Paper - The effect of the heart-beat upon the development of the vascular system in the chick (1918)
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Chapman WB. The effect of the heart-beat upon the development of the vascular system in the chick. (1918) Amer. J Anat. 23: 175.
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The Effect of the Heart-beat upon the Development of the Vascular System in the Chick
W. B. Chapman
Anatomical Laboratory of the University of Missouri
At the time the circulation begins in the chick, the embryo possesses a number of relatively large blood vessels. Thoma ('93) mentions that the paired dorsal aortae are present, and in a schematic drawing (p. 32) shows them connecting, anteriorly, through capillaries with the heart, and, posteriorly, extending into the capillary net of the extra-embryonic region.
Miss Sabin ('17) in a monograph published recently, states that, in addition to the dorsal aortae, the duct of Cuvier, the cardinals, and several neural vessels have formed. These are represented in plate 1, figures 2 and 3, and in plate 2, figures 1 and 2 of the monograph, which also show the primitive anterior vitelline veins. She describes the vessels of the extra-embryonic region as an extensive capillary plexus which connects with the heart and dorsal aortae of the embryo. Quoting in part, she says: " . . . there is a plexus of vessels covering the entire area opaca and area pellucida which connects with the venous end of the heart and with the entire dorsal aorta of the embryo opposite the zone of the myotomes." That the heartbeat has much to do with the further development of this primitive vascular system has been generally conceded, and Thoma has formulated his three well-known histo-mechanical laws, which, as translated into English by Bruce ('96, p. 266), are as follows: The increase in size of the lumen of the vessel, or what is the same thing, the increase in surface of the vessel wall, depends upon the rate of the blood current." "The growth in thickness pi the vessel wall is dependent upon its tension. Further, the tension of the wall is dependent upon the diameter of the lumen of the yessel and upon the blood-pressure." "Increase of the blood-pressure in the capillary areas leads to new formation of capillaries." In order to test the validity of this view, it was decided to remove the heart by surgical methods before the establishment of a circulation, thereby eliminating most of the mechanical factors mentioned by Thoma, and then to study the further development of the vascular system in the area vasculosa.
II. Review of Literature
Roux ('78) briefly mentions seeing an area vasculosa of a fiveday chick in which the embryo was missing. He describes a simple capillary plexus with a mesh-like arrangement, and without formation of arteries and veins. In a note added later ('95), he mentions seeing further cases of chick embryos in which the embryo had failed to develop, but in which the sinus teminalis had formed. He refers also, in a very indefinite way, to the presence in these embryos of large vessels which corresponded somewhat in position and in direction to the normal arteries and veins. He gives no illustrations.
Loeb ('93) tested the effect of solutions of potassium chloride on Fundulus eggs, and observed that the living embryos developed without a heart-beat, the heart being thrown into a state of tetanus by the potassium. In spite of this some vessels were formed. This work was later confirmed, in a general way, by Stockard ('06), although he states that the vascular development was far from normal.
Knower ('07) studied the effects of the early removal of the heart and arrest of the circulation on the development of frog embryos. In this experiment the heart was removed by cutting. Some of the embryos lived and continued to develop for as long as fourteen days. Many of the main blood vessels developed, although they were irregularly distended and abnormal.
Patterson ('09) studied the development of the extra-embryonic vascular system in chicks in which the embryo had been prevented from forming by making injuries on the unincubated blastoderm. He states (pp. 87-88), although there is not the slightest trace of the embryo proper present, yet the vascular system of the area opaca is well laid down, and even large vessels are seen to pass inwards to the center of the pellucid area." This is illustrated in figure 7, p. 89, of his article.
Stockard ('15) made extensive studies, using chemical methods somewhat similar to J. Loeb's on Fundulus embryos. He found that in embryos in which there had been no heart-beat, the aorta developed into a vessel of considerable size, and he mentions that other vessels had also developed. He pictures only a crosssection of the aorta, however, and his reference to the other vessels is very meager.
It seems clear, then, from the observations of Thoma, and Sabin, that before the circulation of blood commences, a considerable development of the vascular system has taken place. This includes, in the embryo proper, the development of definite aortae, of short stretches of the two vitelline veins, of some of the dorsal segmental arteries, of part of the cardinal veins, and certain neural vessels in the head region, while, in the extraembryonic area, there is present an indifferent net-work of capillaries, to which we might add (Lillie, '08, pp. 87-88) the sinus terminalis. This much obviously develops as the result of hereditary influences. Soon after the circulation starts, certain other arteries and veins begin to be marked out, and extensive further changes take place within a few days.
