Book - Contributions to Embryology Carnegie Institution No.67
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Doan CA. The circulation of the bone-marrow. (1922) Contrib. Embryol., Carnegie Inst. Wash. Publ. 70 14: 27-45.
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The Circulation of the Bone-Marrow
By Charles A. Doan, Anatomical Laboratory of the Johns Hopkins University, (1 plate, 3 text-figures) 27-45
Introduction
In considering the varied functions of the vascular system of the body, attention has been riveted in the past almost solely on the grosser arterio-venous circulation and the observable changes associated with these vessels in health and disease. Only comparatively recently has the tendency to overlook the connecting link between afferent and efferent systems been noticeably changing, and from many different sources there are now various evidences of an awakening realization of the importance of the capillaries, the real structural medium of body nutritive exchange. As has been strikingly stated by a recent writer, the cardio-vascular system exists only to regulate the blood-flow through the capillaries, for here takes place the exchange of gases necessary for internal respiration and the exchange of materials necessary for metabolism.
This failure to devote more direct consideration to the function of the capillaries has probably been due in large part to their unobtrusive and rather obscure existence in the larger functioning unit and to the technical difficulties which observations on these, the smallest vessels of the circulation, involve. Especially has the latter factor operated in reference to the circulation in the marrow of the bone. The methods of direct observation, recently so ingeniously evolved for a study of the capillary circulation in many of the other tissues of the body, are manifestly incapable of application when it comes to a study of the tissues inclosed within a thick, bony shell. Still another factor has hitherto influenced the lack of interest in a careful analysis of the circulation of the marrow, viz, the fascination which investigators have found in attempts to classify and relate the various precursors of the different circulating blood-cell elements known to have their origin and development in the red marrow of the long and flat bones. The result has been a most thorough morphological study of the cells of the marrow. Ehrlich (1891), Pappenheim (1919), Maximow (1909), Bunting (1906), Danchakoff (1908), Dickson (1908), Ferrata (1918), and many others have studied minutely the cytology of the hemopoietic tissues, leaving little to be desired so far as gross morphological description is concerned. There are fundamental points of difference, however, in the theories as to the original or parent cell type or types. This difference of opinion among investigators has led to the formation of two schools — the monophyletic school, with strong adherents in Dominici, Pappenheim, Weidenreich, Maximow, Danchakoff, and Ferrata, and the dualistic or polyphyletic school, supported notably by Ehrlich, Naegeli, Schridde, and Morawitz. Both the monophyletic and the polyphyletic interpretations have arisen out of a study of normal and pathological tissues fixed and stained with identical methods in an identical manner, but by different investigators. From the careful analysis of fixed tissues we have gained much in our understanding of the blood and its formation, but it has become increasingly evident that the problem of the original type or types of parent blood-cells still remains, with a necessity for the development of further methods of attack. Until further progress toward this fundamental comprehension of first principles has been made, by means of studies along different lines of approach than hitherto employed, we shall still be without the basis for a rational therapy.
Within the past two decades exceedingly valuable contributions toward solving the problem of the origin and development of individual types of blood-cells have been made through embryological studies. The most representative work on the embryology of the blood is that carried out by Danchakoff (1908, 1909) and Sabin (1920, 1921) on birds and by Maximow (1909, 1910) on the mammal. Both Maximow and Danchakoff recognized the relationship between endothelium and blood-cells, not only in the stage of the primitive blood-islands but also in somewhat later stages; both have thought, however, that endothelium gives rise only to indifferent blood-cells. Schridde (1907), on the other hand, has described the direct transformation of endothelium into erythroblasts in early human embryos. Maximow believed that although the early erythroblasts of mammalian embryos are intravascular in origin and derived indirectly from endothelium, the ultimate erythroblasts of the adult are a group of cells extra-vascular in origin. This may be said to be the prevailing view to-day. The question lias been reopened recently, however, by the work of Sabin (1920, 1921). It was not until she had actually seen, by direct observation on living chick embryos during the second day of incubation, the differentiation of the red cell from early endothelium and later the origin of the monocyte cell-series and clasmatocytes from the same source in chicks of the third and fourth days, that the etiological importance of the endothelium, and hence the significance of the exact pattern of the vessels of the marrow in the mature organism, was fully understood. Thus the whole blood problem receives a new impetus in a different direction. This work places an emphasis upon the importance, not hitherto adequately appreciated, of a more comprehensive and exact knowledge of the endothelial content of adult marrow. It is not a purely morphological standpoint to which the importance attaches now, nor are we interested in it solely as a means by which the blood-cells gain entrance into the circulation. The important question, stimulated by the work of Sabin, is the very suggestive one as to the possible direct relationship between the endothelium of the hemopoietic tissues and the blood-cells of the mature organism.
