Clinical Science and Molecular Medicine (1974)46,375-391.
GROWTH O F T H E H E A R T AND L U N G S I N HYPOXIC
R O D E N T S : A MODEL O F H U M A N HYPOXIC DISEASE
CAROLIN HUNTER, GWENDA R. BARER, J. W. SHAW
AND
E. J. C L E G G
Department of Human Biology and Anatomy and Department of Medicine,
University of Shefield
(Received 6 November 1973)
SUMMARY
1 . Rats and mice were kept in a decompression chamber at 52 kPa (390 mmHg) for
1-4 weeks and their hearts and lungs were compared with littermate control animals.
In both species growth was retarded in the hypoxic environment.
2. In both species small peripheral lung vessels became thickened, developing two
elastic laminae with a muscular coat between. A method was developed for assessing
these changes in large numbers of animals. The number of thick-walled vessels was
still high after 4 weeks’ recovery in a normal environment. Pulmonary vascular
resistance, measured by a perfusion method, increased in animals kept in the decompression chamber.
3. Mouse lungs became heavier than controls; the increase was not due to a greater
fluid content. Rat lungs were heavy in relation to body weight but not heavier than
controls; there may have been slight thickening of alveolar walls. Chest areas,
measured from radiographs, were large relative to body weight in hypoxic rats.
4. The relationship between right and left ventricular weight and body weight was
studied in normal rats and mice. The left ventricle grew about four times more quickly
than the right. Changes in ventricular weights during exposure in the decompression
chamber and subsequent recovery in a normal environment were related to these
normal growth curves.
5. In both species the right ventricle grew abnormally fast in the decompression
chamber. It was absolutely heavier than that of controls and relative to body weight
was extremely heavy. After 4 weeks’ recovery the relationship between right ventricular weight and body weight was nearly normal; this was achieved by retarded growth
or actual loss of weight.
6. In mice the left ventricle grew normally in the decompression chamber and was
heavy in relation to body weight. In rats its growth was retarded in the chamber and
was normal in relation to body weight.
Correspondence: Dr G. R. Barer, Department of Experimental Medicine, The New Medical School, Beech
Hill Road, Sheffield SlO 2RZ.
375
376
Carolin Hunter et al.
7. Morphometry of the hypertrophied right ventricle showed that muscle fibre size
and total muscle mass had increased in hypoxic rats. There had been no increase in
nuclear mass, but the perinuclear sarcoplasm had increased. All layers of the myocardium participated in the hypertrophy.
Key words : hypoxia, right ventricular hypertrophy, pulmonary hypertension,
pulmonary arterioles.
In several hypoxic states in man the right ventricle and pulmonary arterioles are thick-walled
in comparison with those of normal adults. This is true in the mature foetus, in normal people
born in high mountain regions, and in patients with emphysema, kyphoscoliosis or the Pickwickian syndrome (O’Neal, Ahlvin, Bauer & Thomas, 1957; Naeye, 1961a, b, 1962, 1966;
Arias-Stella & Saldaiia, 1962; Arias-Stella & Recavarren, 1963; Hasleton, Heath & Brewer,
1968). Similar changes have been produced in animals kept under hypoxic conditions or in
decompression chambers and are present in cattle with high mountain disease (Naeye, 1962,
1965b; Abraham, Kay, Cole & Pincock, 1971; James & Thomas, 1968; Alexander, 1962).
Hypoxia of moderate but not severe degree has been shown to cause changes in lung growth of
functional significance (Burri & Weibel, 1971; Bartlett, 1970; Bartlett & Remmers, 1971). In
the present study we have compared the growth rates of the lung and the two ventricles in
normal and hypoxic conditions and the changes which result from return to a normal environment. Preliminary accounts of some portions of the work have been published (Abbott, Barer,
Clegg & Shaw, 1968; Cook, Barer, Shaw & Clegg, 1970; Barer, Cook, Clegg & Shaw, 1970).
MATERIALS A N D METHODS
The growth of randomly bred male albino mice (Tucks no. 1) or albino Wistar male rats living
in a hypoxic environment (air at 52 kPa, 390 mmHg) in a decompression chamber, was
compared with the growth of littermate control animals living in air at atmospheric pressure.
The temperature and humidity of the hypoxic environment was slightly higher than that of the
control animals. Food and water were provided freely to both groups, but the animals in the
chamber ate and drank less than controls (Clegg & Harrison, 1968). Details of maintenance
are described by Hunter & Clegg (1973).
Growth of the lungs and right and left ventricles. Animals were killed with ethyl chloride.
The right ventricle was separated from the left, leaving the septum as part of the left; atria and
vessels were removed and the left ventricle was opened. Lungs and ventricles were washed in
saline, blotted and weighed, a time-schedule being kept. Normal growth curves of the lungs
and ventricles were constructed from nine litters, each of six mice; one mouse from each litter
was killed at 22,29,44,58,72 and 86 days of age; fourteen further mice were killed at 230-251
days of age. Similar curves were obtained from six litters of six rats, one from each being killed
at 22, 56, 83, 110, 200 and 244 days of age and also five further litters of six, one animal from
each being killed at 24, 31,45,59,69 and 91 days of age. The number of animals used in the
different experiments is given in Table 1.
Estimation of number of thick-walledperipheral pulmonary vessels. Several methods have been
devised to estimate the degree of thickening of pulmonary arterioles in man and animals in
hypoxic conditions (ONeill, Thomas & Hartroft, 1955; Naeye, 1962, 1965a; Wagenvoort,
377
Heart and lungs in experimental hypoxia
1960; Hasleton, Heath & Brewer, 1968). Inherent problems arise from the difficulty of comparing analogous vessels if some have changed size, and the difficulty of comparing wall
thickness if some vessels are in a state of constriction or contraction (Short, 1962). We categorized all vessels by their position and the nature of their elastic coat.