The question now arises, whether this further development of the vascular system, which takes place after the circulation is established is dependent upon the mechanical factors concerned with the circulation or not. According to Thoma, it is, while the observations of Roux and of Patterson indicate that the sinus terminalis will develop in the absence of a heart-beat, and that possibly the same may be true of other yolk-sac vessels. The observations of Loeb, Knower, and Stockard indicate the development of some main vessels when no circulation is present, but do not give a complete picture. It therefore still remains to be determined, precisely, how much vascular development takes place independently of the circulation.
III. Method of Investigation
The chick was selected for this study on account of its rapid development, because it is possible to secure material for experimentation during most of the year, and because of the ease of injecting and studying the extra-embryonic vessels, which lie in a single plane, and which early develop a very characteristic pattern. After some experimentation, I decided upon the thirtythird hour of incubation as the proper time for operation, and afterwards, endeavored to arrange my operations so that they would come as nearly the hour mentioned as possible. At this time, the chick has about twelve somites. Miss Sabin has observed that the heart begins beating at the ten somite stage, but that the circulation does not start until after fifteen or sixteen somites have formed, which would mean about the thirty-eighth hour of incubation.' Therefore, any time between the thirtieth and thirty-eighth hour of incubation would have sufficed for accurate results in this experiment.
The technic employed in this work was suggested by Professor Clark and modified to suit the experiment. It is as follows:
The egg is taken from the incubator and placed in a warmbox. The shell is sterilized at the point to be opened by wiping with a small cloth saturated with alcohol. As soon as the alcohol has evaporated, a small hole is scraped through the shell with the point of a scalpel using care not to puncture the shell membrane.
If a drop of warm water or Locke's solution is dropped upon the opening at this time, it will help to loosen the membrane from the shell, and will also prevent to some extent, long cracks and the breaking off of large pieces of shell. The shell is removed over an area about one centimeter or less in diameter and the shell debris washed off with warm sterile Locke's solution. The shell membrane is carefully stripped back with the forceps and the blastoderm exposed and flooded with Locke's solution. If the embryo is eccentrically placed, it may be necessary to rotate the egg slightly or remove more of the shell. The heart, if not visible, may be brought into view by pulling on the covering membrane with the forceps and turning the embryo slightly to one side.
The next procedure is cutting a small opening through the vitelline membrane and lateral plate and, catching the heart with the forceps, pulling it well out from the embryo and severiDg its connections, both anteriorly and posteriorly, with the scalpel or scissors. For this work I have used knives made from dissecting needles or by grinding down old scalpels, but I have found the small iridectomy scissors much easier to handle and more reliable. After removing the heart, the embryo is again covered with the sterile Locke's solution warmed to about 100 degrees Fahrenheit, the hole in the shell covered with a piece of isinglas ., and sealed with a mixture of wax and resin (Rabaud). The egg is now returned to the incubator and allowed to develop further.
The operation is carried out in the warm-box at incubator temperature. If moderate aseptic precautions are observed there is little danger of infecting the embryo. The work is done under the binocular microscope, and is not difficult if the instruments are sharp, but if they are dull, especially the forceps, it is next to impossible to secure accurate results.
On several occasions, at stages of from sixteen to twenty-four hours incubation, I injured the embryo by burning with the cautery or by cutting sufficiently to prevent its further development. The extra-embryonic area, however, continued to grow in a manner similar to that in the experiments in which the heart itself was removed, and the changes in its blood-vessels were similar.
The chicks were incubated varying lengths of time from twenty-four hours up to and including ten days after the operation. I was unable to keep any of the chicks alive longer than nine days, that is, about eight days after the operation. The mortality for chicks up to the end of the ninth day of incubation was about fifty per cent. After the egg has been incubated as long as desired, it is again opened and the vessels injected with India ink. The specimen is fixed in Bouin's solution, which may be applied to the chick while on the egg, or, preferably, after the embryo has been removed and flattened out on a slide. The specimens are stained faintly and cleared in oil of wintergreen (Spalteholz method), or, if desired, mounted permanently in damar. After being studied, the embryo has, in some cases, been embedded and sectioned.
