle, where the tissular 0, tension may be temporarily reduced to levels incompatible with optimal muscle function. There is evidence obtained primarily in rodents, that tissue changes, specifically Accepted
for publication
’ Supported
25 April
by U.S. Public
1976.
Health
Service Grants
# HL-14317 305
& HL-12679
N. BANCHERO
306
et d.
increased capillary density, are involved in the process of adaptation to hypoxia (Cassin et al., 1971; Valdivia, 1958). Also, increased capillary density associated with smaller muscle fiber diameters has been found in skeletal muscle of dogs in hypoxia (Banchero, 1975; Eby and Banchero, 1976). We have studied the effects of changes in skeletal muscle and in the cardiocirculatory system on 0, transport in dogs in hypoxia at rest and during moderate treadmill exercise. Previous reports on the circulatory effects of chronic hypoxia in dogs indicate either a normal cardiac response over a 26 day sojourn (Vogel and Hannon, 1966) or a depression represented by lower cardiac output, heart rate and stroke volume during a 21 day exposure (Hansen et al., 1962).
Materials
and methods
Mongrel dogs weighing from 16 to 39 kg were studied awake and unsedated at two different levels of barometric pressure (PB): 635 mm Hg, in Denver, at 1,610 m and after 3 weeks of continuous exposure to 435 mm Hg in a hypobaric chamber (Colorado State University, Ft. Collins, Colo.). At both levels of Pa the animals were studied at rest as well as during treadmill exercise on a 15 y0 incline. Preparation of the animals
Healthy dogs, vaccinated for distemper, rabies and hepatitis, and quarantined for three weeks, were selected for their ability to run on a treadmill. The dogs were maintained in individual cages at a temperature of 22 + 2 “C. A tracheostomy was performed (Thilenius and Borquez-Vial, 1963) under general anesthesia (sodium pentobarbital 30 mg/kg i.v.) using sterile surgical technics. One of the two sternothyroid muscles was removed at this time for histological studies. 600,000 IU of penicillin were administered upon completion of this procedure. A minimum of one week was allowed for postsurgical recovery, before the animals were exercised on a treadmill, 3 times a week, at a 15 y0 incline, at increasingly higher speeds, up to 10 mph. The dogs were run for 3 minutes at each speed or until refusal occurred. We had previously found that dogs reach a ventilatory steady state after 3 minutes of exercise as judged from the measurements of respiratory function. Half of the maximal speed reached in the training sessions was then chosen and the animals were trained to run at that speed for about 20 minutes. Two to three days before the experiment, under general anesthesia, a catheter (PE 190) was introduced percutaneously into the right jugular vein and advanced into the pulmonary artery. A second catheter (PE 160) was inserted in the left carotid artery and advanced to the thoracic aorta. The external portions of these catheters were sutured to the skin using a piece of rubber tubing to prevent kinking. These catheters were used for blood pressure recording and blood sampling. After completion of the 3 week sojourn at simulated altitude the dogs were anesthetized and the contralateral sternothyroid muscle was removed.
EFFECTS OF ALTITUDE ON DOGS
307
Protocol
The dogs were studied following the sequence shown in fig. 1. The exercise extended over 17 min with the dogs running at an incline of 15 “/, at half the maximal speed reached during training. At a PB of 635 mm Hg seven dogs were studied twice,
i
iBIb
6 i EXERCISE
TIME (mini
Fig. 1. Temporal sequence of measurements and blood sample collections in conscious dogs studied standing at rest and during treadmill exercise at an incline of 15T,,.The collection of expired air and the measurement of cardiac output during exercise began after steady state was achieved as tested in preliminary experiments.
breathing room air and a mixture of 14% 0, in N, . At 14% 0, the levels of PI*, were similar to those in the hypobaric chamber while breathing room air at a PEZ of 435 mm Hg. At a PB of 435 mm Hg eight dogs were studied in exactly the same fashion, only while breathing room air. In addition, dogs breathed 99 74 0, at both levels of PB during which time only blood samples were collected. Animals were brought to the laboratory at least one hour before the experiment and preparations were made with the animal standing quietly on the treadmill. A fan was placed in front of the animal to improve air circulation. The dogs were studied only once a day. The experiments were conducted at room temperature, close to the thermoneutral point for dogs (2325C) (Hammel et al., 1958). In Denver the temperature varied from 20-25 “C and from 20.8-24.5 “C in the hypobaric chamber.
308
N. BANCHERO
et al.
Measurements Blood pressures were measured in the pulmonary artery and the aorta using Statham pressure transducers (Model P23Db). The zero reference level was set at the level of the shoulder joint with the animal standing at rest. Heart rate was calculated from the pressure recordings. Blood samples obtained, anaerobically and simultaneously in heparinized glass syringes, were analyzed for Po, , Pco, and pH using Radiometer electrodes and corrected to the dog’s rectal temperature at the time of sampling. Lactic and pyruvic acid, hemoglobin and hematocrit were also measured in these samples. Lactic and pyruvic acids were analyzed by the enzymatic method of Marbach and Weil (1967). Resting blood oxygen saturations were determined in an American Optical oximeter. After indocyanine green had been injected into the animal blood oxygen saturations (exercise values) were calculated with a Severinghaus rule (Severinghaus, 1966). No statistically significant differences were found, in animals in whom no indocyanine green was injected, between the values for 0, saturation measured in the oximeter and those calculated with the rule. In vivo hemoglobin-oxygen dissociation curves were constructed using the resting 0, saturations (oximetry) and the partial pressure of 0, , corrected to 38 C, measured in arterial and mixed venous blood samples obtained from dogs while at different levels of inspired 0, concentration, and using an equation reported by Hellegers et al. (1959): log Po, = K, -K, (7.4-pH)+K, log S/(100-S), where K,, K, and K, are constants, and S = saturation; K, is the logarithm of P,,, K, is the Bohr constant and K, = l/n (n is Hill’s constant). The mean Hb-0, curves for all dogs, at the two different levels of barometric pressure were then plotted on double logarithmic scales, at 38 “C and pH of 7.4. An endotracheal tube with an inflatable cuff (dead space 30 ml) connected to a two way Hans-Rudolph plastic valve (Warren E. Collins, Inc.) (dead space 18 ml) was used for collection of expired air. The volume of expired air (fin*& and the respiratory frequency (f) were continuously recorded using a Parkinson-Cowan gas meter fitted with an electrical potentiometer. Samples of expired air were collected at set intervals (fig. 1) in rubber bags for analysis of 0, and CO, content using a Beckman paramagnetic 0, meter and a Beckman LB-l CO, meter, respectively. These instruments were calibrated with known gas mixtures before and after each measurement. The concentrations of 0, and CO, in the calibrating mixtures were analyzed by the Scholander method. Body and spirometer temperatures were measured continuously using thermistors (Yellow Springs Instruments, Model 130 STD). Alveolar ventilation was calculated using the equation: VA = K vc,J Paco2 (Rahn and Fenn, 1955). Cardiac output (Q) was measured at rest and during exercise (fig. 1) by the dye dilution technic (Cardiogreen@) using a Waters (Model XC-250) densitometer. Injections were made in the pulmonary artery while sampling from the aorta with a Harvard infusion withdrawal pump. Blood was immediately reinfused into the animal after each curve. Four to live dye curves were obtained in rapid succession
EFFECTS OF ALTITUDE ON DOGS
309
in each condition and an average value calculated. The densitometer was calibrated at the end of each experiment using two to three known blood-dye mixtures. Histological measurements Immediately after removal the muscle was fixed in buffered formalin, processed and cut transversely in 7 pm sections and stained with the PAS reaction. An average of 75 sections were stained from each muscle sample. Diameters of capillaries and fibers were measured in 30 fields, selected at random, using a calibrated eyepiece micrometer (Weibel et al., 1966). The number of capillaries per mm2 was counted in the same fields. A detailed description of these measurements has been given (Banchero, 1975).
Red ts
The average body weights of these dogs were 24.9 and 25.1 kg at PB'S of 635 and 435 mm Hg, respectively. The maximal weight loss was 3.0 kg while the maximal gain was 2.5 kg after a 3 week exposure to 435 mm Hg. Because the respiratory parameters and blood gas values obtained during the 5th and 13th minutes of exercise were not statistically different, they have been averaged, and therefore, only one set of values is reported for the exercise condition, with the exception of data for lactic and pyruvic acids.