TABLE
1. Covariance analysis of lung and right ventricular weights in control and hypoxic animals. Mean values
+SEMareshown.Statisticalsignificanceof comparedmeans: * P<O.O5, ** Pc0.02, *** P<O*Oland**** P <
0.001. NS, not significant. The numbers of animals in each group are given in parentheses after the lung weights
and, if different, after the right ventricular weights.
Experiment
Group
Adjusted mean
lung Wt. (g)
Compared
means
Adjusted mean
right
ventricular
wt. (g)
Compared
means
Mice
Hypoxia
28-56 days,
28 days
recovery
Hypoxia
202-230
days,
28 days
recovery
Hypoxic HE
Control HC
Recovery RE
Control RC
Hypoxic HE
Control HC
Recovery RE
Control RC
0.205f 0.008 (6)
0-149f0.005 (6)
0.148 f O*OO3(1 1)
0.137+0.005 (11)
0.269fO.001 (8)
0*179f0*010 (8)
0.198 f0.011 (6)
0.197+.0*011 (9
HE/HC****
RE/RCNS
HE/RE****
HC/RCNS
HE/HC****
RE/RCNS
HE/RE****
HC/RCNs
0.055f0.038 (15)
0.028 kO.002 (15)
0*035+0*002(22)
0.026f 0.002 (22)
0.055f0*004
0.032f0.003
0.041 fO.003
0035 & 0003
HE/HC****
RE/RC**
HE/RE****
HC/RCNS
HE/HC****
RE/RCNS
HE/RE***
HC/RCNs
Rats
Hypoxia
2 8 4 5 days,
28 days
recovery
Hypoxia
35-50 days,
28 days
recovery
Hypoxia
65-83 days,
28 days
recovery
Hypoxic HE
Control HC
Recovery RE
Control RC
Hypoxic HE
Control HC
Recovery RE
Control RC
Hypoxic HE
Control HC
Recovery RE
Control RC
1.312t- 0.096 (5)
1-127+0*036 (5)
1.213 f0.086 (3)
1.021 f 0.100 (3)
HE/HCNS
RE/RC**
HE/RENS
HC/RENS
HE/HC*
RE/RC*
HE/RENS
HC/RCNs
HE/HC****
RE/RC****
HEIRE****
HC/RCNs
0.281 0.024
0.146t 0.009
0-105f0~022
0071 f0,025
0.267f0.021
0.174 0.010
0.164 0.013
0*123+0*017
0.361 f 0.021
0.182 f0.012
0.249 0.014
0.168 f0.015
Species
~
~
1-366fO-133 (4)
1*13OfO*O64 (4)
1.288 k 0.084 (4)
1-105+0-109 (4)
2.131 20.096 (4)
1.411 k0.055 (4)
1-497fO.064 (4)
1.249 f0.067 (4)
+
~~
HE/HC****
RE/RCNS
HE/RE****
HC/RC*
HE/HC****
R.E/RC***
HE/RE***
HC/RCNs
HE/HC****
RE/RC***+
HEIRE***
HC/RCNs
The lungs were fixed in 10% formalin-saline. Complete transverse sections of one lobe in
mice and two or three lobes in rats were cut and stained for elastic tissue with Gomori's aldehyde fuchsin stain. The whole section was systematically examined at x 40 objective magnification. All small vessels with a definite elastia coat adjacent to alveoli or alveolar ducts were counted
and the proportion of these having a double elastic lamina was calculated. Small veins were
included to avoid the difficulty of distinguishing them from arterioles. A double elastic lamina
was said to be present when two laminae with a space between were visible for at least half the
diameter in cross section, or the length of wall in longitudinal section (three-quarters of the
G
378
Carolin Hunter et al.
diameter in a few early experiments). In some of the hypoxic rats the inner elastic layer was
fainter than the outer and fragmented. Haematoxylin and eosin staining confirmed that there
was a layer of smooth muscle between the elastic laminae in rats. In mice the medial layer
between the elastic laminae was thinner and only occasional muscle nuclei were seen; the space
may have contained either muscle or amorphous connective tissue. Both arterioles and
venules with two elastic laminae were designated ‘thick-walled peripheral lung vessels’ (TWPV)
and recorded as a % of the total peripheral vessels. The results, of an ‘all or none’ nature, did
not estimate the degrees of thickening. Placing the vessels in categories was not always easy.
These shortcomings were outweighed by the possibility of examining large numbers of animals,
and the method was vindicated by the clear-cut results obtained. All sections were counted
‘blind’. Results were repeatable by one individual, with good agreement between the two
individuals who did the counting.
Chest size. This was measured in rats from dorsi-ventral radiographs in a standard position
under ether anaesthesia at intervals during exposure to hypoxia and also during recovery. The
area enclosed between the first and tenth ribs was planimetered.
Morphometry of the heart. Hearts were perfused through the coronary arteries with 10%
formalin-saline at a pressure of 13 kPa (1 00 mmHg). Paraffin sections (1 0 pm) through the base,
middle and apical regions were stained with Mallory’s and Heidenhain’s stain. The relative
sizes of the different muscle layers of the wall of the right ventricle and the proportions of the
different tissue elements were estimated by point-counting, a Wild 10 BK graticule being used.
Absolute areas of the sections of right ventricle were measured on camera lucida drawings.
Cell-division rates in hearts and lungs. Four litters of six male mice, and two litters of six and
one litter of four male rats were used, all animals being 28 days old when half of them were put
in the chamber. Comparisons of cell-division rates in control and experimental animals were
made after either 1 or 4 weeks’ hypoxia.
