The Blood Pressure of the Chick Embryo During Development

This investigation was made as part of a wider inquiry into the developmental physiology of chick circulation in relation to the histology of the developing vascular system.

One observation on the blood pressure of the chick embryo has already been made by Hill & Azuma (1927) who measured the pressure in the arteries of the chick blastoderm after 2 days’ incubation and found it to be $212$ cm. of water.

Their method, like that used in the present work, is a sphygmomanometric one. To the blastoderm freed from its surroundings they applied an external air pressure, transmitted through an elastic transparent membrane thereby compressing the blastoderm against a glass plate ; the least external pressure necessary to collapse the arteries was then measured.

Hill & Azuma’s method as it stands cannot be readily applied to later stages of incubation because of the difficulty of freeing the larger area vasculosa of older embryos from yolk and white without injury.

The method described below has been developed for measurements on the arteries of the chorio-allantois and does not involve interference with the yolk sac. It can only be used, however, when the chorio-allantois has reached a certain size which prohibits measurements earlier than the sixth day of incubation.

Thirty-three successful measurements have been made oh embryos of 6–19 days’ incubation, the chorio-allantoic arteries ranging in diameter from 0·25 to 0·75 mm. The final curve of arterial pressure during development (Fig. 4) includes the value for the 2-day chick obtained by Hill & Azuma.

The artery in which the blood pressure is to be measured is compressed between a glass probe, inserted through an incision in the vascular membrane, and a capsule, 2 cm. in diameter, to which air is admitted under pressure and of which the floor is formed by a membrane of thin rubber with a hole in it (Fig. 1). This hole is smaller in diameter than the glass probe. The hole and probe are placed concentrically when measurements are made, and the vertical distance between them is adjusted with great care. If they are too close, the artery is compressed without any air pressure being applied to the chamber, but if they are too far apart, bubbles of air escape.^* The artery is compressed directly by the air in the chamber.

If a continuous rubber membrane was employed, any curvature imparted to it would introduce an error into the measurements, for the pressure on the two sides of the membrane would not be the same, and it would be almost impossible to use the device with no curvature of the membrane. The errors thus introduced would be serious as the pressures to be measured are small in comparison with those usually measured by sphygmomanometric methods. The earliest trials of this apparatus were made with continuous membranes and although the thinnest rubber sheeting obtainable was used, no uniform readings could be obtained.

The probe and the brass tube attached to the capsule are both carried by special, micro-manipulator heads, which give a coarse movement over a wide range in all three dimensions. In the upper end of the glass probe a 6 V. ‘flash-lamp’ bulb is mounted The probe thus serves as a glass rod illuminator, and the artery is clearly seen under a low-power microscope through the hole in the rubber membrane, and the cover-slip forming the roof of the pressure capsule. The inside of this cover-slip needs moistening with glycerine to prevent condensation.

The vessel containing the saline, in which egg and device are immersed, has a capacity of about 250 c.c. and is heated by a compact form of water-bath, controlled to give the necessary temperature of 38–39° C.

The procedure is as follows. A hole is made in the air space of the egg which is then immersed in the warm saline, and with extreme care the shell is gradually picked off, using an outward motion. When most of the shell is removed, the shell membrane is peeled off, with even greater care. The last third of shell and shell membrane can usually be floated away from their contents. The whole chorio-allantoic membrane is then exposed, covering nearly all the yolk sac and albumen. In stages up to 12 days or so, it is advisable to free the chorio-allantois from the yolk sac by careful tearing at the edge of the former where the two embryonic membranes are attached to each other. When the operation is successful, a preparation can be made of the embryo in its amnion with yolk sac and chorio-allantois intact on each side.

Next, an accessible chorio-allantoic artery is selected, and a small incision made in the membrane near it, through which the probe is inserted. The capsule is cautiously lowered, and the artery made to lie suitably across the device, by gently pulling on the membrane. The probe and membrane are brought near together to compress the artery, and air pressure is admitted to the capsule. Probe and membrane are now separated until air bubbles, are on the point of escaping from the hole in the membrane, and the air pressure is varied until the artery can be compressed by this agency alone. The pressure is then adjusted until a point midway between systolic and diastolic pressure is found, so that the blood pulsates across the hole from the side nearest the heart with each heart beat, being squeezed out during diastole. Separate measurements for systolic and diastolic pressures are not attempted, as only in this intermediate position is a clear end-point obtainable. The air pressures applied are measured on a water manometer, and pressure and time of observation are noted down over a period of 10–30 min. The arterial pressure gradually falls as the preparation deteriorates (Fig. 2). Up to the tenth day of incubation, the fall in arterial pressure with time under observation is very gradual. This is due to the extreme ease with which eggs of this age can be decanted, as described above, with almost no haemorrhage and with the chorio-allantoic circulation almost unaffected. In the second half of the incubation period, however, increasingly more damage is done by this treatment. Since the chorio-allantoic capillaries go into stasis with the slightest mechanical stimulus and haemorrhage is unavoidable with any manipulation at all, conditions for the measurements of the arterial pressure become less favourable, as the eggs become older. This is reflected in the curves of Fig. 2 in which the arterial pressure in embryos of different ages is plotted against time of observation; the curve at 17 days is much steeper than at earlier stages. Fig. 3 expresses the slope of these curves for twelve sets of observations, and it is seen that they usually, though not always, become much steeper in later stages.