Before injecting, a careful search is always made for remnants of the heart or any pulsating blisters, such as I will describe a little further on in this paper.
IV. Description of the Extra-Embryonic Vascular System of Embryos Studied
The appearance of the embryo at the stage of operation is shown fairly well in figure 1, which represents an embryo slightly beyond the operative stage At this period the blood cells have not acquired their hemoglobin and no vessels are visible under the binocular. The embryo appears to be floating free within the clear area pellucida, and the area opaca is apparent only as a wide milky-white band around the area pellucida. The blood vessels in the embryo proper of chicks of this age have been studied by Miss Sabin (T7) through injectiods of India ink into the aorta and larger vessels, and her results have already been referred to.
As previously mentioned, and as shown in figure 1, the vessels of the extra-embryonic region consist of a dense capillary net extending from the embryo to the sinus terminalis at the time the circulation begins. Anteriorly, the sinus terminalis breaks up into a plexus of venous capillaries through which, at a later stage, the blood from the sinus is returned to the heart. This venous plexus gradually becomes denser and evolves into the primitive right and left anterior vitelline veins as the heart is approached. Near the posterior end of the embryo, each aorta breaks up into capillaries, which are continued outward onto the extra-embryonic area by a dense capillary meshwork, in which there is as yet, no vessel marked out, as the omphalo-mesenteric artery — which develops here soon after circulation commences. There are the well-known 'clear' areas, without blood capillaries, surrounding the head region, others on either side of the body of the embryo between the anterior vitelline yeins and the plexus in the omphalo-mesenteric region, in which, however, a single very narrow capillary is present in the specimen drawn, while there is a large oval non- vascular region including and surrounding the caudal undifferentiated end of the embryo. The capillaries bordering the clear areas next the body of the embryo are especially narrow, while near the border vein, particularly in the posterior portion of the area, they are very wide. This condition represents that immediately before the effects of circulation begin to be exerted, for, as stated in the note, circulation had already started on the other side, and a suggestion of the om phalo-mesenteric arteries could be made out there.
Fig. V Camera lucida drawing of an injected, normal chick of 35 hours incubation, 16 somites, cleared in oil of wintergreen, viewed from above. The left half of the area is shown. Enl. 12't X.
1 The chick shown in figure 1 was incubated 3.5 houi's, but in development has reached the stage usually seen in chicks of about 38 hours incubation. It is, therefore, slightly older than most of the chicks at the time of operation. The ink was injected into the sinus terminalis and forced through the capillary net, or, as I have termed it in the text, the venous plexus of capillaries anterior to the embryo, to the rudimentary vitelline veins. Ink was also introduced into the heart. The heart pumped the ink back and forth into the sinus and vitelline veins for several minutes, but soon particles of the ink began to shoot through the arches into the right dorsal aorta. This was followed quickly by a rapidly growing stream of ink which moved forward as propelled* by the pulsating heart and spread out into the capillary net at the termination of the dorsal aorta several
In normal development, soon after circulation commences, the omphalo-mesenteric arteries differentiate out of the capillary plexus, the anterior vitelline veins grow together anterior to the embryo, forming a single large vein, and, somewhat later, the posterior and omphalo-mesenteric veins develop. The fate of the border vein in normal embryos will be referred to later. These changes have been fully described by Thoma ('93) and Popoff C94).
Let us now turn to the fate of the vessels deprived of the action upon them of the factors concerned with the circulation.
minutes before granules of ink began to appear in the left aorta. In a number of embryos studied, the circulation was invariably established upon the right side first. It will be noted that the aortae pass immediately into the capillary plexus in the posterior portion of the embryo, and that no large branches have as yet been formed out upon the yolk-sac.
In all of the chicks operated upon, with the exception of the ones that failed to survive the operation, the vessels of the extraembryonic region were filled with red blood cells, which made them clearly discernible before the injection. These vessels, during the first two or three days after operation, were injected without especial difficulty, the injection mass spreading out uniformly from the point of injection. In later stages, however, increasing difficulty was encountered, the ink often going but a short distance. This difficulty proved to be due to the irregular narrowing and retraction of the vessels which occurs in later stages.
That the area vasculosa continues to grow and expand after the operation is certain, and the results of a number of measurements upon operated chicks are reported in tabulated form below as compared with normals at the time of operation and later.
