J. Embryol. exp. Morph. Vol. 19, 3, pp. 347-62, May 1968
With 3 plates
Printed in Great Britain
347
A time-lapse photographic study of chick embryos
exposed to teratogenic doses of hypoxia
CASIMER T. GRABOWSKI 1 & ROBERT E. SCHROEDER 1
From the Department of Biology, University of Miami
Continuous, long-term observations of amniote embryos have always been
difficult. Special culture techniques for young avian and mammalian embryos
have been developed (New, 1967) and these have helped to visualize the early
stages of development. But studies of normal and abnormal development during
the major period of organogenesis have been made largely by tedious indirect
methods, such as the examination of a series of embryos preserved at different
time intervals. Transitory responses to toxic stimuli have been particularly
difficult to detect in this manner. To observe the visible initial effects of teratogenic agents, a photographic time-lapse study of chick embryos in their natural,
in ovo, state was initiated. This report compares the changes in normal and
hypoxia-treated embryos during the third day of development.
Of the many agents which produce abnormal development, oxygen deficiency
is one of the better known, since (1) it is readily induced in the laboratory by
a variety of means, and (2) it is generally considered to be a significant cause of
spontaneously occurring anomalies (Rubsaamen, 1952; Ingalls, 1952). Dareste,
in 1877, and many others after him, produced oxygen lack in the chick embryo
by covering the egg with impervious material. More recently, the effects of
hypoxia on the chick embryo have been studied utilizing partial vacuum and
gaseous mixtures (Gallera, 1951; Naujoks, 1953; Biichner, 1955; Grabowski&
Paar, 1958; Grabowski, 1961, 1964). The last two studies in particular indicated
that the effects of hypoxia were more varied than generally suspected. For
instance, some of the abnormal development induced by hypoxia is caused by
cytotoxic effects, but most is mediated through a complex 'edema syndrome'.
In our photographic study, the edematous state, its development and some of
its consequences are described in detail.
1
Authors' address: Department of Biology, University of Miami, Coral Gables, Florida
33124, U.S.A.
23
JEEM 19
348
C, T. GRABOWSKI & R. E. SCHROEDER
MATERIALS AND METHODS
Eggs of the Kimber strain of white leghorn chickens were incubated at 39 °C
for 3 days prior to use. Observations of the embryo were made through an
overlying coverglass window sealed with paraffin to the shell (see New, 1967).
The eggs were kept in a transparent chamber maintained at 39 °C ± 1° through
which pre-warmed air or a mixture of nitrogen and air flowed. Photographs
were taken on Kodak Plus-X film with a Nikon F 35 mm camera, equipped
with either a 55 mm Micro Nikkor lens and bellows attachment or a 500 mm
Medical Nikkor lens. The definition obtained with both lenses was equally good,
but the latter, a modified telephoto lens, was more convenient to use because of
the greater working distance. A green filter (Wratten B) was used to enhance
the visibility of the circulatory system. An electronic flash gun beneath the egg
provided illumination. The initial magnification was x 2-5; the prints for study
purposes were uniformly made at x 15. With this system observations could be
made on embryos 2-5 days old with exposures taken as rapidly as 7 sec. apart.
Many soft structures such as the brain and spinal cord, eyes, somites, limb
buds, and even visceral arches were clearly seen on these photographs even
though no vital stain was used (Plates 1-3). The embryonic and extra-embryonic
blood vessels were particularly clear. Aplanimeter measured the area of the whole
embryo or parts of it. Such a measurement essentially represents the area of an
optical saggital section. In some cases, changes in shape were studied by comparing tracings made from successive prints (Text-fig. 1). In several experiments
measurements were taken of the angle between the head and trunk of the embryo,
using the dorsal aorta of both regions as a guide.
The development of normal 3-day embryos was compared to those exposed
to 4-6 % oxygen for 4-8 h and then returned to 21 % oxygen. The usual period
of observation was 18 h with photographs taken every 15-30 min. Additional
photographs of survivors were made at 24 h. Usually 30-40 successive photographs were available for each embryo. On every photograph of a series,
measurements were taken of: (1) crown-rump length, measured from the top
of the mid-brain to the tip of the tail; (2) the diameter of the aorta a short
distance below the aortic arches; (3) the diameter of the aortic arches at their
mid-points; (4) the width of the anterior cardinal vein at a point just before it
crosses over the dorsal aorta; (5) the diameter of the anterior vitelline vein just
above the head of the embryo; and (6) the diameter of the vitelline arteries and
veins at a point close to the embryo, whenever separated enough to be distinguishable. These points of measurements are shown in Plate 1, fig. B.