Immediately on removal of animals from the chamber, [3H]thymidine (specific radioactivity
5 Ci/nmol; 40 pCi in mice and in rats removed after 1 week and 250pCi in rats removed after 4
weeks) was injected intracardially under ether anaesthesia; 45 min later the animals were killed
with ether and the organs were removed and fixed in 10% formalin-saline. Three sections were
cut from each heart as described above and were layered with Kodak AR stripping film at
23°C; they were stored for 64 days at 0°C and then developed by the method of Rogers (1967).
In haematoxylin-stained sections point counting was performed on five fields of the inner
muscle layer on each of the three sections ( x 100 objective magnification) to determine the %
division rate; this equals the percentage of cells undergoing DNA replication, as indicated by
labelling. For the lungs, three blocks were cut from each of two lobes in rats, and one block
was cut from each of six lobes in mice. After treating the sections in the same way as those from
the hearts, point counting was used to estimate the percentage of labelled cells in a unit area,
from five fields from each lobe in the rat, and three fields in the mouse.
Lung perfusions. Pressure-flow relationships of the lung vasculature were measured by a
modification of the column method of Nichol, Girling, Jerrard, Claxton & Burton (1951)
described by Barer & Shaw (1971). The lungs were perfused from a reservoir with dextran,
which was allowed to flow out of the left atrium, or by a closed-circuit method with a pump,
using rat blood. A column placed immediately proximal to the pulmonary artery cannula was
filled with blood or dextran to measure pressure-flow relationships. It was allowed to empty
into the lung while pressure was measured at its base.
Heart and lungs in experimental hypoxia
379
Packed cell volume (PCV). This was estimated in samples taken by cardiac puncture after
ethyl chloride anaesthesia.
Statistical analysis of the results. MeanskSEM are given in text and tables. Differences
between means were tested by the unpaired t test and recorded as significant when P was equal
to or less than 0.05. To compare pulmonary vascular resistance in control and hypoxic animals
all the values for pressure and flow obtained during perfusions were pooled for each group and
regression lines calculated. Regression coefficients were compared by using the t test.
One of the principal effects of hypoxia is a marked reduction in food and water intake,
resulting in a fall in body weight (Clegg & Harrison, 1968). Hence, when organ weights in
hypoxic and control animals are to be compared, some technique must be adopted which takes
into account the differences in body weight between the different groups. The technique of
covariance analysis (Snedecor, 1956) was used for this purpose, in such a way that not only
could regression coefficients of organ weights on body weights be compared, but also the organ
weights themselves, after adjustment to allow for between-group differences in body weights.
RESULTS
Development of thick-walled peripheral lung vessels (T WP V ) in chronic hypoxia
In normal rats and mice arteries accompanying terminal and respiratory bronchioles have a
muscular coat bounded by two elastic laminae. More peripheral vessels accompanying alveolar
ducts and alveoli, both arterioles and venules, are usually thin-walled. They have one elastic
lamina and almost no muscle (Fig. la). In the animals exposed to hypoxia an increased
proportion of these latter vessels were muscularized and had two elastic laminae (Fig. lb).
Most of the thickened vessels were arterioles, distinguished by their position in relation to
alveolar ducts and continuity with muscular arteries. A few could have been venules since in
cross section they could not be distinguished from arterioles by the counting method used.
In six experiments thirty-eight rats (total) were put in the chamber at 52 kPa (390 mmHg)
at ages ranging from 28 to 65 days for periods ranging from 13 to 29 days; on removal they
were compared with forty-three littermate control animals. In every experiment the count of
TWPV was significantly higher in the hypoxic animals than in the controls [Table A, which is
deposited together with Tables B and C with the Librarian, Royal Society of Medicine, 1
Wimpole St, London WlM 8AE, from whom copies may be obtained on request (Clinical
Science and Molecular Medicine Table 731243, A-C)]. For example, in one experiment there
were 24-1& 4.1 % TWPV in four experimentalrats exposed to hypoxia from 28 to 49 days of age
and 9.6+0-9% in their littermate controls (P<O-O2). All results are summarized in the upper
part of Fig. 2. In three of the experiments half of the rats from the chamber were allowed to
recover for 4 weeks in a normal environment. In all three experiments the count of TWPV was
still significantly raised (Table A). These results are shown in the lower half of Fig. 2.
In five experiments fifty-two mice (total), either 28 days old or ‘adult’, were placed in the
chamber at 52 kPa (390 mmHg) for times ranging between 8 and 33 days, and compared on
removal with fifty-seven littermate control animals; the number of TWPV was again significantly higher than in the control groups in every experiment and the difference was similar in
magnitude to that found in rats. From one group, 28 days old when put in the chamber, nine
experimental mice were killed after 8 days, ten mice after 18 days and six mice after 28 days,
each with littermate control groups. The percentages of thick-walled vessels at these three
‘I
Carolin Hunter et al.
380
(a)
4
P
+
Lc
v)
2
i
18
24
I 18
24
30
36
0
.s
‘Et
4
2-
n
6
ll
n
12
I 30
Thick-walled peripheral vessels “70)
FIG.2. Histogram showing the percentage of thick-walled peripheral lung vessels. (a) Hypoxic
rats killed immediately on removal from the decompression chamber (m) and littermate controls
killed on the same day (0).(b) Hypoxic rats killed 4 weeks after removal from the decompression
chamber (m) and littermate controls killed on the same day (0).
times for control and hypoxic groups respectively were 1*0+0*4% and 10.3+2-6% (P<O-OI),
3.7&0-9%and 10 1+2.0% (P<O-OI), 2-8+0.8% and 13-6,2-1% (P<O*OOI).
A local strain of mice was used in three of these experiments and Tucks no. 1 strain in the
other two. In the latter the counts of TWPV were much lower in both control and hypoxic
groups than in the local strain; the differences between control and hypoxic groups were
similar in both strains.