VENTILATORY MEASUREMENTS
(tables 1 & 2)
Regardless of the level of partial pressure of 0, in inspired air our dogs at rest had high respiratory rates and minute ventilations with relatively little variation around the mean. This panting was not related to changes in room and/or body temperature. Jennings et al. (1973a) found similar degrees of panting in conscious dogs well accustomed to the laboratory, whose hypothalamic temperature did not change consistently. Resting 0, consumptions and CO, productions in dogs at a Pn of 635 mm Hg breathing air were generally high and reflected the panting and associated pulmonary hyperventilation. The resting Paco2’ s were accordingly low. This respiratory condition was similar to that of awake dogs studied by other investigators (Flandrois et af., 1971; Jennings et al., 1973a; Vogel and Hannon, 1966). On the average, Vo, rose during treadmill exercise about 3.5-fold as a result of more effective ventilation with significant increases in VT and a 4.4-fold increase in VA. No systematic changes in f were observed. During inhalation of 14% 0, at a Pn of 635 mm Hg there was a moderate increase in the resting VE, \j~ and VT, but no significant changes in Vjoa. This additional hyperventilation was associated with a decrease in PaLco2and marked respiratory
310
N. BANCHERO
t?t ai.
TABLE Average
ventilatory
values
+ 1 SD in conscious
levels of barometric
1
dogs at rest and during
pressure
and inspired
0,
treadmill
Rest PB (mm Hg)
635
7jE(L/Blin BTPS)
ir, (Ljmin
635
STPD)
(ml/min kg
oco2 (ml/min
f (min-
STPD)
kg STPD)
‘)
VT(ml)
Seven dogs were studied
435
0.14
635
435
0.14
0.21
0.21
41.2
30.8
64.0
65.5
66.9
5.6
4.4
9.6
6.6
7.2
15.6
7.6
10.2
16.4
33.7
43.5
52.9
1.2
1.8
7.8
7.7
13.”
13.2
307
287
276
1069
918
870
80
37
103
155
90
223
12.4
11.5
11.4
43.8
36.8
36.0
2.5
2.0
3.9
7.2
5.1
8.5
10.0
10.3
10.8
37.7
33.0
34.6
0.9
1.4
3.8
6.0
4.1
9.2
168
157
101
156
146
119
24
25
47
27
20
32
209
267
337
399
455
578
43
45
125
46
52
190
113
143
112
59
71
77
at 635 mm Hg and 8 were studied TABLE
Blood gas values in conscious
635
0.21
34.8
BTPS)
V,, (ml/min
at different
Exercise
0.21
F’o,
exercise
concentration
dogs at rest and during
at 435 mm Hg.
2 exercise at different
levels of barometric
pressure
and FI,* Rest
PB(mm Hd Fro,
0.21
PI,, (mm W pro> (mm Hg)
Pa,,
635
(mm Hg)
Sao, (%)
Cao, (~01%)
121
Exercise 635 0.14
435 0.21
635 0.21
635 0.14
435 0.21
80.7
78.4
80.3
77.8
0.7
0.8
0.4
0.8
0.7
0.4
86.6
56.6
61.9
92.0
59.3
62.9
7.3
6.5
4.9
6.2
7.5
2.4
82.0
53.4
55.5
85.9
53.6
53.6
3.8
8.6
5.3
5.5
6.2
6.5
94.5
82.6
83.5
94.2
81.6
79.9
0.8
4.3
3.7
0.9
3.4
5.5
18.3
14.9
18.8
19.2
15.7
18.0
2.7
1.7
2.7
2.3
2.0
2.8
121
EFFECTS OF ALTITUDE
Pace,(mm W
@a fro,(mm W svo,(%I Go2 W%) pHV
(a -J)O,
(vol”YO)
Coefficient
0,
Utilization ~.,_
(%)
Average
values
ON DOGS
311
28.4
22.2
16.6
24.6
17.9
14.9
3.5
3.8
4.9
4.6
5.5
3.0
7.415
7.468
7.453
7.419
7.479
7.436
0.018
0.027
0.016
0.022
0.024
0.028
41.5
30.5
36.3
33.8
26.6
28.3
3.6
5.4
3.5
2.9
5.2
5.0
64.6
47.5
57.6
47.3
35.4
39.9
5.1
7.4
5.5
6.3
9.2
9.7
12.1
8.7
13.0
9.6
6.8
9.2
2.1
1.7
2.7
1.8
1.7
2.7
7.377
7.442
7.426
7.353
7.413
7.392
0.024
0.021
0.017
0.026
0.048
0.025
6.16
6.28
5.84
9.51
8.90
9.02
1.08
1.23
0.99
I .48
1.71
1.77
33.7
42.2
31.5
49.2
57.1
52.5
4.1
8.4
6.3
6.4
10.7
11.3
f 1 SD.
alkalemia. During exercise the changes in ventilation were quantitatively similar to those in dogs breathing room air. \jo2 increased 3.2-fold. After three weeks of exposure to a PB of 435 mm Hg the resting %‘E was slightly lower than in normoxia but the VA was significantly higher than at 635 mm Hg, causing the Paco2 to drop to 16.6 mm Hg. The respiratory frequency was significantly lower (P < 0.005) and the VT was higher (P < 0.01) than in normoxia. VT increased during exercise, increasing \j~ and VA, but the vo, was less than in normoxia (P < 0.05). The frequency of breathing was also lower than at a PB of 635 mm Hg. BLOOD MEASUREMENTS
An average decrease of 4 mm Hg was observed in Hb-0, affinity as a result of exposure to simulated altitude for 3 weeks (fig. 2). This observation is in agreement with earlier findings in severely anemic dogs (Cropp and Gee, 1972). Hemoglobin and hematocrit (fig. 3) No systematic differences were found between the resting hemoglobin concentration or the resting hematocrit obtained under the two levels of Floz at a PB of 635 mm Hg. Hemoglobin and hematocrit increased significantly (P < 0.05) after three weeks of exposure to a PB of 435 mm Hg with no systematic changes in mean corpuscular hemoglobin concentration. The values for [Hb] and Hct at a PB of 435 mm Hg were almost identical to those found in dogs native to 4,350 m (Banchero et al., 1975). During exercise mild increases in hemoglobin and hematocrit were observed at a PB of 635 mm Hg. They were absent at a PB of 435 mm Hg.
312
et al.
N. BANCHERO
Blood gases (table 2)
In Denver, at a Pn of 635 mm Hg, the values for P,, and So, in arterial blood were similar to those found in dogs at sea level, both at rest as well as during exercise. This was probably due to the pulmonary hyperventilation and associated hypocapnia and alkalemia observed in these dogs, which per se causes Hb-0, affinity to increase. Approximately the same degree of hypoxia (PI,~) was achieved at a PB of 635 mm Hg by administering a 14% 0, mixture as by decompressing the chamber to 435 mm Hg while maintaining room air. Because the alveolar ventilation was significantly greater in the chronically hypoxic dogs, as seen above, they had higher PA~*‘s. Larger alveolar arterial 0, differences in these dogs eliminated this small advantage, thus PaG2 and Sao2 were essentially the same in dogs in acute and in chronic hypoxia. The pulmonary arterial-venous shunting calculated in these condi1.0 0.8 .
0.6. 0.4. ? 0.2.
e8 0.0 VI-0.2% J -0.4 -0.6. -0.6.
-
t
-1.0 .. . ". A. 1.0 1.2 IliP I.6 W 02(mmHg)
*
0’0 . * 20
2.0
’
*
SEA LEVELDOG *
’
’
*
40 60 P trnz.1 4
’
I
’
100
0.8.
0.6.
80
z
0.4-
5
o 60 F
FORT COLLINS ~43smmtig
i :40 a u) 2q 0 0
.
’ 20
*
’ 40
SEA LEVEL DOG *
. 60
’ . GO
IO0
Po2bamH~) Fig. 2. Average
H&O,
dissociation
curves
of 7.4 and 38 C plotted on logarithmic on HbO, coordinate systems (right
in conscious
dogs at a Pa of 635 and 435 mm Hg at a pH
scales (left panels) and values for blood saturation and Po, plotted panels). The average curve for sea level dogs is also shown. The
P,,‘s obtained by interpolation were 30.4 mm Hg at a Pa of 635 mm Hg and 34.3 mm Hg after three weeks at a Pa of 435 mm Hg. The values of K, , K, and K, were 1.48,0.42,0.31 and 1.54,0.41and 0.25 at 635 and 435 mm Hg, respectively.
EFFECTS OF ALTITUDE
313
ON DOGS
60
50 l-
40 I2 Iz
3o
0” G
20 ,
/-
-
3E i!