PLATE 1
Time sequence photos of a normal embryo, at stage 17 at start of observations, taken (A) at
start (0 time); (B) at 7i h; (C) 14 h; (D) 23 h. The arrows in (B) indicate the points at which
blood vessel measurements were made. Note particularly the clarity of the vascular system,
the increase in size of the embryo and its blood vessels and the progress of cephalic flexure.
J. Embryol. exp. Morp/i., Vol. 19, Part 3
PLATE 1
-•••*
C. T. GRABOWSKI & R. E. SCHROEDER
facing p. 348
/. Embryol. exp. Morph., Vol. 19, Part 3
C. T. GRABOWSKI & R. E. SCHROEDER
PLATE 2
Hypoxia-induced teratogenesis
349
Measurements of the total area of the embryo were made on every second or
third print of a series. Measurements of other blood vessels, of the angle between
head and trunk and other structures also were made, but not on every embryo.
Presenting all the data in detail is neither necessary nor desirable, and only the
averages of the measurements made at the start of the experiment (zero hours),
at 7 h, and at 13 h of the area of the embryo and diameters of several blood
vessels are presented in Tables 1 and 2. Some data on other structures are
included. Altogether, this study is based on 10 control and 34 treated embryos
for which complete records were available, and is supplemented with observations on 18 additional embryos that were either younger or older than the
majority, or for which only partial records were available.
Hematocrit values were obtained on blood samples drawn with glass microneedles from the vitelline arteries of normal 3-day embryos and embryos exposed
to 6 % O2 for 6 h. From the latter group the blood was drawn within 1 h
following treatment. All samples were then placed in glass capillaries and spun
in a Micro-hematocrit centrifuge for a standard length of time. Relative volumes
of serum and packed cells were recorded. The rate of heart beat was measured
by tapping a blood cell counter in synchrony with the heart.
RESULTS
Observations on normal embryos
Since the 18 h period of observation is one of considerable growth and
morphogenesis, the development of treated embryos must be carefully compared with that of controls. Embryos would normally progress by 1-1-5
Hamburger & Hamilton (1951) stages in the first 13 h of observation. Further,
the measurements of embryos at stage 17 at the onset of observations were
significantly different than those of stage 18 at the start (Tables 1,2).
Two major changes observed in the neural tube were a gradual increase in
size and progressive development of cephalic flexure. Progress of cephalic
flexure could be followed by measuring the angle between the head and trunk.
In embryos at stage 17 this angle is approximately 100-110°; at stage 18 it is
85-90° and becomes reduced to 60-70° by stage 19 (Text-fig. 2). In the photographs numerous changes in the circulatory system could be followed, such as
the rapid development of the capillaries of the anterior cardinal system and
steady increases in size of the aorta, anterior cardinal vein, and other embryonic
and extra-embryonic blood vessels (Table 2). In the aortic arch region the
PLATE 2
Time sequence photos of a chick embryo exposed to 6 % O2 for 6 h and then returned to air.
Photos taken at (A) start of experiment, (B) 6 h, (C) 9J h, (D) 12 h. See text for explanation
(embryo 'B', p. 356). Note particularly the large changes between (A) and (B), and compare
with Plate 1, figs. A and B. Note also the development of the tail hematoma in (C) and
extra-embryonic bleeding in (D).
23-2
350
C. T. GRABOWSKI & R. E. SCHROEDER
appearance of the fourth arch and, somewhat later, disappearance of the second,
could be followed with ease (Text-fig. 1 and Plate 1). Other structures that
exhibited changes during this period of observation were the heart, limb buds,
and tail bud. The allantois became visible in the prints usually toward the end
of the third day.
An estimate of the embryo's growth was made by measurements of the crownrump length and of the area of the embryo on the prints. The area of all normal
embryos increased 25 % in the first 7 h and 43 % by 13 h (Table 1). Despite this
increase in embryo size, the crown-rump length actually decreased slowly, since
the progressive development of cephalic flexure gradually brings the brain
closer to the tail. The crown-rump length characteristically decreases in waves
(Text-fig. 2).
Table 1. Average changes in total area (in sq. mm) of normal
and hypoxia-treated embryos
13 h
7h
No. of
embryos
Normal
6 h a t 6% O2
4
8
Normal
6 h a t 6% O2
6
11
Start.
Area
(mm2)
/o
Area
Stage 17
14-3(1-5)*
17-8 (1-8)
14-6 (20)
22-1 (40)
17-2 (1-7)
180 (2-7)
0/
0/
Stage 18
21-5 (20)
25-4 (3-3)
/o
increase
Area
24
57
20-1 (1-6)
20-3 (3-6)
40
39
25
41
25-0 (2-4)
23-5 (3-5)
45
31
increase
* Figures in parentheses are standard deviations.