Increased pulmonary vascular resistance after chronic hypoxia
Fig. 3 shows pressureflow lines for one experiment in mice (dextran perfusion) and one in
rats (blood perfusion). In both experiments, the slope of the group regression line was significantly less in the hypoxic animals, indicating a greater pulmonary vascular resistance. Two
other rat experiments gave similar results although in one of them the difference was not significant. Comparison of vascular resistance is only valid if the vascular bed is similar in size in
the two groups, which appears probable for there were no significant differences between the
mean lung weights of control and hypoxic groups.
Growth of the lung in hypoxic and control animals
Fig. 4 shows the regression lines and 95% confidence limits relating lung weight to body
Heart and lungs in experimental hypoxia
(Facing p . 380)
Carolin Hunter et al.
Heart and lungs in experimental hypoxia
381
weight in a large series of rats and mice; the logarithm of body weight was used for rats since
untransformed lung weights had a curvilinear relationship with body weight. Against these
standard lines are plotted the mean values ( 5SEM) of the lung weights for groups of animals
subjected to hypoxia at different ages and for varying periods, together with those of their
littermate controls.
In each group half the animals (called ‘hypoxic’) were killed immediately on removal from
the chamber, together with their controls, and half (called ‘recovery’) were killed after 4 weeks
of recovery in a normal environment, again with their controls.
Pulmonary artery pressure (mmHg)
FIG. 3. Post-mortem pressure-flow relationships of the lungs. (a) Regression lines of grouped
pressure-flow (dextran perfusion) relationships of ten control (curve C) and ten hypoxic mice
(curve E) are compared (P<0.001). Hypoxic mice were in the chamber 18-33 days. (b) Pressureflow regression lines from four control and six hypoxic rats (blood perfusion; P< 0.05). Hypoxic
rats were in the chamber 13-18 days. The broken line in (a) shows resistance of the apparatus,
which was negligible in (b).
In mice the lungs of both hypoxic groups were significantly heavier than those of their controls and in relation to body weight were extremely heavy. The young mice had gained body
weight more slowly than their controls, while the adult mice had lost weight. The gain in lung
weight in the adult group was surprising and suggested the possibility of accumulated fluid in
the lung. However, there were no significant differences in water content between either of the
experimental groups and their controls. In rats all three hypoxic groups had lungs which were
heavy in relation to body weight (which again was less than controls), but only in the oldest
group (A) were the lungs absolutely heavier than those of controls.
Lung weights of ‘recovery’ mice had diminished and were now similar to controls. Lungs of
‘recovery’ rats were still relatively heavier than their controls. In some experiments adjustment
in lung weight had been achieved by a deceleration of growth and in others by an actual loss of
weight.
To allow for differences in body weights, the lung weights of the different groups were compared by covariance analysis (Table 1). In all but one experiment in rats the adjusted mean
weights of the hypoxic lungs were significantly greater than those of controls. In mice and in the
oldest group of rats the ‘recovery’ groups had lungs which were significantly lighter than the
Carolin Hunter et al.
382
I.!
1.4
1.4
13
1.:
1.2
1.2
RE
T
1.1
IogBody w t ( g )
1
0.14
I
35
45
Body wt. ( g )
FIG.4. Effect of hypoxia and recovery on lung weight. Experimental results are plotted against the
normal growth curves relating lung weight to body weight (in rats log body weight) obtained from
a large series of control animals, these growth curves being shown as regression lines and 95%
confidence limits. 0 , Mean values (1SEM) are shown for: HE (hypoxic experimental), animals
killed immediately on removal from the decompression chamber; HC, littermate controls killed
on the same day; RE (recovery experimental), animals which had been in the chamber but allowed
to recover for 4 weeks; RC, littermate controls killed on the same day. The ‘recovery’groups were
littermates of those killed immediately on removal from the chamber. (a) Rats (group A): HE, in
the chamber from 65 to 82 days of age. (b) Rats (group B): HE, in the chamber from 28 to 45 days
of age. (c) Rats (group C): HE, in the chamber from 35 to 48 days of age. (d) Mice: young HE group
(to the left of the broken line), in the chamber from 28 to 56 days of age; adult H E group (to the
right of the broken line), in the chamber from 202 to 230 days of age. Numbers of animals in each
group are given in Table 1.
Heart and lungs in experimental hypoxia
383
hypoxic groups but in the two groups of young rats there had been no significant fall in the
adjusted lung weight during the recovery period. The adjusted lung weights of none of the
‘recovery’ mice but all the ‘recovery’ rats were significantly greater than their controls. There
was no significant difference between the adjusted weights of the lungs of the two control
groups in any experiment.
Cell-division rates in normal and hypoxic lungs
There were no significant differences in % division rates between hypoxic and control lungs
in either mice or rats after 1 or 4 weeks hypoxia (Table B; deposited data, see note above).
Control rates were 0-65&0.14at 35 days of age and 0.37k0.40 at 56 days of age for mice;
control rates for rats at these two ages were 1.75 +0-15 and 0-31f0.07.
Chest shape and size in normal and hypoxic rats
Areas of the dorsi-ventral projection of the thoracic cage were measured at intervals from
radiographs in eight rats kept in the chamber from 28 to 56 days of age and compared with
those of eight littermate controls. Up to and including 45 days of age there were no significant
differences between the areas of the two groups although at 45 days the mean body weight of
the hypoxic animals was only 129k2.3 g whereas that of the controls was 191k4.2 g. At 56
days of age chest areas were smaller in the hypoxic rats. This was due to a lesser Iongitudinal
measurement, the width being similar; the chests looked ‘barrel-shaped‘. The slope of the
regression line relating chest area to body weight was significantly steeper in the hypoxic
group (hypoxic 3-3 x 10-4_f0*3x 10-4; controls = 2.3 x 10-4+0.8 x 10-5; P<0.02). These
results suggest that the lungs of the hypoxic rats were being held at a higher volume than those
of controls, at least when anaesthetized.