Area vasculosa, .33 hours Area vasculosa, 106 hours Area vasculosa, 105 hours Area vasculosa, 8§ days. Area vasculosa, 8 J days.
Embryo, 4^ days
Embryo, 4| days
Embryo, 41 days
711 m .
Normal chick Operated chick Normal chick Operated chick Normal chick Operated chick Operated chick Normal chick
It is apparent from this table that while there is a surprising amount of peripheral extension of the area vasculosa in the operated chicks, still the growth is less than half as rapid as in the normal chicks. The embryos showed a similar difference. A number of operated chicks measured approximately 6.5 mm. in length as compared with a normal embryo of the same age, which was 13 mm. in length.
The area vasculosa of the operated chicks did not always expand uniformly in each direction, as in the normal, but often grew out at certain points, usually posteriorly from the embryo, in advance of other portions. Figure 2 is a diagram of one of these specimens. The effect of this irregular growth of the area is apparently shown in th6 shape of the capillary plexus^thus, in figure 13 the peripheral capillaries, formed by the breaking up of the border vein, have grown out so rapidly that the inner capillaries have assumed a radial appearance.
The general picture presented is shown in figure 3. The area vasculosa appears, at first glance, to form a homogeneous network of intercommunicating capillaries, extending from the embryo proper to the border vein, which forms a wide rim around the outside — (somewhat wider than shown in the illustration). Examined more closely, it is noted that, anterior to the embryo
Fig. 2 A diagrammatic sketch (actual size) of the blastoderm of an operated chick of 73 hours incubation, showing the distorted shape of the area vasculosa.
there is a depression in the border vein, behind which a vessel, wider than most of the capillaries, extends toward the embryo — the anterior vitelline vein. It is also to be noted that, in the region surrounding the embryo, in the area pellucida, the spaces between the vessels are larger than in the area opaca. One looks in vain for any vessel which might be interpreted as omphalomesenteric artery, or posterior vitelline vein. To the left of the embryo there are two isolated blisters, and several blind ending vessels. Fuller reference to this will be made later.
Fig. 3 Camera lucida drawing of area vasculosa of an operated chick of 60 hours incubation (30 hours after operation). The capillaries in the area pellucida have begun to break up. H, a hole torn in the blastoderm when mounting, X, at this point the ink was forced out of the tiny vessels by too much pressure, and settling in the tissues, so obscured the capillaries that they could not be distinguished. Veil. Vit. Anl., Anterior vitelline vein.
Figure 4 is a drawing of an unoperated chick which had been incubated 48 hours and had developed abnormally. The embryo appears as an oval membrane with two little splotches of protoplasm underneath it. There was no circulation, and a fairly uniform capillary plexus was present throughout the area, extending under the oval membrane. Only at one place — anterior to the membrane — is there any sign of a vessel larger than a capillary, which is very probably the rudimentary heart and anterior vitelline veins. There is no trace of any vessel which could be interpreted as omphalo-mesenteric artery. It will be noted that, as in figure 3, the continuity of the plexus has begun to be lost in the area to the left of — and posterior to — the embryonic rudiment.
In all the operated chicks which were studied, by injection and by combined injection and staining, there was never found any definite vessel which could be interpreted as omphalo-mesenteric artery or vein, or posterior vitelline vein. There were, however, certain characteristic changes, which were repeated in all specimens, some of which resembled the changes in normal chicks, and which will be taken up separately.
The anterior vitelline veins
As shown in figure 1, there is a space between the right and left anterior vitelline venous plexus at the time circulation commences. In normal chicks, as PopofT has shown, this space is encroached upon by the two plexus, until it is obliterated, the two veins fusing in the mid-line, forming a single vein, which, for a time, returns most of the blood of the extra-embryonic area. It was most interesting to find that in the absence of a heart-beat, the vessels in the proamnionic region continue to develop in an almost normal manner for a time. During the second day after the removal of the heart (third day of incubation) the right and left vitelline veins fuse across the mid-line anterior to the embryo, and a single vessel, which corresponds to the left anterior vitelline vein of the normal chick, is formed (figs. 5, 6 and 7, cf. also fig. 3). During the fourth day, however, this anterior vessel begins to break up into capillaries and soon disappears. This is the only vessel larger than a capillary that is formed in the extra-embryonic region after the time when the circulation should begin, and with its disappearance and the breaking-up of the sinus terminalis into capillaries, which will be described in detail below, the condition becomes uniform throughout the extra-embryonic region.