Effects of moderate hypoxia (6 h at 6 % O2) on 3-day embryos
The embryos in this group were exposed to 6 % oxygen either continuously
for 6 h or discontinuously, i.e. 4 h of hypoxia, 1 h of room air and another 2 h
of hypoxia. The effects of the discontinuous treatment tended to be somewhat
more pronounced and prolonged than those of the continuous treatment, but
since these differences were relatively minor, all the embryos in these two groups
were considered together. Good records on eight embryos at stage 17 at onset
of the experiment and on 11 embryos at stage 18 were obtained. Of these
19 embryos, six (32%) died within 24 h after the treatment was started; most
of the remainder survived to the fifth day when they were sacrificed. Some were
PLATE 3
Time sequence photos of an embryo exposed to 6 % O2 for 6 h (discontinuously). (A) Taken
at start of experiment ;(B) at 7 h ; (C)at 18 h. This is embryo ' C of p. 356. Note particularly
the increase in size of embryo and blood vessels, 'straightening', and hemorrhage over
forebrain in ' B ' and the hematoma in right leg bud in ' C
J. Embryol. exp. Morph., Vol. 19, Part 3
PLATE 3
C.T.GRABOWSKI& R. E. SCHROEDER
facing p. 350
Hypoxia-induced teratogenesis
351
allowed to live until the seventh or eighth day of development. Although a slight
degree of retardation in growth and development was apparent in the treated
embryos within the first 24 h, this difference usually was still not apparent by
the fifth day.
Changes in size and shape
A pronounced swelling of the entire embryo was a conspicuous and consistent
effect of the treatment. This was apparent not only on casual inspection of the
photographs (Plates 2 and 3), but also from measurements of area (Table 1).
The average increase in area of all 6 h treated embryos over the first 7 h of
observation was 41 or 57 % (according to age) compared to 25 % in the controls. By 13 h the experimental embryos were nearly normal in size. As measured
from prints, it is apparent that hypoxia produces a transient but significant
increase in the area of the embryo which, in turn, reflects a volume increase in
the embryo. This increase can be detected 30 min to 1 h after the treatment
begins. It usually reaches a maximum shortly after the treatment is terminated
and is followed by a gradual return to normal. Occasional embryos, however,
continued in the edematous state for as long as 5 or 10 h after termination of
treatment.
The crown-rump length of experimental embryos always increases approximately 10-15 % in the first 2 h of hypoxia, remains constant, and then starts to
diminish after the treatment is ended (Text-fig. 2). This increase, which is in
marked contrast to the steady decrease found in normal embryos, may be due
partly to the general size increase of treated embryos. To a greater extent, however, it is caused by transitory reversal of normal flexion movements, since
these changes in C.R. length are closely paralleled by changes in the angle between
the trunk and head. In the control embryos this angle gradually becomes reduced
as flexion progresses. In hypoxia-treated embryos, the angle increases by as
much as 10° during the first or second hour of treatment and normally does
not begin to decrease again until treatment is terminated (Text-fig. 2). This
' straightening' is also seen in the photographs (Plates 2 and 3). In a few embryos
measurements of the area of the forebrain and mid-brain were made. In the
treated embryos this area increased or decreased along with the over-all increase
or decrease of the rest of the embryo, indicating that hypoxia also induces
swelling within the neural tube. Since the neural tube at this stage is a closed
system, it would seem likely that this transitory reversal of normal flexion movements is due to an increase in turgidity within the distended neural tube. Whether
this temporary interference with a normal morphogenetic movement can have
any permanent effect has not yet been ascertained.
Effects of hypoxia on the circulatory system
The most conspicuous feature of these observations was the dramatic increase
in the size of the heart and diameter of the embryonic blood vessels (Text-fig. 1;
Plates 2 and 3). Although the over-all size increase in hypoxia-treated embryos
352
C. T. GRABOWSKI & R. E. SCHROEDER
was only 16-33% above normal, the major embryonic blood vessels always
increased to double, triple, or in some individual cases, even quadruple their
initial diameter. Extra-embryonic vessels were only slightly affected. These
increases were first detectable 0-5-1 h after treatment was started and continued
for the duration of exposure to hypoxia. Return to 21 % oxygen usually, though
not always, started a gradual but irregular return to normal diameter (Text-fig. 2).