ICl-
,-
0. PSmmHq
h
CONDltlON Fig. 3. Average
values
r
+
635
435
REST
(+ 1 SEM) for hematocrit rest and during
P 730 8 7 P 5 - 20s s -10 p w I -0 635
435
EXERCISE
and hemoglobin concentration exercise at two levels of PB.
in conscious
dogs at
tions at rest were not different. The average resting PVo,was significantly higher at a PB of 435 mm Hg (FI,, = 0.21) than at 635 mm Hg (FI,~ = 0.14), apparently due to the hematologica1 changes that had taken place after three weeks of hypoxia. There was no change in arterial 0, saturation during exercise in acute hypoxia but there was a fall in Saoz in chronic hypoxia, which was more evident in the second exercise measurement 13 min after the exercise had begun, and which was associated with a 12% pulmonary arterial-venous shunting. A drop in Paol! of 2 mm Hg was measured at a PB of 435 mm Hg. A decrease in Sao2 has been reported in man exercising in hypoxic conditions {Banchero et al., 1966; Grover, 1965). Exercise produced a decrease in PCo, of 5 mm Hg in acute hypoxia, while in chronic hypoxia the drop was 8 mm Hg. .Differences in cardiac output during exercise between these two conditions, in the pH of blood and in Hb-0, affinity accounted partly for these differences. Lactic and pyruvic acids (fig. 4)
Resting lactic acid increased slightly during 14% 0, inhalation at a PB of 635 mm Hg but pyruvic acid did not change. Both lactic and pyruvic acid were significantly higher in resting dogs after 3 weeks of exposure to hypoxia. It is possible that lactic acid was primarily higher due to increases in pyruvic acid. Lactate increased during exercise at ail Ievels of PQ,,, the changes were generally more marked during the second determination, 13 min after the exercise had begun.
314
N. BANCHERO
et al.
2-
I-
OA
0.2-
0.1-
O-
Pe mmtlg
635 635 435
63s 636 435
63S63S
FI
0.21 0.14 0.21
0.21 0.14 0.21
0.21 0.14 0.21
02 CCUDITION Fig. 4. Average
values (k
REST
CARDIACOUTPUT
EXERCISE
I SEM) for lactic and pyruvic acid in conscious and 13th minute
of exercise at different
AND BLOOD OXYGEN
435
TRANSPORT
dogs at rest and during
the 5th
levels of Pa.
(table 3)
It is well established that the ventilatory condition contributes importantly to modifying the parameters of cardiovascular function. This is especially noticeable in the dog (Adrogue et al., 1970; Kontos et al., 1965). Jennings et al. (1973b) found tachycardia and elevated cardiac output associated with increased minute ventilation in conscious dogs. The average cardiac output (0) at rest breathing air at a PB of 635 mm Hg was normal and heart rate (HR) was elevated, so the stroke volume was slightly low. The resting arterio-venous 0, difference was 6.16 ~01%. Cardiac output increased about two-fold during exercise primarily because of increased heart rate with a smaller contribution (35%) of stroke volume; however, individual variability was considerable. The arterio-venous 0, content difference widened to 9.51 ~01% during exercise and the coefficient of 0, utilization increased (table 2). Resting 0 and HR increased moderately during acute hypoxia. The magnitude of these changes was closely related to the fall in arterial 0, content. Therefore, the O2 transport by arterial blood, S,,T (i.e. Q x Ca&, remained the same as in normoxia (table 3). The arterio-venous 0, content difference was maintained at 6.28 ~01% in acute hypoxia by increasing the coefticient of 0, utilization. After three weeks at a PB of 435 mm Hg the values for 0, HR and Caoz were similar to those found at a PB
EFFECTS OF ALTITUDE ON DOGS
315
TABLE 3 Cardiac function values and systemic 0, transport in conscious dogs at rest and during treadmill exercise at different levels of barometric pressure and inspired 0, concentration Exercise
Rest PB
(mm Hg) F’o,
635 0.21
635 0.14
435 0.21
635 0.21
635 0.14
435 0.21
Q (ml/min kg)
157 25
177 IO
155 45
336 69
357 26
325 102
HR (mm-‘)
126 16
157 22
133 28
192 18
204 I9
164 28
SV (ml)
31 8
28 4
30 I4
42 9
44 4
43 I7
S,,T (ml/min . kg)
27.5 5.2
26.4 3.1
30.4 11.1
65.8 16.2
56.7 5.9
58.6 17.5
Average values k I SD.
of 635 mm Hg (FI,, = 0.21). This observation agrees with that of Vogel and Hannon (1966) in dogs exposed to 3,500 m and with our previous observations in dogs native to the Andes (Banchero et al., 1975). Exercise produced the same relative increases in Q in both acute and chronic hypoxia that it did in normoxia. HR was, however, significantly higher in acute hypoxia than in chronic hypoxia (P < 0.025) while stroke volume was not different. The absolute values for 0 were, therefore, higher in acute hypoxia than in chronic hypoxia but the difference was not significant. The S,,T was lower during exercise in hypoxia than in normoxia. TABLE 4 Blood pressures in dogs at rest and during exercise at different levels of barometric pressure and FI,, Rest PB
(mm Hg) FIo,
635 0.21
Exercise 635 0.14
435 0.21
635 0.21
635 0.14
43E 0.21
163k22 91*20 123+22
I71 +_32 98kl7 129+22
BLOOD PRESSURES (mm Hg) Aorta
systolic diastolic mean
144k 18 99+ 9 118k15
135+16 92+12 110+13
151+18 104,12 122+13
170*19 92+16 126k 17
33* 4 4* 3 l6+ 3
39+14 13* 5 21+11
42+ 8 14* 4 21+ 7
56+10 5+ 4 22* 7
Pulmonary artery
systolic diastolic mean
Average values + I SD.
53+15 6k 5 21+ 9
68, 9 8+ 5 27, 8
316 BLOOD PRESSURES
N. BANCHERO et a/.
(table 4)
No differences in resting aortic blood pressures were found in the different experimental conditions. Resting pulmonary artery blood pressure increased moderately during acute and chronic hypoxia because of an increase in pulmonary vascular resistances. Moderate increases in aortic and pulmonary arterial blood pressures were measured during exercise at both levels of barometric pressure, despite decreases in resistances to blood flow.
HISTOLOGICAL MEASUREMENTS (table
5)
The number of capillaries per unit area in skeletal muscle doubled after 3 weeks of exposure to a PB of 435 mm Hg. This increased capillary density appeared to be caused by a significant decrease in the average muscle fiber diameter from 65 to TABLE 5 Morphological data in canine skeletal muscle before and after exposure to simulated altitude for three weeks _~
_
Natives
to 1610 m
(635 mm Hg) Number of dogs
~_ After 3 week exposure to 4880 m (435 mm Hg)
5
5
617 120
1245 168
Capillary density (cap/mm’)
m SD
Capillary diameter (Itm)
m SD
4.5 0.8
4.5 0.7
Fiber diameter (pm)
ni SD
65.4 8.4
45.2 6.8
Relative fiber surface area
tii
84.2
114.8
SD
20.1
21.4
(pmZ!lOOO pm3)
45 pm when the, dogs were exposed to hypoxia. The cross-sectional area of the average muscle fiber decreased to half its value at a PB of 635 mm Hg, while the number of capillaries per muscle fiber remained unchanged. The capillary diameter did not change (4.5 pm) but in the denser capillary bed, the relative surface area for capillaries doubled.
Discussion
Our dogs in normoxia had high respiratory
rates, minute ventilations and 0, con-
EFFECTS OF ALTITUDE
ON DOGS
317
sumptions, with relatively little scatter around the mean. Panting commonly occurs in conscious resting dogs, as found by Jennings et al. (1973a). Panting, associated with hyperventilation, can be suspected in the dogs studied by Vogel and Hannon (1966) and Flandrois et al. (1971) who found, as we did, resting Pacoz’s of about 30 mm Hg and relatively high arterial pH’s. Wide variations in respiratory patterns are known to occur in awake dogs which affects the cardiocirculatory function of these animals in a predictable fashion depending on the respiratory behavior at the time of measurement (Jennings et al., 1973b). Dogs tend to change from normal breathing to panting, with no intermediate patterns (Schmidt-Nielsen, 1964). Ventilation in our animals was fairly stable during each experiment, with occasional day to day variations. This basal hyperventilation in the control study (\j~/\j~ = 0.22) with an average respiratory equivalent of 113 liters of ventilation per liter of O,, tends to complicate the interpretation of the ventilatory findings in acute and chronic hypoxia and during exercise. During acute hypoxia the changes in ventilatory output were characterized by moderate increases in tidal volume and in alveolar ventilation but no changes in the frequency of breathing, which remained high. The arterial pH was markedly alkalotic. After three weeks of continuous exposure to hypoxia the alveolar ventilation was more than twice that measured in normoxia. This increased ventilatory output was due to the combined effects of increased tidal volumes and reduced respiratory rates, resulting in much greater PA/~~E ratios (average 0.57) and considerable hypocapnia. The average respiratory equivalent was no different than the one measured in dogs in normoxia. The respiratory equivalent, however, is not a good index of ventilatory effectiveness since in hypoxia the number of molecules of 0, per liter of air is less, and therefore, more air must be moved to obtain the same amount of 0,. After a three week sojourn at 4,880 m, the dogs hyperventilated more than Andean dogs (Banchero et al., 1975). Treadmill exercise of moderate degree, compared to the controls at rest, was associated with four-fold increases in alveolar ventilation and doubling of the tidal volume but only small changes in the frequency of breathing. These effects were similar at all levels of inspired PO, producing further decreases in PacOl. That exercising dogs have lower Paco2 ‘s than resting dogs had been reported previously by Vogel and Hannon (1966) and by Flandrois et al. (1971). These findings disagree with the data of Whaten et al. (1962) who found relative hypoventilation during treadmill exercise in dogs whose Pacoz ‘s increased over resting values of about 40 mm Hg. The cardiocirculatory data in our dogs when at a Pn of 635 mm Hg, FIN* = 0.21, were similar to those found in normal conscious dogs studied by others (Vogel and Hannon, 1966), at 1,610 m above sea level. Hence, our cardiocirculatory findings at other levels of inspired PO, have been compared to these normoxic values. During 14% 0, inhalation, before significant changes in hemoglobin concentration could occur, cardiac output increased due to a proportional increase in heart rate to maintain systemic 0, transport in the presence of reduced arterial blood 0,
318
N. BANCHERO
et af.