Growth changes in the two ventricles during hypoxia: cross-sectional areas of right and left
ventricles
Fig. 5(a) and Fig. 5(b) show that in a hypoxic rat the wall of the right ventricle was greatly
thickened when compared with that of a littermate control rat. In a group of six rats kept in the
chamber from 28 to 44 days of age, the ratio of left/right ventricular areas (measured from two
sections per rat by planimetry) was 2.7+0-1, compared with 6.3rtO-5 for their littermate
controls (P<O-Ol). It was difficult to obtain reproducible results in mice and we therefore
studied weight changes in the two ventricles during hypoxia and recovery relative to the normal
weight changes during growth in both rats and mice.
Normal growth curves of the two ventricles
Fig. 6 shows the relationship between right or left ventricular weights and body weight in a
large series of control mice and rats. In both species the growth rate of the left ventricle relative
to body weight was about four times that of the right ventricle. In mice the right ventriclejbody
weight relationship appeared to be linear throughout the period studied. In rats the relationship was curvilinear. In mice, left ventricular weight was linearly related to body weight up to
86 days of age; at greater ages it was no longer related to body weight, suggesting the influence
of other factors in its control. In rats the relationship between left ventricular weight and body
weight was curvilinear.
384
t
o*20[
L
-3
.-U
+
L
C
3
Carolin Hunter et al.
(b)
(0)
0.8 -
0.6 -
0.4
-
m B
0.04
0
I
I
I
I
I
I
10
20
30
40
50
60
0
100
300
500
Body wt. ( g )
FIG.6. Normal growth curves of left (L) and right (R) ventricles in mice (a) and rats (b). Mice:
22-86 days old (a);230-257 days old ( 0 ) .Rats: 22-200 days old ( 8 ) ;244 days old (0).
Right ventricle in hypoxia and recovery
Fig. 7 shows regression lines and 95% confidence limits relating right ventricular weight to
body weight for the control animals, derived from the data shown in Fig. 6. In the rats right
ventricular weight is plotted against the logarithm of the body weight, SO as to produce a
linear relationship. Mean values for experimental groups and their littermate controls are
plotted against these growth lines for the control animals, as described for the lungs in Fig. 4.
The control groups in both species were very close to the regression lines. The experimental
animals had right ventricles which were absolutely heavier than those of control animals, and,
in relation to body weight, extremely heavy. Overall body growth was either retarded or
showed an actual loss, as already described in the section on lung growth, but there was still
an accelerated growth of the right ventricle.
After recovery from hypoxia the mean weights of the right ventricles in mice were closer to
the normal regression line owing to an actual loss of weight. In the ‘recovery’ rats only group A
had right ventricles still notably heavier than their controls; right ventricles of groups A and B
had probably lost weight whereas group C had undergone a period of decelerated growth.
To allow for differences in body weight the right ventricular weights of different groups were
compared by covariance analysis, as in the lung experiments, and results are shown in Table 1.
In all the experiments the adjusted mean weights of the right ventricles of the hypoxic groups
were significantly greater than those of controls and after recovery the ventricles were
significantly lighter than those weighed immediately after removal from the chamber. However,
0.3 -
--
IE
((1)
RE
0
al
;.c
HE
0.2
+
0.3-
0.3-
(b)
[c)
HE
-
O"-
HE
C
m
>
c
-
S
01
:0.1
0.1
0
s
0
I
I
I
0,
I
I
I
0
I
I
I
._
k
0.03
f
0.02
I
35
25
1
45
Body wt. (g)
FIG.7. Effect of hypoxia and recovery on right ventricular weight. Experimental results are plotted
against regression lines and 95% confidence limits obtained from the normal growth curves shown
in Fig. 6. log Body weight is used for rats. Groups of animals, symbols and lettering are as
described for Fig. 4. Numbers of animals in each group are given in Table 1.
they were still signscantly heavier than their controls, except in one experiment in each
species.
Left ventricle in hypoxia and recovery
In all the rat experiments, and in those with adult mice, left ventricular weights, after covariance adjustment, were not significantly different in experimental animals from their
controls. However, in the young mice the adjusted mean ventricular weights of animals killed
immediately after the period of hypoxia were significantly greater than in their controls
(P<0-01). There was also a significant fall in adjusted mean weights in experimental animals
over the period of recovery (Pt0-05).The relationship between left ventricular weight and body
weight for mice is shown in Fig. 8.
Eflect of hypoxia on muscle layers and tissue components of the rat right ventricle
The measurements of the areas occupied by the inner, middle and outer layers of the myocardium in sections taken from the base, middle and apex of the ventricles from four control
386
Carolin Hunter et al.
RC
R$Hc
HE
&
Body wt. (9)
FIG.8. Effect of hypoxia and recovery on left ventricular weight as a function of body weight in
mice. Symbols, lettering and groups are as described for Fig. 4(d). The regression line and 95%
confidence limits for the growth of the normal left ventricle (obtained from Fig. 6 ) are not con
tinued beyond 39 g body weight as there was no relationship between left ventricular weight and
body weight beyond this point.
and four experimental rats (in the chamber from 28 to 43 days of age) showed that in the
hypoxic groups all layers were thickened at all levels, except the outer layer at the apex; the
inner layer was most affected (Table C, deposited data, see note above).
The diameter of right ventricular muscle fibres was greater in hypoxic than control rats (Figs.
5c and 5d). The proportion of the right ventricular wall occupied by different tissue elements
was assessed by point counting on the sections described in the last paragraph. The number of
points lying over muscle fibres, muscle nuclei, perinuclear sarcoplasm, connective tissue fibres,
connective tissue nuclei and blood vessel lumina was determined in twelve fields within the
inner muscle layer. The percentage of total points counted occupied by each tissue element
(amount of this element per unit area of right ventricle) is given in Table C (deposited data, see
above); the mean proportions of each tissue element in control and hypoxic rats were compared by unpaired t tests. Although the total quantity of muscle fibres and connective tissue
nuclei had increased in hypoxic rats they were in the same proportion as in control animals.