Table 2. Average changes in blood vessel diameter in normal
and hypoxia-treated embryos
All dimensions in mm
No.
of
Conditions cases
Normal
4
6h, 6% O2
8
8h, 6%O 2
6
Normal
6
6h,6%0 2
11
Third aortic: arch
Aorta
7h
0
0-23
(0009) *
0-24
(0051)
0-21
(0036)
13 h
0
7h
13 h
Anterior cardinal vein
0
7h
13h
Stage 17
0-37
010
015
012
0-29
019
017
019
(0041) (0038) (0014) (0028) (0013) (0022) (0025) (0019)
0-35
0-44
008
0-23
015
010
0-22
0-31
(0068) (0103) (0030) (0046) (0042) (0030) (0132) (0123)
0-33
007
018
018
0-21
008
0-34
0-21
(0037) (0083) (0028) (0029) (0063) (0019) (0050) (0126)
Stage 18
0-45
013
019
019
0-21
0-38
0-25
0-33
013
(0049) (0059) (0044) (0-007) (0004) (0018) (0016) (0028) (0036)
012
0-38
0-29
017
012
0-29
0-47
0-32
0-23
(0032) (0055) (0029) (0023) (0038) (0044) (0025) (0045) (0030)
Figures in parentheses are standard deviations.
The aorta, third aortic arch, and the anterior cardinal vein in normal embryos
increase in diameter approximately 25 % in the first 7 h of observation, and
another 25 % by 13 h. On the other hand, the average increase of the aorta in
the hypoxia-treated embryos was 73 % in the first 7 h (Table 2). In some individuals the aorta increased as much as 2\ times its original diameter. The caudal
aortae, which are clearly seen in the photographs, showed proportional increases
(Plates 2 and 3). By 13 h these vessels usually returned to nearly normal diameter
(Table 2).
The third aortic arch is relatively stable in contrast to the second arch, which
normally disappears. It is a small artery compared to the aorta, but displays
the same pattern, i.e. a maximum increase at 7 h and a return to normal diameter
at 13 h (Table 2; Text-fig. 1). The average increase of this vessel over the first
7 h was from 2\ to 3 times its initial diameter.
The anterior cardinal vein is very sensitive to hypoxia, especially its large
sinus dorsolateral to the aorta. The average increase in this vessel was 3 times
normal and, in some individuals, this vein increased fivefold over the first 7 h
Hypoxia-induced teratogenesis
353
of observation (Table 2). The capillaries of the anterior cardinal system also
became engorged (Plate 3).
The extra-embryonic vessels on the other hand, did not follow the same
pattern of change as the embryonic vessels. The major vitelline arteries and veins
showed a slight to moderate degree of distention, but accurate measurements
were seldom possible because these vessels overlap each other. The best measurements were made on the relatively small anterior vitelline vein, and this vessel
Text-fig. 1. Hypoxia-induced enlargement of heart and adjacent blood vessels.
Tracings taken from time-sequence photographs of hearts at same stage of beat,
i.e. atrium contracted, ventricle just starting to contract andfillingthe truncus arteriosus. All three embryos at stage 17 at start of observations. Embryo A is a control;
embryos B and C were exposed to 6 % O2 for 6 h. Note enlargement of all parts
in treated embryos still evident at 9 h, 3 h after return to air. In embryo B the heart
has returned to near-normal size by 13 h. Note also that aortic arch 4 is not open in
the first drawing of each series, but is present by 7 h. Aortic arch 2 shows signs of
closing by 13 h. Abbreviations: v.v., vitelline vein; v., ventricle; t.a., truncus
arteriosus; 2, 3, 4, aortic arches; atrium is hidden behind ventricle.
showed no significant change in diameter during hypoxia beyond that found in
the controls. Perhaps the thicker, and presumably stronger, walls of the extraembryonic vessels help to prevent their distention. However, the embryonic
(terminal) portion of the vitelline vein showed considerable distention (Text-fig. 1).
The rapid increases in blood vessel size, which are not accompanied by any
corresponding decrease in size of other vessels, demonstrate that exposure to
354
C. T. GRABOWSKI & R. E. SCHROEDER
moderate hypoxia produces a sharp increase in blood volume (hypervolemia).
This was further checked by measuring changes in hematocrit. In normal 3-day
embryos, the blood cells composed an average of 22 % of the total volume of
blood (8 determinations, range 20-0-26-6 %). In blood samples obtained from
embryos that had just been exposed to 6 % O 2 for 6 h, this hematocrit value had
l
10
12
14
l
16
18
Time (h)
Text-fig. 2. Effects of moderate hypoxia (6% O2) on c.R. length and diameters of
aorta and anterior cardinal vein (A.C.V.) of 3-day chick embryos. Embryos A
(control) and C were at Hamburger & Hamilton stage 18 at start of study, embryo
B at stage 17. Photographs taken every 30 min. All measurements made on x 15
prints. The figures shown associated with the C.R. length curves are measurements
of the angle between head and trunk. See text for details. Photographs of embryo B
are shown in Plate 2, of embryo C in Plate 3.