content. After three weeks at a PB of 435 mm Hg moderate increases in hemoglobin concentration (average 18 %) were enough to maintain arterial 0, content at values similar to those measured in dogs at a PB of 635 mm Hg, breathing air. Cardiac output and thus, systemic 0, transport, were similar to those in normoxic dogs. An additional advantage associated with the increase in hemoglobin concentration is that the same differences in 0, content between arterial and mixed venous blood are maintained with a smaller drop in 0, saturation than is necessary with normal hemoglobin concentration. Independently, the ‘S’ shape of the HbO, dissociation curve allows the delivery of the same amount of 0, per milliliter of blood with a much smaller difference in the partial pressure of 0, between the arterial and mixed venous blood, because when the Po, in inspired air decreases the animal operates in the middle, shoulder portion of the Hb0, curve. Oxygen delivery to the tissues was also helped by a small reduction in HbO, affinity. The benefits of this lower Hb-0, affinity in the in oivo situation were partly offset by the alkaline pH of the blood in these dogs when at simulated altitude. These data with the exception of the reduced HbO, affinity and alkaline arterial pH are very similar to those from Andean dogs native to 4,350 m (Banchero et al., 1975) suggesting that the cardiocirculatory adjustments that occur in the dog after exposure to hypoxia are well developed after only a few weeks in that environment. Resting Q and arterio-venous 0, difference in dogs in normoxia were similar to those reported by other investigators in dogs under similar experimental conditions (Barger et al., 1956). However, stroke volume was slightly low due to the mild tachycardia commonly observed in panting dogs and which is related to the increased respiratory rate (Jennings et al., 1973b). We found a mild increase in Q and heart rate in acute hypoxia and no evidence of cardiac depression, i.e. the same Q, HR and SV after 3 weeks at simulated altitude of 435 mm Hg. Arterial blood pressures and systemic peripheral resistances were not affected by hypoxia of three weeks. These data support previous observations in Andean dogs chronically adapted to hypoxia (4,350 m), who also showed no evidence of cardiac depression (Banchero et al., 1975) and in dogs exposed to 3,500 m for 3 weeks (Vogel and Hannon, 1966). Furthermore, we found no cardiac depression during treadmill exercise at moderate work (36 ml of O,/min . kg) in dogs in hypoxia. These data agree with results in dogs in acute hypoxia (Piiper et al., 1966) and in chronic hypoxia (Vogel and Hannon, 1966) and with data in Andean men at 4,540 m (Banchero et al., 1966). They are in conflict with the results of Vogel et al. (1974) and Hartley et al. (1967) in men. No differences in mean aortic pressures were measured in the dogs exposed to chronic hypoxia, suggesting that the changes in capillary density did not appreciably change systemic vascular resistance. On the other hand, mean pulmonary artery pressure was higher in hypoxic conditions reaching values similar to those measured in Andean dogs (Banchero et al., 1975). The variability of mean pulmonary artery pressure was also similar to that of Andean dogs. Traditionally, the modest increases in lactic and pyruvic acids in hypoxic dogs could be explained by a decrease in tissue perfusion and/or the accompanying tissue
EFFECTS OF ALTITUDE ON DOGS
319
hypoxia. Our data on cardiac output and on muscle capillarity seem to contradict this line of thinking unless changes in systemic blood flow distribution are invoked. However, the marked hyperventilation of our dogs in hypoxia, and the tissue and blood cell alkalosis that accompany the resulting hypocapnia, may stimulate the glycolytic pathway by means of an effect on phosphofructokinase, as postulated by Minakami and Yoshikawa (1966). The further increase in the levels of these metabolic products in the blood of these animals during exercise is probably due to an increased production rate, even though the removal rate may be higher as shown by Eldridge (1974). The changes in capillary density and in muscle fiber diameter observed in the sternothyroid muscle of the dogs after 3 weeks in hypoxia seem to be important in the process of adaptation to a low P,, environment. Furthermore, we have found that dogs native to the Andes, at 4,350 m have smaller muscle fiber diameters and denser capillary networks than the dogs studied at 435 mm Hg for three weeks (Eby and Banchero, 1976). It is well established that 0, reaches the cells by the process of diffusion. Fick’s law states that the rate of transfer of a substance is directly proportional to the pressure gradient if the diffusion distance remains constant. If the distance is reduced to half while maintaining the same concentration gradient the rate of transfer of the substance will double. A more dense capillary network and shorter diffusion distances would therefore be, other factors remaining constant, a very efficient mechanism to compensate for decreases in the partial pressure of oxygen in arterial blood (Kety, 1957). If it is assumed that the changes we observed in the sternothyroid muscle, where the average decrease in muscle fiber diameter was from 65 to 45 pm, is indicative of changes in the whole muscle mass, we can estimate the consequences of such a change on the P,, levels of the ‘lethal corner’ of the average muscle fiber. Using Krogh’s formula (Kety, 1957) for a capillary-tissue cylinder and the values for P,, and intercapillary distances obtained in the sternothyroid muscle we have calculated the partial pressure of 0, that would exist in the ‘lethal corner’ of the average skeletal muscle fiber at the venous side of the capillary at the 2 levels of barometric pressure if factors such as metabolic rate of the tissue and the diffusion coefficient were to remain unchanged. The average mixed venous PO2 measured at these 2 levels of Pn were used in these calculations. No corrections were introduced to account for the larger surface area of the capillary bed after three weeks of exposure to hypoxia. At a PB of 635 mm Hg, a condition similar to that found at sea level, the Po, at the ‘lethal corner’ would be 25 mm Hg at rest, while at a Pn of 435 mm Hg, despite the drop in arterial P,, the tissue P,, would be 30 mm Hg because of the decrease in muscle fiber cross-sectional area and denser capillary bed. If no changes in intercapillary distance and muscle fiber diameter were to develop after exposure to hypoxia calculations indicate that the P,, in muscle, at a Pn of 435 mm Hg, would be only 20 mm Hg. More importantly, during exercise, when the 0, consumption of muscle increases, the P,, at the ‘lethal corner’ was always much lower at a Pn of 635 mm Hg than after three weeks at 435 mm Hg, if the parameters involved in these
320
N. BANCHERO
et al.
calculations, other than muscle fiber size and intercapillary same for both levels of barometric pressure.
distances, remain the
Acknowledgements During this study, Dr. M. Gimenez was on leave from the Unite 14, INSERM, at Nancy, France. Dr. A. Rostami was on leave from the University of Isfahan, Iran, and was supported by the World Health Organization. The authors wish to acknowledge the hypobaric facilities provided by the Department of Physiology and Biophysics, Colorado State University in Fort Collins, and especially the cooperation of Mr. John Fitch.
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EFFECTS
Physiology
(1976) 27, 305-321;
OF SIMULATED
NATAL10
Department
have been studied
exercise at
exposure
marked
635 and
animals,
associated
outputs.
in P,,
[Hb]
transport
(617 to 1245 cap/mm’) about
30”,. Calculations
in the hypoxic
IN DOGS’
AHMAD ROSTAMI
qf Medicine,
Dmrer,
Cola. 80220.
moderate
animal
remained
chamber.
hyperventilation
significantly
At both levels of PB the dogs
and low Pa,,>‘s
(28 mm Hg at
ml:min kg) reflecting
O2 content.
Capillary
after 3 weeks at a Pa of 435, whereas indicate because
density
and hypoxic
in the hypoxic 0, content
in the sternothyroid
the average
dogs
difference
muscle doubled
muscle fiber diameter
decreased
of the muscle fiber is relatively
in tiber size and intercapillary
Altitude
higher
distances.