Obviously, before attempting to determine this relationship, a thoroughly comprehensive understanding of the extent and distribution of the endothelium in the marrow of the long and flat bones is essential. But here again we find in the literature a wide difference of recorded observation on the part of various workers. The views held may be classified into three groups, together with their respective supporters. (1) The earliest observations followed close upon the first recognition of the bone-marrow as a hemopoietic tissue. Hoyer (1869) could detect no endothelial walls in the so-called capillaries or blood-channels in observations on the marrow of injected rabbits. Rindfleisch (1880), using a gelatin injection mass, interpreted the regularly outlined channels in his sections of bonemarrow (very well illustrated in one of his plates) as indicative of tissue spaces filled with blood and limited only by the medullary parenchyma, that is to say, entirely devoid of endothelial lining. This earlier view, however, has been quite clearly shown to have been based upon erroneous observations, and the later conceptions, while being divided by two different interpretations, nevertheless agree on the presence of endothelium-lined blood-vessels as the essential basis of the circulation. (2) Langer (1877) was among the first to advance the opinion that the vascular system of the bone-marrow is a closed system lined throughout with a continuous endothelial layer. Bizzozero (1891), a few years later, after more extensive investigations than had hitherto been made, reported as follows:
- "In the marrow of birds one is able to affirm that the venous capillaries are limited by a thin nucleated membrane, consequently they are not the simple hollow spaces in the tissue of the marrow as so many have maintained."
On the other hand, Bizzozero was not so positive about the circulation in mammals and was rather prone to doubt the completeness everywhere of the vascular walls in mammalian marrow. Denys (1887-1888), also drawing his conclusions from experiments on the bird, concurred in the observation that the vascularization of the marrow is that of a single closed system of vessels lined with endothelium. Again, Van der Stricht (1892) differentiated between avian and mammalian marrows, in the former observing only closed venous capillaries possessing an endothelial wall throughout their extent, in the latter describing non-continuous vascular walls. Minot (1912) questioned the adequacy of proof for the contention that there are direct openings into the parenchyma from the blood-vessels. Schafer (1912) contented himself with stating that there were two theories, frankly withholding any opinion in the controversy.
Finally, Drinker, Drinker, and Lund (1922), in a recent analysis of a very extensive series of splendidly controlled injections of marrow, state their belief that the "capillaries conducting blood in the bone-marrow of the mammal in a condition of normal blood formation are closed structures lined throughout with endothelium and not in communication with the marrow parenchyma." (This coincides with my own [1922] observations on mammalian marrow.) They further advance a most interesting explanation of the marrow condition during active hyperplasia.
- "Under conditions of active red-blood-cell formation the extremely delicate walls of these capillaries [venus sinusoids] are grown through by irregularly placed red cells in varying stages of maturity. The capillaries are thus, for a period of varying length, open structures, but the opening presented does not result in flooding the marrow parenchyma with blood, because of the packing of the immature blood-cells, which is an essential phase in the process of encroachment upon the capillary wall."
(3) As has been suggested above, the third view is that there is an incomplete endothelial lining to the venus sinuses with openings directly into the parenchyma for the exit of blood plasma and the entrance of mature cells. Weidenreich (1903, 1904), in his researches on the marrow as a hemopoietic organ, found that so-called "cell-nests" constitute the blood-forming tissue, that they are appendages of the venous capillaries, and that the endothelium of the latter is deficient in the region of these "cell-nests." Venzlaff (1911) maintained that erythrocytic differentiation takes place within the venous sinuses of avian marrow from lymphocytes that have passed out of the "Leukoblastershaufen" (the "cell-nests" of Weidenreich), in the region of which he also believed the endothelium of the sinuses to be lacking. Brinckerhoff and Tyzzer (1902), in studies on the uninjected marrow of rabbits, described places in which the blood-stream is not confined within endothelial walls but wanders through channels in the reticulum and the masses of cells. More recently, Bunting (1919) describes the marrow vascularization as follows:
- "The circulation as revealed by natural injections of the rabbit's marrow is unlike that of any other organ but resembles superficially that of the spleen pulp." 1
He further states that there is no capillary network and describes slender arterioles originating near the center of the marrow and proceeding, without capillary side branches or anastomoses, to the periphery, where they open directly into wide, thin-walled sinuses.
Desiring to investigate the relationship which endothelium might bear to the supply of red blood-cells in the mature organism, it became necessary to know its distribution at first hand. The interesting results which have attended these studies are presented with the belief that they open up a new field of possibilities, only vaguely hinted at heretofore, but now having a definite basis in anatomical
Materials and Method
The conclusions reached in this paper are based largely on a series of investigations on about forty adult pigeons. Further experiments of a similar character, conducted on the dog, cat, rabbit, and white rat, seem to substantiate and corroborate the gross findings in the pigeon, so far as I have been able to observe in a limited series. A larger number of observations on mammals will be necessary before a complete report can be made.
An attempt has been made to get complete injections of the vascular system of the bone-marrow. This has not been easy, the difficulties being fourfold: (1) to secure a satisfactory medium for injection, (2) to keep the pigeons alive sufficiently long during the preliminary insertion of the cannula, etc., (3) to secure and maintain just the right pressure for perfusing, and (4) to wash out and inject under conditions as nearly physiological as possible and for the optimum length of time.
It has been found, in general, that pigeons are peculiarly susceptible to chloroform. All operations have been done on anesthetized birds, and a light ether anesthetization has been found entirely satisfactory. It is desirable to have the animal alive during the first stage of the washing-out process.