, A;
, B;
, C; ©, hypoxia
started; A, return to air; H, hematoma; E, exsanguination.
decreased to an average of 17% (9 determinations, range 15-7-22-0%). Assuming that the number and size of red blood cells has remained constant in
the embryo during the 6 h of treatment, the fluid portion of the blood would
Hypoxia-induced
teratogenesis
355
have to increase by at least 37% to account for this increase in the relative
volume of red blood cells. (See Grabowski, 1966, p. 202, for explanation of
calculations. In that previous study a plasma volume increase of 60 % was found
in 5-day embryos exposed to 10 % O2 for 5 h.) It should be noted that extraembryonic vessels expand very little, so that most of this increase in blood
volume would be contained within the embryonic blood vessels. This hypoxiainduced hypervolemia is a reversible effect since return to 21 % oxygen usually
starts a gradual return to normal blood vessel diameter, and, presumably, to
normal blood volume.
Hemorrhage was the most common visible result of exposure to hypoxia.
Of the embryos exposed to 6 h of hypoxia, 8 out of 19 bled to some significant
degree. Typically, hemorrhage does not begin until several hours after termination of treatment (Text-fig. 2). The most common site of bleeding in the embryo
was from capillaries of the anterior cardinal vein in the brain region (Plate 3).
Almost as frequent was the occurrence of bleeding in and near the tail and
extremities, where the escaped blood usually formed distinct hematomas (Plates
2 and 3). Such hematomas are teratogenically important since, if they persist,
they can lead to the abnormal development of adjacent structures (GluecksohnSchoenheimer, 1945; Jost, 1951; Giroud, Lefebvres, Prost & Dupuis, 1955;
Grabowski, 1964). Some of the hematomas formed abruptly and remained
constant in size (Plate 2, fig. C); others gradually increased in size (Plate 3). Not
infrequently, hematomas and areas of diffuse bleeding were reabsorbed. Of the
19 embryos treated for 6 h, five developed extensive bleeding at embryonic or
extra-embryonic sites, resulting in exsanguination and death (Plate 2, fig. D).
Such bleeding was the most common cause of death following exposure to
moderate hypoxia.
Conspicuous increases in the size of the heart accompanied the blood vessel
changes in treated embryos. This change is shown in the silhouette tracings of
Text-fig. 1 in which several hearts are shown at the same stage of contraction.
Exposure to hypoxia also affects the rate of heart beat. The normal rate in 3-day
embryos is 164 beats/min (12 cases, s.D. 19-4). After 1 h of hypoxia, the rate in
these same embryos decreased to 126 beats/min. (s.D. 11-9; decrease significant
at 1 % level, /-test). During the last 3 h of treatment, the average rate was 121
beats/min (s.D. 20-9) and in some embryos it dropped to 80/min. On return to
2 1 % O2 the rate immediately increased in every embryo, and after £ h in
a normal atmosphere the rate increased to 174/min (s.D. 24-7). During treatment
the rate of heart beat in some embryos became erratic, alternating between
periods of fast and slow rates. Cardiac arrest sometimes occurred during the
last 2 h of treatment, usually followed after 5-30 s by spontaneous recovery.
Atrio-ventricular arhythmias (e.g. three beats of atrium to one of the ventricle)
were occasionally observed during treatment. It is evident that the heart is
profoundly affected in several different ways by exposure to moderate hypoxia.
356
C. T. GRABOWSKI & R. E. SCHROEDER
Specific case histories
The foregoing generalizations can be illustrated by two case histories. Embryo
B, solid line in Text-fig. 2 (see also Plate 2), was exposed to 6 % O2 for 6 h.
A rapid increase in C.R. length accompanied the 5° increase in the angle between
head and trunk. After a 45 min lag, the size of the anterior cardinal vein gradually increased 2\ times, the aorta almost twofold (Plate 2, fig. B). Blood vessel
diameter, C.R. length, and the angle between head and trunk all began to decrease
as soon as the embryo was returned to 21 % oxygen, indicating a restoration of
the homeostatic mechanisms for maintenance of fluid balance. However, the
decline in blood vessel diameter was irregular and at %\ h a moderately large
(0-5 mm) hematoma abruptly appeared in the tail (Plate 2, fig. C). At l l ^ h
a large extra-embryonic vessel ruptured and rapid exsanguination occurred
(Plate 2, fig. D). Although most blood vessels collapsed at this time, the tail
hematoma persisted.
Embryo C, the dotted line in Text-fig. 2 (see also Plate 3), was exposed to
6 % oxygen for 4 h, returned to air for 1 h, and then exposed to hypoxia for 2 h.
The parameters measured reflected this pattern inasmuch as they showed a slight
dip after 4 h and then continued to increase again after return to hypoxia.