Hypoxia
Capillary Cardiac
P,, ‘s decreased arterial-venous
that the Paz in the ‘lethal corner’
of the decrease
the
after 3 weeks at 435 mm Hg with a 4 mm
and mixed venous
but arterial
unchanged.
treadmill
in Denver at a Pa of 635 mm Hg and after 3 weeks
\io>’ s were high (12.4 and 11.4
exercise. Arterial
U.S.A
muscle and on the cardiocirculatory
Q, HR. SV and systemic blood pressures were normal in both normoxic
(82 to 56 and 42 to 36 mm Hg. respectively) 0,
Amsterdam
TRANSPORT
on skeletal
with pulmonary
and Hct increased
at rest as well as during
and systemic
Company,
dogs (16-39 kg) at rest and during
were obtained
I7 mm Hg at 435 mm Hg). Resting
Hg decrease
School
to a Pa of 435 mm Hg using a hypobaric
panting,
high respiratory
ON 0,
altitude
conscious,
IS”; incline. Measurements
of continuous showed
qf Colorado
to simulated
on awake,
Publishing
MANUEL GIMENEZ, and SHEILA H. EBY
University
Abstract. The effects of exposure system
ALTITUDE
BANCHERO,
qf Physiology,
Norrh-Holland
Muscle
density output
0,
Hemoglobin
fiber size
transport
Ventilation
Acclimatization to chronic hypoxia involves several cardiopulmonary adjustments which contribute to supply 0, to the body tissues. Resting 0, consumption in hypoxia is the same as in normoxia but the partial pressure of 0, in the cells is assumed to be somewhat lower. It is during exercise in hypoxia that the maximal 0, consumption, ix, the maximal aerobic power is reduced in relation to normoxic standards (Balke et al., 1965: Buskirk et al., 1967; Klausen et al., 1966; Piiper et al., 1966). Buskirk (1969) suggested that the factors limiting aerobic capacity reside in skeletal muscle, where the tissular 0, tension may be temporarily reduced to levels incompatible with optimal muscle function. There is evidence obtained primarily in rodents, that tissue changes, specifically Accepted
for publication
’ Supported
25 April
by U.S. Public
1976.
Health
Service Grants
# HL-14317 305
& HL-12679
N. BANCHERO
306
et d.
increased capillary density, are involved in the process of adaptation to hypoxia (Cassin et al., 1971; Valdivia, 1958). Also, increased capillary density associated with smaller muscle fiber diameters has been found in skeletal muscle of dogs in hypoxia (Banchero, 1975; Eby and Banchero, 1976). We have studied the effects of changes in skeletal muscle and in the cardiocirculatory system on 0, transport in dogs in hypoxia at rest and during moderate treadmill exercise. Previous reports on the circulatory effects of chronic hypoxia in dogs indicate either a normal cardiac response over a 26 day sojourn (Vogel and Hannon, 1966) or a depression represented by lower cardiac output, heart rate and stroke volume during a 21 day exposure (Hansen et al., 1962).
Materials
and methods
Mongrel dogs weighing from 16 to 39 kg were studied awake and unsedated at two different levels of barometric pressure (PB): 635 mm Hg, in Denver, at 1,610 m and after 3 weeks of continuous exposure to 435 mm Hg in a hypobaric chamber (Colorado State University, Ft. Collins, Colo.). At both levels of Pa the animals were studied at rest as well as during treadmill exercise on a 15 y0 incline. Preparation of the animals
Healthy dogs, vaccinated for distemper, rabies and hepatitis, and quarantined for three weeks, were selected for their ability to run on a treadmill. The dogs were maintained in individual cages at a temperature of 22 + 2 “C. A tracheostomy was performed (Thilenius and Borquez-Vial, 1963) under general anesthesia (sodium pentobarbital 30 mg/kg i.v.) using sterile surgical technics. One of the two sternothyroid muscles was removed at this time for histological studies. 600,000 IU of penicillin were administered upon completion of this procedure. A minimum of one week was allowed for postsurgical recovery, before the animals were exercised on a treadmill, 3 times a week, at a 15 y0 incline, at increasingly higher speeds, up to 10 mph. The dogs were run for 3 minutes at each speed or until refusal occurred. We had previously found that dogs reach a ventilatory steady state after 3 minutes of exercise as judged from the measurements of respiratory function. Half of the maximal speed reached in the training sessions was then chosen and the animals were trained to run at that speed for about 20 minutes. Two to three days before the experiment, under general anesthesia, a catheter (PE 190) was introduced percutaneously into the right jugular vein and advanced into the pulmonary artery. A second catheter (PE 160) was inserted in the left carotid artery and advanced to the thoracic aorta. The external portions of these catheters were sutured to the skin using a piece of rubber tubing to prevent kinking. These catheters were used for blood pressure recording and blood sampling. After completion of the 3 week sojourn at simulated altitude the dogs were anesthetized and the contralateral sternothyroid muscle was removed.
EFFECTS OF ALTITUDE ON DOGS
307
Protocol
The dogs were studied following the sequence shown in fig. 1. The exercise extended over 17 min with the dogs running at an incline of 15 “/, at half the maximal speed reached during training. At a PB of 635 mm Hg seven dogs were studied twice,
i
iBIb
6 i EXERCISE
TIME (mini
Fig. 1. Temporal sequence of measurements and blood sample collections in conscious dogs studied standing at rest and during treadmill exercise at an incline of 15T,,.The collection of expired air and the measurement of cardiac output during exercise began after steady state was achieved as tested in preliminary experiments.
breathing room air and a mixture of 14% 0, in N, . At 14% 0, the levels of PI*, were similar to those in the hypobaric chamber while breathing room air at a PEZ of 435 mm Hg. At a PB of 435 mm Hg eight dogs were studied in exactly the same fashion, only while breathing room air. In addition, dogs breathed 99 74 0, at both levels of PB during which time only blood samples were collected. Animals were brought to the laboratory at least one hour before the experiment and preparations were made with the animal standing quietly on the treadmill. A fan was placed in front of the animal to improve air circulation. The dogs were studied only once a day. The experiments were conducted at room temperature, close to the thermoneutral point for dogs (2325C) (Hammel et al., 1958). In Denver the temperature varied from 20-25 “C and from 20.8-24.5 “C in the hypobaric chamber.
308
N. BANCHERO
et al.
Measurements Blood pressures were measured in the pulmonary artery and the aorta using Statham pressure transducers (Model P23Db). The zero reference level was set at the level of the shoulder joint with the animal standing at rest. Heart rate was calculated from the pressure recordings. Blood samples obtained, anaerobically and simultaneously in heparinized glass syringes, were analyzed for Po, , Pco, and pH using Radiometer electrodes and corrected to the dog’s rectal temperature at the time of sampling. Lactic and pyruvic acid, hemoglobin and hematocrit were also measured in these samples. Lactic and pyruvic acids were analyzed by the enzymatic method of Marbach and Weil (1967). Resting blood oxygen saturations were determined in an American Optical oximeter. After indocyanine green had been injected into the animal blood oxygen saturations (exercise values) were calculated with a Severinghaus rule (Severinghaus, 1966). No statistically significant differences were found, in animals in whom no indocyanine green was injected, between the values for 0, saturation measured in the oximeter and those calculated with the rule. In vivo hemoglobin-oxygen dissociation curves were constructed using the resting 0, saturations (oximetry) and the partial pressure of 0, , corrected to 38 C, measured in arterial and mixed venous blood samples obtained from dogs while at different levels of inspired 0, concentration, and using an equation reported by Hellegers et al. (1959): log Po, = K, -K, (7.4-pH)+K, log S/(100-S), where K,, K, and K, are constants, and S = saturation; K, is the logarithm of P,,, K, is the Bohr constant and K, = l/n (n is Hill’s constant). The mean Hb-0, curves for all dogs, at the two different levels of barometric pressure were then plotted on double logarithmic scales, at 38 “C and pH of 7.4. An endotracheal tube with an inflatable cuff (dead space 30 ml) connected to a two way Hans-Rudolph plastic valve (Warren E. Collins, Inc.) (dead space 18 ml) was used for collection of expired air. The volume of expired air (fin*& and the respiratory frequency (f) were continuously recorded using a Parkinson-Cowan gas meter fitted with an electrical potentiometer. Samples of expired air were collected at set intervals (fig. 1) in rubber bags for analysis of 0, and CO, content using a Beckman paramagnetic 0, meter and a Beckman LB-l CO, meter, respectively. These instruments were calibrated with known gas mixtures before and after each measurement. The concentrations of 0, and CO, in the calibrating mixtures were analyzed by the Scholander method. Body and spirometer temperatures were measured continuously using thermistors (Yellow Springs Instruments, Model 130 STD). Alveolar ventilation was calculated using the equation: VA = K vc,J Paco2 (Rahn and Fenn, 1955). Cardiac output (Q) was measured at rest and during exercise (fig. 1) by the dye dilution technic (Cardiogreen@) using a Waters (Model XC-250) densitometer. Injections were made in the pulmonary artery while sampling from the aorta with a Harvard infusion withdrawal pump. Blood was immediately reinfused into the animal after each curve. Four to live dye curves were obtained in rapid succession
EFFECTS OF ALTITUDE ON DOGS
309
in each condition and an average value calculated. The densitometer was calibrated at the end of each experiment using two to three known blood-dye mixtures. Histological measurements Immediately after removal the muscle was fixed in buffered formalin, processed and cut transversely in 7 pm sections and stained with the PAS reaction. An average of 75 sections were stained from each muscle sample. Diameters of capillaries and fibers were measured in 30 fields, selected at random, using a calibrated eyepiece micrometer (Weibel et al., 1966). The number of capillaries per mm2 was counted in the same fields. A detailed description of these measurements has been given (Banchero, 1975).