Fig. 4 Unoperated embryo of 48 hours incubation which developed without a heart. The sinus terminalis was present but is not shown in the drawing. Enl. 35 X.
Fig. 5 A camera lucida drawing of tho anterior vitelline vein in an operated chick of 60 honrs incubation (30 hours after operation).
Fig. 6 Camera lucida drawing of the anterior vitelline vein in an operated chick of 63 hours incubation (30 hours after operation).
Fig. 7 A camera lucida drawing of the anterior vitelline vein in an operated chick of 80 hours incubation (48 hours after operation). The vessel has grown much narrower and is beginning to break up into capillaries nearer the embryo.
The vessels of the area opaca and the area pellucida
Soon after the operation a difference in the arrangement of the capillaries of the area pellucida and of the area opaca begins to appear, which becomes quite marked about the sixtieth hour of inculcation (twenty-seven hours after the operation). In the former the spaces between the vessels are seen to be growing larger and the capillaries less numerous than in the latter (fig. 8). A trace of this is noticeable in figures 3 and 4. In the older specimens this condition is more pronounced. Figure 9 shows an embryo of four and one-half days in which the difference in the appearance of the capillaries is becoming decidedly marked and the two areas are separated by a rather definite border around the peripherv of the area pellucida. In still older embryos the capillaries within the area i:)ellucida become smaller in diameter and more scattered, and in embryos of seven or eight days (fig. 10) are broken up and appear as little streaks and puddles of blood, which are, in reality, isolated endothelial lined vesicles and spaces. A somewhat similar, though less intense, change occurs in the area opaca. Here the process is slower, and consists chiefly of the narrowing of many of the capillaries, some of which become solid and separate in the middle. Figure 11 shows a small portion of the area opaca in an eight day chick (seven days after operation), in which there are a great number of solid cords, some of which have broken in twain and begun to retract. The beginning of the process of narrowing and retraction of the capillaries in the area opaca is apparent by the fifth or sixth day, and is well demonstrated by the increasing difficulty of injection, which has been mentioned above.
The sinus terminalis
The sinus terminalis, or vena terminalis, as it is termed by Popoff, will now be considered. This relatively large embryonic vessel is present before the heart is formed (Lillie, '08, pp. 8788). Popoff has described it as beginning to 'degenerate' (breakup) in chicks of forty somites, which would be somewhere around the eighty-fifth or nintieth hour of incubation, the breaking-up continuing progressively until the sinus is resolved into capillaries by about the tenth day of incubation. I tested this out on a few normal chicks and found that the process begins about the time mentioned, but that it is often complete by the sixth day of incubation. I was much surprised to find that the sinus terminalis in the operated chicks followed this general rule and began to break up into capillaries at about the same time as the normal. Figure 12 shows the sinus at forty-four hours, before breaking-up has started. Figure 13 is from a chick of eighty hours incubation, and figure 15 is from a section of a chick one hundred and five hours old where the vessel has been replaced by a richly growing capillary plexus, which, judging by the peripheral sprouting, is highly vegetative. This was a somewhat extreme case and in the same embryo portions of the sinus had not yet begun to break up, although it had extended some distance from the embryo after the operation. Figure 1(3 is a camera lucida drawing of the sinus region of a normal chick of the same age. This drawing more nearly represents the conditions found in both normal and operated chicks of this age.
Fig. 8 A camera lucida drawing of an operated chick of 90 hours incubation (57 hours after operation).
Fig. 9 A free-hand drawing of a chick of Ak days incubation in which the breaking-up of the cai)illaries in the area pellucida is quite pronounced. Operated at 33rd hour of incubation.
Fig. 10 A camera lucida drawing of a section of the area pellucida of a chick of 8 days incubation (7 days after operation) which shows small lakelets of blood formed by the breaking-iip and retraction of the capillaries. Enl. 24 X.
Fig. 11 Camera lucida drawing of capillary net in area opaca during eighth day of incubation. Operated chick. Enl. 52 X.
Fig. 12 A section of the sinus terminalis of <i chick of 44 hours incubation in which the heart was destroyed by burning with the cautery in the region of the heart rudiment. Operated at twentieth hour of incubation.