The proportion of connective tissue fibres was consistently lower in hypoxic than in control
rats but the difference was not significant, nor was the proportion occupied by blood vessel
lumina. However, muscle nuclei did form a significantly smaller proportion of the right ventricular wall at the base, mid-zone and apex in the hypoxic rats. Perinuclear sarcoplasm formed a
larger proportion in hypoxic rats, this difference being significant at the apex and base (Fig. 5c)
but not in the mid-zone. This is the area of sarcoplasm round the nucleus which is devoid of
myofibrils and contains fat droplets and pigment granules. The absolute areas occupied by
Heart and lungs in experimental hypoxia
387
different tissue elements was obtained by multiplying the amount of an element per unit area
by the total area of the appropriate muscle layer obtained by planimetry. The absolute areas
occupied by muscle nuclei were similar in control and hypoxic rats in spite of the great increase
in muscle fibre in the latter group. Absolute areas of perinuclear sarcoplasm were increased in
hypoxic rats. Measurements of tissue elements were also made on the outer muscle layer with
results which differed only in detail. The relative area of muscle nuclei was decreased in hypoxic
rats, and that of perinuclear sarcoplasm was increased though to a smaller extent than in the
inner layer. Thus during the period of right ventricular growth in the chamber there had been
a great increase in muscle mass and fibre size with no parallel increase in muscle nuclear mass,
although the amount of perinuclear sarcoplasm had increased.
Cell-division rate of the right ventricle in rats and mice
Table B (deposited data, see above) shows the cell-division rate measured in the inner muscle
layer of the right ventricle in rats and mice exposed to hypoxia for 1 and 4 weeks, as compared
with their controls. At 1 week there was a significant increase in the division rate in hypoxic rats
in the middle and apical regions; for the middle muscle layer the division rate was 1.71 &0*46%
for controls and 3.06f0-49% for hypoxic rats. At 4 weeks the rate was not significantlyraised;
for the same layer the division rate was 0-34+0.19% for controls and 0-82f0.36% for the
hypoxic rats (not significant).
In mice, the results were similar to rats but the number of dividing cells was less at both times;
the difference between hypoxic and control groups was not significant. Determination of cell
types was not possible.
Packed cell volume
This was increased in all hypoxic animals. For example, the mean value for the three groups
of rats shown in Figs. 4, 7 and 8 was 82%, but was not significantly different from controls
after 4 weeks’ recovery. In a group of forty-eight mice exposed in the chamber from 28 to 56
days of age, the mean PCV was 67% on removal from the chamber, and remained higher than
the mean control value (41%) for 11 days.
DISCUSSION
In our experiments both of the species studied rapidly developed right ventricular hypertrophy
and muscularization of pulmonary arterioles when exposed to hypoxia. Electrocardiographic
changes characteristic of right heart hypertrophy were also observed (Cook, 1971). Naeye
(1965a), James &Thomas (1968) and Abraham et al. (1971) showed that hypoxic rats and mice
develop right ventricular hypertrophy, which can be reversed (Abraham et al., 1971). Widimsky,
Urbanova, Ressl, Ostadal, Pelouch & Prochazka (1 972) studied the effect of intermittent
severe hypobaric hypoxia in rats at a simulated altitude of 7000 m. Right ventricle weight relative to body weight was increased after twenty-four exposures and left ventricular weight
relative to body weight was increased after seventy exposures. These rats also developed focal
necroses in the myocardium. By relating right ventricle weight to its normal growth curve, we
showed that it grew abnormally fast in a hypoxic environment, although general growth was
retarded. In spite of the greatly increased amount of muscle tissue there was no increase in the
amount of muscle nuclei; the increased number of dividing cells must have been in the supporting tissues. During recovery the growth of the right ventricle was retarded or weight was actually
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Carolin Hunter et al.
lost until a normal relationship with body weight was restored. After the 4 weeks recovery
period right ventricular weights were nearly normal relative to body weight; in most rats the
ECG no longer showed right ventricular preponderance (Cook, 1971). In young hypoxic mice
the growth of the left ventricle showed the same characteristics as that of the right ventricle,
but in older animals and in rats of all ages left ventricular growth was retarded and its weight
remained in proportion to body weight. The complex control mechanisms determining these
relative growth changes remain to be explored.
The rats and mice from the decompression chamber were lighter than controls. Other
studies under similar conditions showed that the animals grow more slowly than littermate
controls, sometimes after a period of initial weight loss (Hunter & Clegg, 1973). Since they
eat and drink less than controls it is important to know whether some of the organ changes
observed are secondary to the reduced diet and not direct consequences of hypoxia. This
question has been studied by Harris, Gibson & Gloster (1972), who compared rats kept in a
hypobaric chamber at 53 kPa (400 mmHg) with both free-fed and pair-fed controls. They
found that right ventricular weight was higher in hypoxic rats than in either control group.
Left ventricles of the hypoxic group were lighter than those of free-fed controls but heavier
than those of pair-fed controls. However, as well as impairing food intake, hypoxia causes
greatly reduced fluid intake (Fregly 8z Waters, 1966; Clegg & Harrison, 1968). Preliminary
unpublished work (E. J. Clegg & C. Hunter) suggests that when the food and water intake of
hypoxic and pair-fed animals are compared, the hypoxic group show no relation between food
and water intake and in the latter group water intake is dependent to a highly significant extent
on food intake. In practice it was found impossible to ensure that hypoxic and littermate pairfed animals received the same amounts of food and water over a given period. This difficulty
impairs the comparison between hypoxic and pair-fed controls.