However, the increases continued for several hours after the second treatment
was concluded, and return to normal size did not begin until 10 h after observation had begun. At this time the anterior cardinal vein was 4 times larger than at
the start of the experiment and the aorta 2\ times larger (Plate 3,fig.B). Bleeding
over the forebrain occurred at 6 h (Plate 3, fig. B) and over the midbrain at
13 h. Small hematomas appeared simultaneously in the right wing and leg buds
at lOf h. Unlike the tail hematoma in embryo B, which appeared abruptly, these
started small and gradually increased in size. The wing hematoma was resorbed
by 18 h, but the leg hematoma at that time was 0-35 mm in diameter (Plate 3,
fig. C). The embryo died a few hours later.
Effects of various other degrees of hypoxia on the chick embryo
Fifteen other cases were studied. These included five embryos exposed to
6 % oxygen for 8 h, six exposed to 4 % oxygen for 6 h, and four embryos
exposed to pure nitrogen for 4 h. Of the embryos exposed to 6 % oxygen for 8 h,
two died at 8 and 9 h from extensive extra-embryonic bleeding. The other four
survived without any apparent ill effects. In general, the distention of the embryo
and blood vessels was comparable to, but somewhat less pronounced than, that
of embryos exposed for 6 h to 6 % oxygen (Table 2). One embryo of this series
deserves special mention. In this specimen the over-all size increase was maintained for 7 h after the treatment ceased. Blood vessel distention persisted and
the vessels continued to increase in diameter up to 15 h. For example, the anterior
cardinal vein showed dimensions of 0-07 mm at the start, 0-27 mm at 7 h and
0-40 mm at 13 h. Similarly, the third aortic arch showed measurements of,
Hypoxia-induced teratogenesis
357
respectively, 0-10, 0-20 and 0-27 mm. At 15 h there were numerous small hemorrhages over the surface of the embryo. The smaller blood vessels had irregular
outlines and the blood within them was dark in appearance, more characteristic
of a dead rather than a living embryo. However, by the next morning, apparently
complete recovery had occurred, and the embryo lived until day 7 when it was
sacrificed and examined. No anomalies were apparent. This embryo and several
similar, though less dramatic, cases vividly illustrate the recovery power of
embryos exposed to a normally teratogenic situation.
Four of the six embryos exposed to 4 % oxygen for 6 h died, three from
extensive hemorrhage. The two survivors of the treatment showed no apparent
ill effects. For the most part, only moderate swelling of the embryo and its
blood vessels occurred. In one specimen, however, the anterior cardinal vein
expanded to 5^ times its normal width by the end of the treatment.
Four embryos were exposed to pure nitrogen for 4 h. Three of these died
within the first 2 h of treatment, all by exsanguination caused by embryonic or
extra-embryonic bleeding. This bleeding was preceded by only slight or moderate
swelling of the blood vessels. The single survivor of this series showed moderate
increase in blood vessel size, i.e. a twofold increase in the size of the anterior
cardinal vein. The C.R. length increased from 95 to 115 mm at 4 h and then began
to decrease. This embryo lived for 30 h after the treatment then died without
any obvious cause.
These experiments showed that maximum swelling of the 3-day embryo and
its blood vessels was usually achieved with an exposure of 6 h to 6% oxygen.
Exposure to more severe conditions of hypoxia can precipitate a variable
amount of swelling, embryonic bleeding, and death.
DISCUSSION
On the basis of the changes induced by moderate hypoxia on the chemistry
of the blood plasma and a consideration of the osmotic relationships of the
embryo to its surrounding environments, Grabowski (1966) concluded that:
(1) the young chick embryo has an osmoregulatory problem, and (2) exposure
to moderate hypoxia interferes with the osmoregulatory activity of the embryo,
producing swelling and concomitant ionic changes in the blood stream. The
teratological consequences of these fluid imbalances have been described to some
extent and the entire sequence referred to as 'the edema syndrome' (Grabowski,
1964). The present study illustrates the initial stages of this edema syndrome on
living specimens. Also revealed are some features previously unsuspected, such
as turgidity effects on flexion, and the extent to which the embryonic heart and
blood vessels are affected.