In operated chicks of seven or eight days of age the sinus terminalis had invariably broken up into capillaries, although the process was often not complete. Figure 14 is a camera lucida drawing of a section of the sinus terminalis as it appeared in a chick during the ninth day of incubation. The vessel had disappeared over half of its length, and, on the side drawn, appeared so thin as to resemble a wide band of endothelium, sending out sprouts from its outer margin and gradually shading off into normal capillaries towards the inside.
A condition somewhat similar to the ones described above was found in one of the eggs that I had been incubating as a normal. In this egg, (incubated six days) for some reason, the embryo had not developed normally and appeared as a small shapeless mass of protoplasm lying in the center of the area pellucida. The area vasculosa, however, was covered with a rich plexus of capillaries which were clearly alive and growing. There was no sign of a border vein around this plexus, and the capillaries were sending out numerous sprouts around their outer margin.
Fig. 13 A camera lucida drawing of a section of the area opaca in the region of the sinus terminalis which has broken up into capillaries. At this point, the capillaries have apparently grown. Operated chick of 3 days' incubation.
Fig. 14 Camera lucida drawing of the sinus terminalis in a chick during the ninth day of incubation (8 days after operation). Enl. 47 X.
Fig. 15 Camera lucida drawing of a section of the outer border of the area opaca where the capillaries are highly vegetative and are sending out nimierous sprouts. Operated chick of 105 hours incubation. Enl. 47 X.
Fig. 16 Camera lucida drawing of the outer edge of the area opaca of a normal chick in which the sinus terminalis has broken up into capillaries. Drawn for comparison with figure 11. Incubated 107 hours. Enl. 44 X.
V. A Short Description of the Embryos
Although this problem deals only with the development of the vascular system, I \\t.11 take the liberty to include a brief description of the embryo. Many of the embryos developed very abnormally, and in some it was impossible to distinguish the anterior from the posterior ends by the shape of the embryo alone, but in chicks where the heart was removed with a minimum of injury to the remainder of the chick, the embryo continued to develop for a time in an almost normal manner. The chick turned upon its left side, the amnion behaved as usual, the eyes were formed, and the wing buds appeared. The most abnormal feature was its failure to grow large. In no case did the embryo exceed 7 mm. in length after the removal of the heart. In a number wherein the heart was injured but not totally destroyed, especiall}^ in the operations with the electric needle, the size of the embrj^o was in proportion to the impairment of the circulation.
An examination of the embryos without hearts, in serial sections, showed very little that could be interpreted as normal. The neural tube and notochord were present, and the general outer contour had a normal appearance, but the embryo contained huge spaces beneath and to the side of the neural tube and notochord and connecting freely with the celomic cavity at many points. These relatively huge spaces are so packed with blood cells that it was often necessary to use the high power in order to tell the wall of the cavity from its contents. Alitoses were numerous both within the substance of the embryo and among the cells inside the open spaces. That several of these spaces were the remains of blood vessels was indicated, but they were very abnormal.
VI. Mention Of Results Obtained In A Number Of Operations Which Were Only Partially Successful
The results obtained in a number of operations that were only partially successful are worth mentioning. In an effort to prevent the formation of the heart, I tried cutting through the lateral plates and dissecting around the neural tube of a number of chicks that were opened before the heart had formed. This operation was often successful, while at other times the heart would later be found developing out in the area pellucida, separate and apart from the embryo or connected with it by a few capillaries. Figure 17 is a case of this kind. The small vesicle shown was pulsating at a normal rate when the egg was opened. By its dilatation blood was sucked in and with its contraction the blood was discharged again into the capillaries, the blood cells moving back and forth over about the same distance each time. That this process had some effect upon the formation of the capillaries directly connected with them is apparent from their radial arrangement as compared with adjoining capillaries to which the circulation did not extend.
Fig. 17 A free-hand drawing of a chick in which the heart had not been destroyed, but developed out in the area pellucida. The appearance of the capillaries through which the feeble circulation flowed is illustrated. Incubated 3 days.