Red ts
The average body weights of these dogs were 24.9 and 25.1 kg at PB'S of 635 and 435 mm Hg, respectively. The maximal weight loss was 3.0 kg while the maximal gain was 2.5 kg after a 3 week exposure to 435 mm Hg. Because the respiratory parameters and blood gas values obtained during the 5th and 13th minutes of exercise were not statistically different, they have been averaged, and therefore, only one set of values is reported for the exercise condition, with the exception of data for lactic and pyruvic acids.
VENTILATORY MEASUREMENTS
(tables 1 & 2)
Regardless of the level of partial pressure of 0, in inspired air our dogs at rest had high respiratory rates and minute ventilations with relatively little variation around the mean. This panting was not related to changes in room and/or body temperature. Jennings et al. (1973a) found similar degrees of panting in conscious dogs well accustomed to the laboratory, whose hypothalamic temperature did not change consistently. Resting 0, consumptions and CO, productions in dogs at a Pn of 635 mm Hg breathing air were generally high and reflected the panting and associated pulmonary hyperventilation. The resting Paco2’ s were accordingly low. This respiratory condition was similar to that of awake dogs studied by other investigators (Flandrois et af., 1971; Jennings et al., 1973a; Vogel and Hannon, 1966). On the average, Vo, rose during treadmill exercise about 3.5-fold as a result of more effective ventilation with significant increases in VT and a 4.4-fold increase in VA. No systematic changes in f were observed. During inhalation of 14% 0, at a Pn of 635 mm Hg there was a moderate increase in the resting VE, \j~ and VT, but no significant changes in Vjoa. This additional hyperventilation was associated with a decrease in PaLco2and marked respiratory
310
N. BANCHERO
t?t ai.
TABLE Average
ventilatory
values
+ 1 SD in conscious
levels of barometric
1
dogs at rest and during
pressure
and inspired
0,
treadmill
Rest PB (mm Hg)
635
7jE(L/Blin BTPS)
ir, (Ljmin
635
STPD)
(ml/min kg
oco2 (ml/min
f (min-
STPD)
kg STPD)
‘)
VT(ml)
Seven dogs were studied
435
0.14
635
435
0.14
0.21
0.21
41.2
30.8
64.0
65.5
66.9
5.6
4.4
9.6
6.6
7.2
15.6
7.6
10.2
16.4
33.7
43.5
52.9
1.2
1.8
7.8
7.7
13.”
13.2
307
287
276
1069
918
870
80
37
103
155
90
223
12.4
11.5
11.4
43.8
36.8
36.0
2.5
2.0
3.9
7.2
5.1
8.5
10.0
10.3
10.8
37.7
33.0
34.6
0.9
1.4
3.8
6.0
4.1
9.2
168
157
101
156
146
119
24
25
47
27
20
32
209
267
337
399
455
578
43
45
125
46
52
190
113
143
112
59
71
77
at 635 mm Hg and 8 were studied TABLE
Blood gas values in conscious
635
0.21
34.8
BTPS)
V,, (ml/min
at different
Exercise
0.21
F’o,
exercise
concentration
dogs at rest and during
at 435 mm Hg.
2 exercise at different
levels of barometric
pressure
and FI,* Rest
PB(mm Hd Fro,
0.21
PI,, (mm W pro> (mm Hg)
Pa,,
635
(mm Hg)
Sao, (%)
Cao, (~01%)
121
Exercise 635 0.14
435 0.21
635 0.21
635 0.14
435 0.21
80.7
78.4
80.3
77.8
0.7
0.8
0.4
0.8
0.7
0.4
86.6
56.6
61.9
92.0
59.3
62.9
7.3
6.5
4.9
6.2
7.5
2.4
82.0
53.4
55.5
85.9
53.6
53.6
3.8
8.6
5.3
5.5
6.2
6.5
94.5
82.6
83.5
94.2
81.6
79.9
0.8
4.3
3.7
0.9
3.4
5.5
18.3
14.9
18.8
19.2
15.7
18.0
2.7
1.7
2.7
2.3
2.0
2.8
121
EFFECTS OF ALTITUDE
Pace,(mm W
@a fro,(mm W svo,(%I Go2 W%) pHV
(a -J)O,
(vol”YO)
Coefficient
0,
Utilization ~.,_
(%)
Average
values
ON DOGS
311
28.4
22.2
16.6
24.6
17.9
14.9
3.5
3.8
4.9
4.6
5.5
3.0
7.415
7.468
7.453
7.419
7.479
7.436
0.018
0.027
0.016
0.022
0.024
0.028
41.5
30.5
36.3
33.8
26.6
28.3
3.6
5.4
3.5
2.9
5.2
5.0
64.6
47.5
57.6
47.3
35.4
39.9
5.1
7.4
5.5
6.3
9.2
9.7
12.1
8.7
13.0
9.6
6.8
9.2
2.1
1.7
2.7
1.8
1.7
2.7
7.377
7.442
7.426
7.353
7.413
7.392
0.024
0.021
0.017
0.026
0.048
0.025
6.16
6.28
5.84
9.51
8.90
9.02
1.08
1.23
0.99
I .48
1.71
1.77
33.7
42.2
31.5
49.2
57.1
52.5
4.1
8.4
6.3
6.4
10.7
11.3
f 1 SD.
alkalemia. During exercise the changes in ventilation were quantitatively similar to those in dogs breathing room air. \jo2 increased 3.2-fold. After three weeks of exposure to a PB of 435 mm Hg the resting %‘E was slightly lower than in normoxia but the VA was significantly higher than at 635 mm Hg, causing the Paco2 to drop to 16.6 mm Hg. The respiratory frequency was significantly lower (P < 0.005) and the VT was higher (P < 0.01) than in normoxia. VT increased during exercise, increasing \j~ and VA, but the vo, was less than in normoxia (P < 0.05). The frequency of breathing was also lower than at a PB of 635 mm Hg. BLOOD MEASUREMENTS
An average decrease of 4 mm Hg was observed in Hb-0, affinity as a result of exposure to simulated altitude for 3 weeks (fig. 2). This observation is in agreement with earlier findings in severely anemic dogs (Cropp and Gee, 1972). Hemoglobin and hematocrit (fig. 3) No systematic differences were found between the resting hemoglobin concentration or the resting hematocrit obtained under the two levels of Floz at a PB of 635 mm Hg. Hemoglobin and hematocrit increased significantly (P < 0.05) after three weeks of exposure to a PB of 435 mm Hg with no systematic changes in mean corpuscular hemoglobin concentration. The values for [Hb] and Hct at a PB of 435 mm Hg were almost identical to those found in dogs native to 4,350 m (Banchero et al., 1975). During exercise mild increases in hemoglobin and hematocrit were observed at a PB of 635 mm Hg. They were absent at a PB of 435 mm Hg.
312
et al.
N. BANCHERO
Blood gases (table 2)
In Denver, at a Pn of 635 mm Hg, the values for P,, and So, in arterial blood were similar to those found in dogs at sea level, both at rest as well as during exercise. This was probably due to the pulmonary hyperventilation and associated hypocapnia and alkalemia observed in these dogs, which per se causes Hb-0, affinity to increase. Approximately the same degree of hypoxia (PI,~) was achieved at a PB of 635 mm Hg by administering a 14% 0, mixture as by decompressing the chamber to 435 mm Hg while maintaining room air. Because the alveolar ventilation was significantly greater in the chronically hypoxic dogs, as seen above, they had higher PA~*‘s. Larger alveolar arterial 0, differences in these dogs eliminated this small advantage, thus PaG2 and Sao2 were essentially the same in dogs in acute and in chronic hypoxia. The pulmonary arterial-venous shunting calculated in these condi1.0 0.8 .
0.6. 0.4. ? 0.2.
e8 0.0 VI-0.2% J -0.4 -0.6. -0.6.
-
t
-1.0 .. . ". A. 1.0 1.2 IliP I.6 W 02(mmHg)
*
0’0 . * 20
2.0
’
*
SEA LEVELDOG *
’
’
*
40 60 P trnz.1 4
’
I
’
100
0.8.
0.6.
80
z
0.4-
5
o 60 F
FORT COLLINS ~43smmtig
i :40 a u) 2q 0 0
.