Thickening of small pulmonary arteries has been produced experimentally under hypoxic
conditions in mice (Naeye, 1965a; James & Thomas, 1968), rats (Abraham et al., 1971), dogs
and calves (Naeye, 1965b). The present work shows that this thickening occurs in small
vessels accompanying terminal air units. An increased proportion of vessels in this situation
developed a double elastic lamina and muscle coat and closely resembled those found by
Hasleton et al. (1968) in human hypoxic states. Since the total number of these vessels was
unaltered there must have been new development of elastic and muscular tissue, either through
hypertrophy or hyperplasia of pre-existing smooth muscle cells or a migration of cells from
more central muscular arteries. Naeye (1965a) found an increased number of medial nuclei in
small pulmonary vessels in hypoxic mice. We found no increase in cell-division rate in the
lungs in our experiments but the number of muscle nuclei in vessel walls in a single lung section
is small and must form a negligible proportion of all lung nuclei. Thickening of the vessel walls
under hypoxic conditions occurred more rapidly than regression on return to a normal environment. After a 4 weeks recovery period the number of thick-walled vessels was still significantly
higher than in controls. However, Abraham et al. (1971) found that the small pulmonary
vessels of hypoxic rats were normal after 6 weeks recovery. The anatomical changes in small
pulmonary vessels probably accounted for the increased pulmonary vascular resistance found
in perfusion experiments.
The vessels which became thickened were separated by only a short diffusion pathway from
the air passages. The cause of the thickening is probably hypoxic vasoconstriction leading to a
work hypertrophy. However, the thickened vessels have so little muscle in the control state that
Heart and lungs in experimental hypoxia
389
it is difficult to visualize them undergoing strong vasoconstriction. That hypoxia, and not a
raised pulmonary artery pressure, is the causal factor is indicated by the work of Naeye (1965b).
In both dogs and calves partial or complete occlusion of a bronchus led to thickening of small
pulmonary arteries (and in calves pulmonary veins as well) in the occluded but not the unoccluded lung. Presumably the vascular pressures were similar in the two lungs, but pulmonary
venous partial pressure of oxygen was lower in the unventilated or poorly ventilated lung. In
cats and dogs a significant relationship was demonstrated between pulmonary vasoconstriction
and pulmonary venous oxygen tension (Barer, Howard & Shaw, 1970). Naeye (1965a) showed
that mice injected daily with a-methyl-DOPA failed to develop thickening of small pulmonary
arteries or right ventricular hypertrophy in a hypoxic environment, suggesting that catecholamine metabolism might be involved in these changes.
We have no explanation for the absolute increase in lung weight found in hypoxic mice and
older hypoxic rats or the relative increase found in young rats. In mice it was not due to increased water content. In rats, morphometric measurements suggested slight alveolar wall
thickening; the proportion of lung parenchyma occupied by alveolar wall relative to alveolar
lumen was increased, but not significantly, in hypoxic rats (Cook, 1971). Burri & Weibel(l971)
found a significant increase in lung volume and alveolar surface relative to body weight in
young rats kept on the Jungfraujoch for 3 weeks (66.5 kPa, 497 mmHg); hyperoxia at normal
barometric pressure had the reverse effect. It was concluded that structural changes of functional significance occur with changes in the environmental partial pressure of oxygen. However, Bartlett (1970), who exposed young rats to a more severe hypoxic stimulus for 15 days
(10.4% 0,), found no significant increase in lung volumes, alveolar surface area or alveolar
number relative to body weight compared with controls. As in our experiments, there was a
significant increase in lung weight relative to body weight, which he thought was due to oedema
fluid. More recently Bartlett & Remmers (1971) exposed young rats to a lesser hypoxic stimulus
for 21 days (Poz, 12.7 kPa, 95 mmHg) and found that they developed a significant increase in
alveolar surface area relative to body weight. It appears that structural adaptation of the lung
follows a moderate but not a severe hypoxic stimulus. Since these deductions depend on the
relationship between lung volume and body weight it is important to establish that these two
variables are related during the period studied. Unpublished results suggest that they may not
have been so related in our experiments (Cook, 1971). In our rats chest shape and size (relative
to body weight) were modified during chronic hypoxia but we cannot say whether this was due
to structural alterations or to breathing from a higher functional residual capacity.
The method of study we have used produces changes in the hearts and lungs of experimental
animals similar to those found in human hypoxic states. The speed with which the changes occur
and the large number of animals which may be employed enables us to make quantitative
analysis of the results. Work in progress in the response to hypoxia of the carotid body of
experimental animals again suggests similarities to the response of hypoxic man (Barer, Edwards & Jolly, 1972). Thus we have a model of human hypoxic disease which should prove
useful for both functional and morphological studies.
ACKNOWLEDGMENTS
We are grateful to Professor Sir Charles Stuart-Harris and Professor R. Barer for their encouragement. We thank Mr D. Bradey for his invaluable help in developing histological
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Carolin Hunter et al.
techniques. Dixie Barraclough, Christine Poad and Derek Fish gave their expert help in many
ways. The Medical Research Council and Sheffield University Research Fund provided money
for apparatus. C.H. and J.W.S. were supported by the Medical Research Council.
REFERENCES
ABBOTT,
J., BARER,G.R., CLEGG,E.J. & SHAW,J.W. (1968) Changes in the small pulmonary blood vessels of
mice exposed chronically to low oxygen tensions. Journal of Physiology, 196, 118~-119~.
ABRAHAM,
AS., KAY,
J.M., COLE,R.B. & PINCOCK,
A.C. (1971) Haemodynamic and pathological study of the
effect of chronic hypoxia and subsequent recovery of the heart and pulmonary vasculature of the rat.
Cardiovascular Research, 5, 95-102.