After a similar operation, I found a pulsating vesicle in the area opaca very close to the sinus terminalis although not connected with the border vein. This vesicle was connected with a relatively huge blood vessel which extended parallel to the sinus terminalis for a short distance and then broke up into radial capillaries through which ink injected into the large vessel passed quickly to the embryo. That this large blood vessel was formed from the capillary net by the functional activity of the pulsating blister can not be doubted. On another occasion I burned away all of an embryo anterior to the first somite. On opening the egg again I found one of these small pulsating vesicles which had developed from a remnant of the heart or the sinus venosus and was pumping away after the manner described for figure 17. I could mention a number of these freaks, but it would have no bearing upon the problem.
It appears, at first, that a number of conflicting factors have been introduced into this experiment. Chief of which are the following :
- The formation of the anterior vitelline vein after the time when the circulation should begin.
- The failure of the large omphalo-mesenteric arteries and veins and the posterior vitelline vein to appear.
- The pecuhar behavior of the sinus terminalis.
- The progressive peripheral growth and spread of the capillary net, in combmaton with the central process of retrogression.
That these factors were not due to accident, but to the enactment of certain simple laws of development that have been long recognized by certain leading scientists can readily be explained.
Roux has divided the formation of the vascular system into three stages, which are as follows :
- A stage of primary differentiation in which the formation of the vessels is governed entirely by certain hereditary principles due to inherited characteristics.
- A transitional stage between the first and third wherein the hereditary formation is gradually supplanted by a process of functional adaptation.
- A stage in which the further formation of all vessels is due entirely to the mechanical forces acting through the circulation.
The early formation of blood vessels which takes place before the circulation begins is clearly not governed by the mechanical forces acting through the circulation, and their formation can be accounted for only as a response to some hereditary principle which as yet is not understood and which most scientists of the present day are unwilling to believe exists. The early onset of the circulation has been a factor which has obscured the cause of the further development of the blood-vessels, and although it was known that there was some formation of blood vessels such as the heart and aorta before the establishment of a circulation, it has only been since the publication of Miss Sabin's work on the subject, that the extent of this early formation of blood-vessels has been appreciated.
It is now clear that by the time the circulation begins, the embryo has a primitive but complete system of blood vessels. With the advent of the circulation, which occurs about the thirtyeighth hour of incubation, we have the beginning of the second stage. In this stage the primary differentiation continues, but is gradually replaced by the functional or mechanical factors due to the heart-beat. In the normal chick, it is impossible to determine just how much of the further formation is due to the heart-beat, but in the operated chicks, the mechanical and functional factors are eliminated and the further development is due entirely to the continuance of the primary differentiation and laying down of blood vessels independent of mechanical influences. That this continues after the time when the circulation should begin is proved by the formation of the large anterior vitelline vein by the fusion of the two, right and left, vitelline veins. It is clear, therefore, that with the failure of the circulation to start at the usual time, the further development of the vascular system is not inhibited, but proceeds for a time in an almost normal manner, that is, it goes a little further and this relatively important embryonic vessel is laid down.
The failure of the omphalo-mesenteric arteries to develop proves that the formation of these vessels is due to mechanical forces acting through the heart-beat, and in the absence of these mechanical forces, due to the circulation, they are not evolved from the capillary net. In view of the fact that I have never at any time observed the slightest indication of the formation of these vessels in my operated chicks, I am at a loss to understand the formation of the large vessels described by Patterson as forming in the region where these vessels are usually formed.
The peculiar cycle of development and retrogression through which the sinus terminalis passes would indicate a further example of this stage of primary differentiation persisting after the time when it is usually thought to have been entirely eliminated. It would also be interesting to know precisely how much further development takes place within the embryo in the absence of a circulation, but this presents a problem within itself, and cannot be taken up in this paper. Roux has stated that this early stage of primary development persists for a considerable length of time, perhaps up until adult Ufe in the human. He thinks the closure of the ductus botalli which occurs at birth or a little later belongs to this stage.
That the mechanical forces very early assert their superiority in the extra-embryonic region is apparent, for with the breakingup of the anterior vitelline vein and the sinus terminalis into capillaries the vessels of this region are reduced to an indifferent capillary plexus. This capillary plexus continues to grow at a gradually decreasing rate for a number of days after the time when it should become functional. With the failure of the circulation to start, we note, about the beginning of the third day of incubation, the regressive changes already mentioned as taking place in the area pellucida. This process is noted in all of the operated chicks, and also in the unoperated chicks that developed without circulation, and is plainly due to the processof retraction and degeneration which has been mentioned by Thoma.