’ 20
*
’ 40
SEA LEVEL DOG *
. 60
’ . GO
IO0
Po2bamH~) Fig. 2. Average
H&O,
dissociation
curves
of 7.4 and 38 C plotted on logarithmic on HbO, coordinate systems (right
in conscious
dogs at a Pa of 635 and 435 mm Hg at a pH
scales (left panels) and values for blood saturation and Po, plotted panels). The average curve for sea level dogs is also shown. The
P,,‘s obtained by interpolation were 30.4 mm Hg at a Pa of 635 mm Hg and 34.3 mm Hg after three weeks at a Pa of 435 mm Hg. The values of K, , K, and K, were 1.48,0.42,0.31 and 1.54,0.41and 0.25 at 635 and 435 mm Hg, respectively.
EFFECTS OF ALTITUDE
313
ON DOGS
60
50 l-
40 I2 Iz
3o
0” G
20 ,
/-
-
3E i!
ICl-
,-
0. PSmmHq
h
CONDltlON Fig. 3. Average
values
r
+
635
435
REST
(+ 1 SEM) for hematocrit rest and during
P 730 8 7 P 5 - 20s s -10 p w I -0 635
435
EXERCISE
and hemoglobin concentration exercise at two levels of PB.
in conscious
dogs at
tions at rest were not different. The average resting PVo,was significantly higher at a PB of 435 mm Hg (FI,, = 0.21) than at 635 mm Hg (FI,~ = 0.14), apparently due to the hematologica1 changes that had taken place after three weeks of hypoxia. There was no change in arterial 0, saturation during exercise in acute hypoxia but there was a fall in Saoz in chronic hypoxia, which was more evident in the second exercise measurement 13 min after the exercise had begun, and which was associated with a 12% pulmonary arterial-venous shunting. A drop in Paol! of 2 mm Hg was measured at a PB of 435 mm Hg. A decrease in Sao2 has been reported in man exercising in hypoxic conditions {Banchero et al., 1966; Grover, 1965). Exercise produced a decrease in PCo, of 5 mm Hg in acute hypoxia, while in chronic hypoxia the drop was 8 mm Hg. .Differences in cardiac output during exercise between these two conditions, in the pH of blood and in Hb-0, affinity accounted partly for these differences. Lactic and pyruvic acids (fig. 4)
Resting lactic acid increased slightly during 14% 0, inhalation at a PB of 635 mm Hg but pyruvic acid did not change. Both lactic and pyruvic acid were significantly higher in resting dogs after 3 weeks of exposure to hypoxia. It is possible that lactic acid was primarily higher due to increases in pyruvic acid. Lactate increased during exercise at ail Ievels of PQ,,, the changes were generally more marked during the second determination, 13 min after the exercise had begun.
314
N. BANCHERO
et al.
2-
I-
OA
0.2-
0.1-
O-
Pe mmtlg
635 635 435
63s 636 435
63S63S
FI
0.21 0.14 0.21
0.21 0.14 0.21
0.21 0.14 0.21
02 CCUDITION Fig. 4. Average
values (k
REST
CARDIACOUTPUT
EXERCISE
I SEM) for lactic and pyruvic acid in conscious and 13th minute
of exercise at different
AND BLOOD OXYGEN
435
TRANSPORT
dogs at rest and during
the 5th
levels of Pa.
(table 3)
It is well established that the ventilatory condition contributes importantly to modifying the parameters of cardiovascular function. This is especially noticeable in the dog (Adrogue et al., 1970; Kontos et al., 1965). Jennings et al. (1973b) found tachycardia and elevated cardiac output associated with increased minute ventilation in conscious dogs. The average cardiac output (0) at rest breathing air at a PB of 635 mm Hg was normal and heart rate (HR) was elevated, so the stroke volume was slightly low. The resting arterio-venous 0, difference was 6.16 ~01%. Cardiac output increased about two-fold during exercise primarily because of increased heart rate with a smaller contribution (35%) of stroke volume; however, individual variability was considerable. The arterio-venous 0, content difference widened to 9.51 ~01% during exercise and the coefficient of 0, utilization increased (table 2). Resting 0 and HR increased moderately during acute hypoxia. The magnitude of these changes was closely related to the fall in arterial 0, content. Therefore, the O2 transport by arterial blood, S,,T (i.e. Q x Ca&, remained the same as in normoxia (table 3). The arterio-venous 0, content difference was maintained at 6.28 ~01% in acute hypoxia by increasing the coefticient of 0, utilization. After three weeks at a PB of 435 mm Hg the values for 0, HR and Caoz were similar to those found at a PB
EFFECTS OF ALTITUDE ON DOGS
315
TABLE 3 Cardiac function values and systemic 0, transport in conscious dogs at rest and during treadmill exercise at different levels of barometric pressure and inspired 0, concentration Exercise
Rest PB
(mm Hg) F’o,
635 0.21
635 0.14
435 0.21
635 0.21
635 0.14
435 0.21
Q (ml/min kg)
157 25
177 IO
155 45
336 69
357 26
325 102
HR (mm-‘)
126 16
157 22
133 28
192 18
204 I9
164 28
SV (ml)
31 8
28 4
30 I4
42 9
44 4
43 I7
S,,T (ml/min . kg)
27.5 5.2
26.4 3.1
30.4 11.1
65.8 16.2
56.7 5.9
58.6 17.5
Average values k I SD.
of 635 mm Hg (FI,, = 0.21). This observation agrees with that of Vogel and Hannon (1966) in dogs exposed to 3,500 m and with our previous observations in dogs native to the Andes (Banchero et al., 1975). Exercise produced the same relative increases in Q in both acute and chronic hypoxia that it did in normoxia. HR was, however, significantly higher in acute hypoxia than in chronic hypoxia (P < 0.025) while stroke volume was not different. The absolute values for 0 were, therefore, higher in acute hypoxia than in chronic hypoxia but the difference was not significant. The S,,T was lower during exercise in hypoxia than in normoxia. TABLE 4 Blood pressures in dogs at rest and during exercise at different levels of barometric pressure and FI,, Rest PB
(mm Hg) FIo,
635 0.21
Exercise 635 0.14
435 0.21
635 0.21
635 0.14
43E 0.21
163k22 91*20 123+22
I71 +_32 98kl7 129+22
BLOOD PRESSURES (mm Hg) Aorta
systolic diastolic mean
144k 18 99+ 9 118k15
135+16 92+12 110+13
151+18 104,12 122+13
170*19 92+16 126k 17
33* 4 4* 3 l6+ 3
39+14 13* 5 21+11
42+ 8 14* 4 21+ 7
56+10 5+ 4 22* 7
Pulmonary artery
systolic diastolic mean
Average values + I SD.
53+15 6k 5 21+ 9
68, 9 8+ 5 27, 8
316 BLOOD PRESSURES
N. BANCHERO et a/.
(table 4)
No differences in resting aortic blood pressures were found in the different experimental conditions. Resting pulmonary artery blood pressure increased moderately during acute and chronic hypoxia because of an increase in pulmonary vascular resistances. Moderate increases in aortic and pulmonary arterial blood pressures were measured during exercise at both levels of barometric pressure, despite decreases in resistances to blood flow.
HISTOLOGICAL MEASUREMENTS (table
5)
The number of capillaries per unit area in skeletal muscle doubled after 3 weeks of exposure to a PB of 435 mm Hg. This increased capillary density appeared to be caused by a significant decrease in the average muscle fiber diameter from 65 to TABLE 5 Morphological data in canine skeletal muscle before and after exposure to simulated altitude for three weeks _~
_
Natives
to 1610 m
(635 mm Hg) Number of dogs
~_ After 3 week exposure to 4880 m (435 mm Hg)
5
5
617 120
1245 168
Capillary density (cap/mm’)
m SD
Capillary diameter (Itm)
m SD
4.5 0.8
4.5 0.7
Fiber diameter (pm)
ni SD
65.4 8.4
45.2 6.8
Relative fiber surface area
tii
84.2
114.8
SD
20.1
21.4
(pmZ!lOOO pm3)
45 pm when the, dogs were exposed to hypoxia. The cross-sectional area of the average muscle fiber decreased to half its value at a PB of 635 mm Hg, while the number of capillaries per muscle fiber remained unchanged. The capillary diameter did not change (4.5 pm) but in the denser capillary bed, the relative surface area for capillaries doubled.