ALEXANDER,
A.F. (1962) The bovine lung: Normal vascular histology and vascular lesions in high mountain
disease. Medicina Thoracalis, 19, 336350.
ARIAS-STELLA,
J. & RECAVARREN,
S. (1963) Right ventricular hypertrophy in native children living at high altitudes. American Journal of Pathology, 41, 55-64.
ARIAS-STELLA,
J. & SALDARA,
M. (1962) The muscular pulmonary arteries in people native to high altitude.
Medicina Thoracalis, 19,484493.
BARER,G.R., COOK,C., CLEGG,E.J. & SHAW,J.W. (1970) Pulmonary vascular and cardiac changes in rats and
mice exposed to chronic hypoxia. Journal of Pathology, 101, Proc. i-ii.
BARER,G.R., EDWARDS,
C. & JOLLY,A.I. (1972) Changes in the ventilatory response to hypoxia and in carotid
body size in chronically hypoxic rats. Journal of Physiology, 2 2 1 , 2 7 ~ - 2 8 ~ .
BARER,G.R., HOWARD,
P. & SHAW,J.W. (1970) Stimulus-response curves for the pulmonary vascular bed to
hypoxia and hypercapnia. Journal of Physiology, 211, 139-155,
BARER,G.R. & SHAW,J.W. (1971) Pulmonary vasodilator and vasoconstrictor actions of carbon dioxide.
Journal of Physiology, 213, 633-645.
BARTLETT,
D., JR (1970) Postnatal growth of the mammalian lung; influence of low and high oxygen tensions.
Respiration Physiology, 9, 58-64.
BARTLETT,
D., JR & REMMERS,
J.E. (1971) Effects of high altitude exposure on the lungs of young rats. Respiration Physiology, 13, 116125.
BURRI,P.H. & WEIBEL,E.R. (1971) Morphometric estimation of pulmonary diffusion capacity. 11. Effect of Poz
on the growing lung. Adaptation of the growing rat lung to hypoxia and hyperoxia. Respiration Physiology,
11,247-264.
CLEGG,E.J. & HARRISON,
G.A. (1968) Some phenotypic modification in rats exposed to reduced atmospheric
pressures. Enoironmental Research, 1,231-239.
COOK,C. (1971) Growth under hypoxic stress. Ph.D. thesis, Sheffield University.
COOK,C., BARER,
G.R., SHAW,J.W. & CLEGG,E.J. (1970) Growth of the heart and lungs in normal and hypoxic
rodents. Journal of Anatomy, 107, Proc. 384.
FREGLY,
M.I. & WATERS,I.W. (1966) Post-hypoxic drinking responses in rats. Federation Proceedings, 25,
1220-1226.
HARRIS,P., GIBSON,
K. & GLOSTER,
J. (1972) Metabolic effects of chronic hypoxia on the myocardium. Bulletin
& Physk-pathologie Respiratoire, 8, 1408-1410.
HASLETON,
P.C., HEATH,
D. &BREWER,
D.B. (1968) Hypertensive pulmonary vascular disease in states of chronic
hypoxia. Journal of Pathology and Bacteriology, 95,431-440.
HUNTER,
C. & CLEGG,E.J. (1973) Changes in body weight of the growing and adult mouse in response to
hypoxic stress. Journal of Anatomy, 114, 185-199.
A.J. (1968) The effect of hypoxia on the heart and pulmonary arterioles of mice.
JAMB, W.R.L. & THOMAS,
Cardiovascular Research, 3, 278-283.
NAEYE,R.L. (1961a) Arterial changes during the perinatal period. Archives of Pathology, 71, 121-128.
NAEYE,R.L. (1961b) Hypoxaemia and pulmonary hypertension. Archives of Pathology, 71,447-452.
NAEYE,R.L. (1962) Hypoxaemia, effects on the pulmonary vascular bed. Medicina Thoracalis, 19,494-501.
NAEYE,R.L.(1965a) Effect of alph-methyl-dopa on heart and pulmonary arteries of hypoxic mice. American
Journal of Physiology, 209,702-704.
Heart and lungs in experimental hypoxia
39 1
NAEYE.
R.L. (1965b) Pulmonary vascular changes with chronic unilateral pulmonary hypoxia. Circulation
Research, 17, 160-167.
NAEYE,R.L. (1966) Development of systemic and pulmonary arteries from birth through early childhood.
Biologia Neonatorum, 10,s-16.
NICHOL,
T., GIRLING,
F., JBRRARD,W., CLAXTON,
E.B. & BURTON,
A.C. (1951)Fundamental instability of the
small blood vessels and critical closing pressures in the vascular beds. American Journal of Physiology, 164,
33cL344.
O’NEAL,R.M., AHLVIN,
R.C., BAUER,W.C. &THOMAS,
W.A. (1957)Development of fetal pulmonary arterioles.
Archives of Pathology, 63,309-315.
O’NEAL,R.M., THOMAS,
W.A. & HARTROFT,
P.M. (1955) The media of small pulmonary arteries in mitral
stenosis. Archives of Pathology, 60,267-270.
ROGERS,
A.W. (1967)Techniques of Autoradiogruphy. Elsevier, Amsterdam.
SHORT,D.S. (1962)The problem of medical hypertrophy in hypertension. Medicina Thoracalis, 19,411-417.
SNEDECOR,
G.W.(1956)Statistical Methods, p. 344. Iowa State University Press, Iowa.
WAGENVOORT,
C.A. (1960)Vasoconstriction and medical hypertrophy in pulmonary hypertension. Circulation,
22, 535-546.
WIDIMSKY,
J., URBANOVA,
D., RESSL, J., OSTADAL,
B., PELOUCH.
V. & PROCHAZKA,
J. (1972) The effect of
intermittant altitude hypoxia on the myocardium of the rat. Bulletin de Physw-pathologie Respiratoire,
8, 1400-1405.