Since the major embryonic vessels increase in diameter from two- to threefold,
it is apparent that the volume of blood must also increase several-fold during
exposure to hypoxia. Hematocrit data support this conclusion. This extreme
hypervolemia may be highly significant to the development of the embryo, since
358
C. T. GRABOWSKI & R. E. SCHROEDER
the cardiovascular system is being molded at this time and hemodynamic factors
are generally considered of primary importance to the process (Jaffee, 1965,
1966; Rychter, 1962). Some vascular anomalies following hypoxia have been
reported (Tedeschi & Ingalls, 1956; Grabowski & Paar, 1958) but these are
difficult to detect unless specifically sought, although the present study suggests
they may be more numerous than heretofore suspected. We have examined our
photographs to see if any abnormal changes in vascular pattern could be
detected as a result of the distention of these vessels. The second aortic arch,
which normally disappears during the period under observation (Text-fig. 1),
was studied to see if the hypoxia-induced distention might have delayed its
closure. Careful scrutiny of many records suggests that in some cases closure
of this arch may be delayed by as much as 2 h. But, at best, this is a slight effect
difficult to establish. In embryos exposed to hypoxia at stages 18 and 19, closure
of the second arch occurred even while this vessel was distended 2 to 3 times
normal size. In such cases the closure was more abrupt than in control embryos.
Even though this particular attempt to find a permanent effect of hypoxiainduced distention on a blood vessel was not successful, a combination of
hypoxia and time-lapse photography may prove useful in studying the significance of hemodynamic factors in the moulding of other parts of the cardiovascular system.
Persistent hematomas in embryonic tissues can cause abnormal development
in adjacent structures (Gluecksohn-Schoenheimer, 1945; Jost, 1951; Waddington & Carter, 1953; Giroud et al 1955; Grabowski, 1964). The initial objective
of these time-lapse studies was to determine, if possible, how and why these
hematomas form as a consequence of hypoxia. It seems reasonable to assume
that the hemorrhage is a result of the distention of the blood vessels beyond
their elastic limits, but the correlation is not precise. Some embryos with great
enlargement of blood vessels seemed to recover completely, and other embryos
with only moderately distended vessels bled to death. It is apparent that more
than simple distention of vessels is involved in the rupturing of blood vessels.
One possibility is that lack of oxygen may cause degeneration of the vascular
epithelium. The high incidence of hemorrhage, preceded by only moderate
swelling, in embryos exposed to pure nitrogen would support this notion.
Another clue may come from the puzzling observation that most hemorrhage
occurs several hours after treatment (Text-fig. 2; see also Grabowski, 1964). It
usually occurs not when blood vessels are maximally distended, but as they are
returning to normal size. A possible explanation of this phenomenon is emerging
from blood-pressure studies. Toben (1967) measured the mean ventricular blood
pressure in normal 5-day chick embryos and in embryos exposed to 10%
oxygen for 5h. The normal level was equivalent to 20-1 mm of water. An
increased blood pressure (to 22-5 mm water) was noted immediately following
treatment and a return to normal levels 5 h afterward. But the maximum increase
(to 25-2 mm of water) did not occur until 2 h following treatment. We have
Hypoxia-induced teratogenesis
359
recently measured the mean ventricular blood pressure in 3-day chicks. Moderate
hypoxia (6 h at 6 %) increases the blood pressure from a normal level of 11-6 mm
of water to an average of 16-5 mm during the second hour after treatment, with
the pressure in some embryos reaching 25-27 mm of water. The relationship
between the tension (T) in a blood vessel wall, the radius (r) of the vessel, and
the pressure (P) within it, is expressed by the formula of Laplace (1841):
T = Pxr. Since both P and r can increase twofold in some embryos during
exposure to moderate hypoxia, the tension within the vessel walls can increase
fourfold. It is possible, therefore, that all three factors, namely, (1) blood vessel
distention, (2) degenerative effects on blood vessel walls, and (3) increased blood
pressure, play a combined role in this teratogenically important process of
hemorrhage and hematoma formation.
The various ways in which hypoxia affects the embryonic heart—distention,
changes in rate of contraction, arhythmias, and arrest—are interesting from the
physiological standpoint. Some of these effects are probably related to the
increased levels of serum potassium, another consequence of exposure to hypoxia
(Grabowski, 1963, 1966). The gross distention of the heart (Text-fig. 1) during
a period of rapid morphogenesis also raises the question of whether or not there
may be permanent effects on heart structure.
The rapid increase in the volume of the neural tube has been correlated with
a temporary reversal of normal flexion movements. These could be the result of
an increase in the volume of cerebrospinal fluid. Were such an increase to persist,
it could theoretically lead to a hydrocephalus-like condition. Such distended
neural tubes may be caused by other agents that produce swelling in embryos,
such as trypan blue in the mouse (Waddington & Carter, 1953; Turbow, 1966).