Discussion
Our dogs in normoxia had high respiratory
rates, minute ventilations and 0, con-
EFFECTS OF ALTITUDE
ON DOGS
317
sumptions, with relatively little scatter around the mean. Panting commonly occurs in conscious resting dogs, as found by Jennings et al. (1973a). Panting, associated with hyperventilation, can be suspected in the dogs studied by Vogel and Hannon (1966) and Flandrois et al. (1971) who found, as we did, resting Pacoz’s of about 30 mm Hg and relatively high arterial pH’s. Wide variations in respiratory patterns are known to occur in awake dogs which affects the cardiocirculatory function of these animals in a predictable fashion depending on the respiratory behavior at the time of measurement (Jennings et al., 1973b). Dogs tend to change from normal breathing to panting, with no intermediate patterns (Schmidt-Nielsen, 1964). Ventilation in our animals was fairly stable during each experiment, with occasional day to day variations. This basal hyperventilation in the control study (\j~/\j~ = 0.22) with an average respiratory equivalent of 113 liters of ventilation per liter of O,, tends to complicate the interpretation of the ventilatory findings in acute and chronic hypoxia and during exercise. During acute hypoxia the changes in ventilatory output were characterized by moderate increases in tidal volume and in alveolar ventilation but no changes in the frequency of breathing, which remained high. The arterial pH was markedly alkalotic. After three weeks of continuous exposure to hypoxia the alveolar ventilation was more than twice that measured in normoxia. This increased ventilatory output was due to the combined effects of increased tidal volumes and reduced respiratory rates, resulting in much greater PA/~~E ratios (average 0.57) and considerable hypocapnia. The average respiratory equivalent was no different than the one measured in dogs in normoxia. The respiratory equivalent, however, is not a good index of ventilatory effectiveness since in hypoxia the number of molecules of 0, per liter of air is less, and therefore, more air must be moved to obtain the same amount of 0,. After a three week sojourn at 4,880 m, the dogs hyperventilated more than Andean dogs (Banchero et al., 1975). Treadmill exercise of moderate degree, compared to the controls at rest, was associated with four-fold increases in alveolar ventilation and doubling of the tidal volume but only small changes in the frequency of breathing. These effects were similar at all levels of inspired PO, producing further decreases in PacOl. That exercising dogs have lower Paco2 ‘s than resting dogs had been reported previously by Vogel and Hannon (1966) and by Flandrois et al. (1971). These findings disagree with the data of Whaten et al. (1962) who found relative hypoventilation during treadmill exercise in dogs whose Pacoz ‘s increased over resting values of about 40 mm Hg. The cardiocirculatory data in our dogs when at a Pn of 635 mm Hg, FIN* = 0.21, were similar to those found in normal conscious dogs studied by others (Vogel and Hannon, 1966), at 1,610 m above sea level. Hence, our cardiocirculatory findings at other levels of inspired PO, have been compared to these normoxic values. During 14% 0, inhalation, before significant changes in hemoglobin concentration could occur, cardiac output increased due to a proportional increase in heart rate to maintain systemic 0, transport in the presence of reduced arterial blood 0,
318
N. BANCHERO
et af.
content. After three weeks at a PB of 435 mm Hg moderate increases in hemoglobin concentration (average 18 %) were enough to maintain arterial 0, content at values similar to those measured in dogs at a PB of 635 mm Hg, breathing air. Cardiac output and thus, systemic 0, transport, were similar to those in normoxic dogs. An additional advantage associated with the increase in hemoglobin concentration is that the same differences in 0, content between arterial and mixed venous blood are maintained with a smaller drop in 0, saturation than is necessary with normal hemoglobin concentration. Independently, the ‘S’ shape of the HbO, dissociation curve allows the delivery of the same amount of 0, per milliliter of blood with a much smaller difference in the partial pressure of 0, between the arterial and mixed venous blood, because when the Po, in inspired air decreases the animal operates in the middle, shoulder portion of the Hb0, curve. Oxygen delivery to the tissues was also helped by a small reduction in HbO, affinity. The benefits of this lower Hb-0, affinity in the in oivo situation were partly offset by the alkaline pH of the blood in these dogs when at simulated altitude. These data with the exception of the reduced HbO, affinity and alkaline arterial pH are very similar to those from Andean dogs native to 4,350 m (Banchero et al., 1975) suggesting that the cardiocirculatory adjustments that occur in the dog after exposure to hypoxia are well developed after only a few weeks in that environment. Resting Q and arterio-venous 0, difference in dogs in normoxia were similar to those reported by other investigators in dogs under similar experimental conditions (Barger et al., 1956). However, stroke volume was slightly low due to the mild tachycardia commonly observed in panting dogs and which is related to the increased respiratory rate (Jennings et al., 1973b). We found a mild increase in Q and heart rate in acute hypoxia and no evidence of cardiac depression, i.e. the same Q, HR and SV after 3 weeks at simulated altitude of 435 mm Hg. Arterial blood pressures and systemic peripheral resistances were not affected by hypoxia of three weeks. These data support previous observations in Andean dogs chronically adapted to hypoxia (4,350 m), who also showed no evidence of cardiac depression (Banchero et al., 1975) and in dogs exposed to 3,500 m for 3 weeks (Vogel and Hannon, 1966). Furthermore, we found no cardiac depression during treadmill exercise at moderate work (36 ml of O,/min . kg) in dogs in hypoxia. These data agree with results in dogs in acute hypoxia (Piiper et al., 1966) and in chronic hypoxia (Vogel and Hannon, 1966) and with data in Andean men at 4,540 m (Banchero et al., 1966). They are in conflict with the results of Vogel et al. (1974) and Hartley et al. (1967) in men. No differences in mean aortic pressures were measured in the dogs exposed to chronic hypoxia, suggesting that the changes in capillary density did not appreciably change systemic vascular resistance. On the other hand, mean pulmonary artery pressure was higher in hypoxic conditions reaching values similar to those measured in Andean dogs (Banchero et al., 1975). The variability of mean pulmonary artery pressure was also similar to that of Andean dogs. Traditionally, the modest increases in lactic and pyruvic acids in hypoxic dogs could be explained by a decrease in tissue perfusion and/or the accompanying tissue
EFFECTS OF ALTITUDE ON DOGS
319
hypoxia. Our data on cardiac output and on muscle capillarity seem to contradict this line of thinking unless changes in systemic blood flow distribution are invoked. However, the marked hyperventilation of our dogs in hypoxia, and the tissue and blood cell alkalosis that accompany the resulting hypocapnia, may stimulate the glycolytic pathway by means of an effect on phosphofructokinase, as postulated by Minakami and Yoshikawa (1966). The further increase in the levels of these metabolic products in the blood of these animals during exercise is probably due to an increased production rate, even though the removal rate may be higher as shown by Eldridge (1974). The changes in capillary density and in muscle fiber diameter observed in the sternothyroid muscle of the dogs after 3 weeks in hypoxia seem to be important in the process of adaptation to a low P,, environment. Furthermore, we have found that dogs native to the Andes, at 4,350 m have smaller muscle fiber diameters and denser capillary networks than the dogs studied at 435 mm Hg for three weeks (Eby and Banchero, 1976). It is well established that 0, reaches the cells by the process of diffusion. Fick’s law states that the rate of transfer of a substance is directly proportional to the pressure gradient if the diffusion distance remains constant. If the distance is reduced to half while maintaining the same concentration gradient the rate of transfer of the substance will double. A more dense capillary network and shorter diffusion distances would therefore be, other factors remaining constant, a very efficient mechanism to compensate for decreases in the partial pressure of oxygen in arterial blood (Kety, 1957). If it is assumed that the changes we observed in the sternothyroid muscle, where the average decrease in muscle fiber diameter was from 65 to 45 pm, is indicative of changes in the whole muscle mass, we can estimate the consequences of such a change on the P,, levels of the ‘lethal corner’ of the average muscle fiber. Using Krogh’s formula (Kety, 1957) for a capillary-tissue cylinder and the values for P,, and intercapillary distances obtained in the sternothyroid muscle we have calculated the partial pressure of 0, that would exist in the ‘lethal corner’ of the average skeletal muscle fiber at the venous side of the capillary at the 2 levels of barometric pressure if factors such as metabolic rate of the tissue and the diffusion coefficient were to remain unchanged. The average mixed venous PO2 measured at these 2 levels of Pn were used in these calculations. No corrections were introduced to account for the larger surface area of the capillary bed after three weeks of exposure to hypoxia. At a PB of 635 mm Hg, a condition similar to that found at sea level, the Po, at the ‘lethal corner’ would be 25 mm Hg at rest, while at a Pn of 435 mm Hg, despite the drop in arterial P,, the tissue P,, would be 30 mm Hg because of the decrease in muscle fiber cross-sectional area and denser capillary bed. If no changes in intercapillary distance and muscle fiber diameter were to develop after exposure to hypoxia calculations indicate that the P,, in muscle, at a Pn of 435 mm Hg, would be only 20 mm Hg. More importantly, during exercise, when the 0, consumption of muscle increases, the P,, at the ‘lethal corner’ was always much lower at a Pn of 635 mm Hg than after three weeks at 435 mm Hg, if the parameters involved in these
320
N. BANCHERO
et al.
calculations, other than muscle fiber size and intercapillary same for both levels of barometric pressure.
distances, remain the
Acknowledgements During this study, Dr. M. Gimenez was on leave from the Unite 14, INSERM, at Nancy, France. Dr. A. Rostami was on leave from the University of Isfahan, Iran, and was supported by the World Health Organization. The authors wish to acknowledge the hypobaric facilities provided by the Department of Physiology and Biophysics, Colorado State University in Fort Collins, and especially the cooperation of Mr. John Fitch.
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