The question thus arises as to how our final estimate of the arterial pressure at each stage is to be made. Up to 10 days of incubation, a simple average of all values obtained in each set of observations is clearly sufficient, but in later stages this gives merely an average value of the arterial pressure of embryos dying from anoxemia. It seems more reasonable to plot at each stage the curve of decrease in arterial pressure with time of observation where the constituent points can obviously be represented by a straight line and to read off from this line the estimated arterial pressure at the beginning of observation. In Fig. 4 the circles express these estimated arterial pressures, and to them most weight has been given in drawing the final curve of arterial pressure against incubation time. The solid points in Fig. 4 represent the averaged values, and where estimated and averaged values are given for the same set of observations, the two points are joined by a vertical line. These lines express the divergence between averaged and estimated arterial pressures.

The arterial pressure in newly hatched chicks was measured by a similar method, except that a continuous rubber membrane and a mercury manometer were used. The chicks were anaesthetized with ether, and the common carotid arteries in the neck were exposed, an illuminated glass spatula was inserted between the arteries and neck muscles, and the capsule applied to the upper surface of the arteries. Six sets of observations were made on chicks in the first week after hatching and others at later stages. The slope of the curve for stages after hatching in Fig. 4 is taken from these observations.

It is proposed to discuss these results at length in a subsequent paper, together with other data from the embryonic circulation. Meanwhile the data on arterial pressure in the chick embryo can be compared with the corresponding results from mammalian foetuses which have been obtained by various authors, notably by Barcroft (1935, 1936).

Arterial pressure for the sheep (Barcroft, 1936) plotted against foetal age gives a curve of the same general shape as mine for the chick embryo, rising, at first gradually, then progressively more steeply. The time scales are respectively 150 and 21 days. It is interesting to compare the sheep and chick embryos when their size and arterial pressure are about the same. The sheep foetus at 44 days and the chick embryos at $1412$ days both have the same arterial pressure (18 mm. mercury) and weight (10 g., Needham, 1931). The arterial pressure of the chick embryo is rising very rapidly at that stage, while that of the sheep, embryo is still in the initial slow rise. The chick embryo also is developmentally much further advanced than the sheep embryo of the same weight.

A further resemblance can be seen between the curves for arterial pressure during development in chick and sheep embryos. In both the rapid rise in arterial pressure in the latter half of development is followed by a period of stationary pressure just before birth. Barcroft gives the arterial pressure of the sheep foetus at 140 days—10 days before term—at 76 mm. of mercury ; in the continuous tracing of arterial pressure during birth by Caesarian section given in the 1935 paper, the pressure at first is 63 mm. of mercury. In the chick embryo the pressure rises very little after 16 days.

Again, at birth, when pulmonary respiration is established, the arterial pressure in both animals rises to a figure which continues the preceding rapid rise, interrupted towards the end of embryonic life. In the sheep foetus the rise in arterial pressure at birth is about 20%, but in the chick embryo the rise appears to be larger and in the data of Fig. 4 lies between 34 and 100%.

The stationary period in arterial pressure at the end of development in the chick embryo is a very interesting problem in developmental physiology. The reason for it certainly does not lie in the heart itself, the weight of which continues to increase at the end of development as rapidly as before and doubles between 16 and 21 days (Olivo, 1930).

On opening an egg at the end of the third week of incubation, one is struck by the extreme darkness of the blood in the chorio-allantoic vessels. The blood in the arteries appears fully reduced and that in the veins far from saturation with oxygen. Unfortunately, no measurements appear to have been made on the oxygen content of the blood of the chick embryo at this stage, a problem which becomes all the more interesting in view of the investigations on mammalian foetuses by Barcroft and co-workers. Appearances, however, suggest that the chick embryo at the end of development is in a state of anoxaemia so far advanced that the action of the heart may be adversely affected.