It is also feasible that this distention of the neural tube could lead to its rupturing
just as distention sometimes leads to rupture in blood vessels. On three occasions
in this study a sudden reduction in the size of distended neural tubes was observed, particularly in the forebrain-midbrain areas. This sudden reduction
was clearly localized in the neural tube, and was not seen in other parts of the
embryo. Presumably it was caused by the rupture of the neural wall and loss of
cerebrospinal fluid, although this could not be established by subsequent
examination of the embryo. However, in a different experiment, an embryo
treated with dimethylsulfoxide (DMSO) at 4 days displayed a marked, persistent
distention of the neural tube. Thirty-six hours after treatment the embryo was
preserved and a jagged wound was visible on the left side of a deflated midbrain,
clearly a ruptured brain wall.
This study also illustrates another important aspect of embryonic life not
always considered in teratological studies, recuperative power. Despite manifestations of physiological and morphological stress in every treated embryo
(swelling, straightening of neural tube, distention of major blood vessels, hypervolemia), almost half of them apparently recovered completely by 24 h after the
start of the experiment.
360
C. T. GRABOWSKI & R. E. SCHROEDER
This study shows that the initial stages of teratogenic action can be examined
by a direct, continuous observation of embryos in their natural environment.
The modified time-lapse technique used here has several advantages over conventional techniques based on the use of movie film, in that (1) detailed measurements on a variety of structures are considerably easier to obtain, (2) the cost
of film and equipment is a fraction of that needed for conventional techniques,
and (3) in the low magnification range, the precision lenses commercially available for 35 mm cameras are easier to use than low power photomicrographic
equipment.
SUMMARY
1. The visible reactions of 3-day chick embryos (in ovo) to moderate hypoxia
(mostly 6 % O2 for 6 h) were quantitatively studied, utilizing a modified timelapse technique. These observations were then correlated with the known teratogenic and lethal effects of this treatment.
2. Considerable swelling of the entire embryo occurs during treatment,
followed by a gradual, but irregular, return to normal size over several hours
after return to 21 % O2. Swelling of the neural tube also occurs and apparently
the increased turgidity within the tube results in a temporary reversal of normal
flexure movements.
3. The major embryonic blood vessels as well as the heart itself become
reversibly distended 2-3 times normal size. This distention suggests the development of a transient increase in blood volume, which was confirmed by a study
of changes in hematocrit. An apparent result of blood vessel distention is their
rupturing which, in turn, leads to the development of teratogenically significant
hematomas and, sometimes, death by exsanguination. Possible effects of this
hemodynamic disturbance on blood vessel formation and heart development
are considered.
4. Despite manifestations of physiological and morphological stress in all
treated embryos (swelling, hypervolemia, reversal of cephalic flexure, etc.) about
hah0 of them recover within 24 h after the experiment, illustrating the recuperative
capacity of embryos exposed to a potentially teratogenic treatment.
RESUME
Etude sur la sequence photographique d'embryons de poulet soumis aux doses
teratogenes de Vhypoxie.
1. Les auteurs etudient quantitativement les effets visibles provoques par une
hypoxie moderee (6 % O2 pendant 6 h dans la plupart des cas) sur des embryons
de poulet ages de 3 jours, in ovo, a l'aide d'une technique modifiee de sequence
photographique. Us comparent ces observations avec les effets teratogenes et
letaux connus, obtenus apres ce traitement.
2. On observe un gonflement de l'embryon entier pendant le traitement;
Hypoxia-induced teratogenesis
361
plusieurs heures apres retour a 21 % d'oxygene, l'embryon reprend progressiveraent sa taille normale. On constate egalement un gonflement du tube nerveux;
la turgescence accrue a l'interieur du tube resulte d'une inversion temporaire
des mouvements normaux de flexion.
3. La plupart des vaisseaux sanguins ainsi que le coeur se dilatent de maniere
reversible et atteignent 2 a 3 fois leur taille normale. Cette dilatation suggere
une augmentation transitoire du volume sanguin; cette hypothese est confirmee
par l'etude a l'hematocrite des modifications visibles. La dilatation des vaisseaux
provoque leur rupture, cause de l'apparition d'hematomes teratologiques significatifs et parfois de la mort par exsanguination. On envisage les effets possibles
de cette alteration hemodynamique sur la formation des vaisseaux sanguins et
le developpement du coeur.
4. Malgre les manifestations d'agressions morphologiques ou physiologiques
observees chez tous les embryons traites (gonflement, augmentation du volume
sanguin, inversion de la flexion cephalique), la moitie d'entre eux recuperent
durant les 24 h qui suivent le traitement. Ces resultats montrent la capacite de
recuperation des embryons soumis a un traitement potentiellement teratogene.
This work was generously supported by grant HD 00641 from the National Institute of
Health, National Institute of Child Health and Human Development. We gratefully acknowledge the technical assistance of Miss M. Milan, Mrs J. S. Bennett, and the'night shift' of
J. Browne and N. Chernoff.
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