In introducing his first histo-mechanical law, Thoma stated that the surface of a vessel wall ceases to grow when the bloodcurrent acquires a definite rate. The vessel increases in size when this rate is exceeded, becomes smaller when the bloodstream is slowed, and disappears when it is finally arrested. The breaking-up of the capillaries in the area pellucida is clearly an application of the latter part of this hypothesis of Thoma's. In this instance, however, there is no circulation into which the contained blood can be pushed by the retracting capillaries, and as these small vessels are filled with blood cells, when they narrow at certain points, break up, and retract, the blood cells are forced into little lakelets of blood surrounded by the endothelium of the retracted capillary which encloses the blood cells as a capsule.
This process continues progressively out over the area pellucida and area opaca, but does not advance so far as in the area pellucida. It manifests itself here by the narrowing of most of the vessels, and the solidification and retraction of some of them — processes which cause an increasing difficulty of injection. By this time, the sinus terminalis is also beginning to offer resistance to the injection mass. As this vessel is usually injected with ease in younger chicks, it is plain that it also under goes a process of narrowing which precedes its breaking-up into capillaries. The blood within these small vessels is contained under some pressure. This can be demonstrated by puncturing them with the injection needle which is usually sufficient to produce an extravasation of blood cells.
The general application of the histo-mechanical laws of Thoma to the factors observed in this experiment are too obvious to permit of much discussion. The failure of the omphalo-mesenteric arteries and vein to develop show that these vessels are entirely dependent upon the blood-stream for their development. Thoma observed this factor and studies the development of these vessels. He states that with the beginning of the circulation, a few channels in the capillary net are selected by the blood-stream in consequence of the general direction which is given to it by the position of the ends of the primitive aorta on the one side and of the venous ostia of the heart on the other. These channels contain the more rapidly flowing streams. They, therefore, dilate and become converted into arteries and veins. Thoma, did not, however, recognize the formation of any arteries or veins in the extraembryonic region as due to other than mechanical forces.
The response of the capillaries to the feeble circulation maintained by the pulsating vesicles in the extra-embryonic region is another factor in support of his description of the formation of larger vessels from the plexus by a circulation.
Thoma states further that, after the beginning of the circulation, some channels which offer resistance to the flow of the blood, and are thus very slowly traversed, atrophy, or disappear altogether. The progressive breaking-iip of the capillaries which, in this experiment, is general throughout the area pellucida, and gradually extends to the area opaca, is clearly this same process on a much larger scale. The little lakelets of blood that are left being due to the inability of the vessels to entirely discharge their contents.
In summarizing the chief factors that have appeared in this investigation, the following are the more important:
1. In chick embryos, in which the heart has been removed before the establishment of the circulation, the embryo and area vasculosa remains alive for seven or eight days after the operation. A limited amount of growth takes place in the embryo proper, while the area vasculosa spreads out over the yolk until it may reach a diameter of approximately 45 millimeters.
2. The development of the blood vessels in the area vasculosa is not entirely inhibited, but the process proceeds in a normal manner for a short period beyond the time at which the circulation usually commences. During this time there are formed in the extra-embryonic region vessels identical with the normal anterior vitelline veins, which fuse anterior to the embryo as in normal chicks, while the sinus terminalis passes through a cycle of development and regresson which markedly imitates the normal. On the other hand, other vessels which normally differentiate early, the omphalo-mesenteric arteries, as well as the omphalo-mesenteric veins, and the posteror vitelline vein, are not formed.
3. With the expansion of the vascular area, there is a continued formation of new capillaries; the property of sprout formation apparently being retained until death. After the third day, however, this is confined to the marginal portions of the area. Near the embryo, in the area pellucida, new formation has ceased at three days, and regressive changes commence; connecting capillaries are retracted, leaving isolated or nearly isolated endothelial blisters distended with fluid or blood cells. This regressive process advances gradually into vessels of the area opaca.
4, The general conclusion seems justified that, while certain large vessels such as the sinus terminalis and the anterior vitelline veins develop as a result of hereditary factors, and continue a normal development for a short time after the circulation starts, 'self-differentiation' of the vascular system is very limited, and the working out of most of the arteries and veins is dependent upon the mechanical factors concerned with the circulation of the blood.
I am indebted to Professor Eliot R. Clark for the assignment of this problem and valuable instructions during its investigation.
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