My most successful injections were made with a pressure of 130 mm. of mercury for both saline and ink. When the pressure was materially increased above this point, rupture and extravasation frequently occurred, whereas with pressures below this level an incomplete injection was apt to result. Both the injection material and the physiological saline were previously warmed to a degree somewhat above body-temperature to insure then reaching the vessels at body-temperature. With a free flow this saline should not be run longer than 8 minutes, preferably a shorter period, judging by the clarity of the venus outflow. The injection mass should be run for about 10 minutes. However, experience only can give one competent judgment in this, as there are many indications, not reducible to writing, which one learns to recognize and be governed by in individual instances. One may get a complete injection of the superficial vessels of the skin and muscle with practically no penetration of the marrow cavities. The optimum condition is to stop as soon as possible after the maximum complete injection of the smallest capillaries of the bone-marrow, which, being manifestly impossible of direct observation, must be a matter of experience.
1 Mollier (1909) has demonstrated openings into the splenic pulp, i. e., fenestrated vessel-walls.
Several injecting solutions were tried. A silver-nitrate solution permeates the vessel walls and, while outlining the larger vessels quite clearly, masks the smaller capillaries completely. Freshly precipitated carmine, even under the best conditions, forms flocculi too large to be carried into the smallest vessels for a complete injection. The best results were obtained from a freshly filtered solution of one part of Higgins india ink diluted with three parts of physiological saline. Very satisfactory injections, which I feel are relatively complete, were secured with this injection mass under the conditions stated above.
The cannula was placed directly into the heart, into the subclavian artery (making ventral incisions), or into one of the iliacs or the abdominal aorta (with a dorsal incision). This latter procedure was used almost exclusively in the later experiments. The antero-posterior incision was made just to the side of the midline; a lateral exposure of the ribs was made and, after removing a section of four ribs, the lung was carefully laid back by blunt dissection, after which the abdominal aorta or common iliac was easily located. The auricle or inferior vena cava was opened for the return-flow outlet. No injections were attempted via the nutrient arteries direct.
After many methods for fixation had been tried, the best results were found to be obtainable by fixing the "marrow pencils" in Helly's fluid at 38° for from 2 to 6 hours and the whole bones in 10 per cent formalin for 24 hours. The former were fixed in the routine manner, dehydrated, cleared, and embedded either in celloidin or paraffin, the celloidin proving better for the study of individual cells when stained. The whole bones were cleared by the Spalteholz (1914) method. As a routine procedure the radius and ulna of one side were fixed and treated for clearing in situ and the "marrow-pencils" of the opposite side were taken out and fixed in Helly's fluid for embedding. 2 It is desirable to fix when fresh and to maintain the "marrow-pencils" in as perfect form as possible. With reasonable care the fresh marrow may be removed intact, and, except in rare instances, there are no spicules of bone in the marrow calling for decalcification. Danchakoff's (1908) modification for the mounting of celloidin sections was used in making serial sections.
For staining sections we have used Giemsa's stain, Wright's blood-stain, methylene-blue-eosin, hematoxylin and eosin, and hematoxylin and carmine. The sharpest differentiation was obtained with a slight modification of the ordinary hematoxylin and eosin stain. A two-minute period in a freshly filtered 1 per cent solution of Ehrlich's hematoxylin, diluted one-half, alkalinization in Ba (OH) 2 solution, and then counterstaining for 2 to 3 minutes in a 5 per cent aqueous eosin, gave a beautiful contrast to the cellular elements. Dr. Sabin found that the addition of orange G to the eosin increased the effectiveness of this combination in the staining of embryonic blood-cells.
- The humerus in the pigeon contains no blood-forming marrow.
Observations
In the earlier incomplete injections the gross architecture of the bone-marrow was plainly evident in the cleared specimens. Figure 4 (plate 1) shows the medullary artery entering the marrow cavity near the center of the diaphasis, perforating the compact tissue obliquely. It divides immediately into two main branches which diverge abruptly, one extending toward each epiphysis. These two main arterial trunks in turn divide about a third of the way to the epiphyses and extend from their point of origin to the limits of the marrow at either end, anastomosing with the vessels entering there. Several small arteries were usually seen at the epiphyses, entering the marrow cavity through the bone, anastomosing with the medullary vessels, and helping to furnish the additional blood-supply to the actively functioning red marrow of these regions.
In addition to this main arterial supply there could be seen numerous small vessels entering along the shaft of the bone (fig. 6), primarily to nourish the cancellous and compact tissue, but anastomosing at the periphery with the arterioles of the central vessels. There was frequent and intimate intercommunication along the entire shaft between the nutrient vessels of the Haversian canals and the circumferential end arterioles and venules of the medulla of the bone. These anastomoses formed a very striking picture in cleared specimens and gave a new insight into the delicacy of the vascular interlacings and the extent of their ramifications. We are not dealing with two more or less separate and distinct systems, one to nourish the marrow, the other the cancellous and compact tissues, but with one interdependent and communicating whole. The subject of the vascular supply of the bone-substance itself has been treated in a recent monograph by Foote (1921) in a most admirable manner, with extensive illustrations.
There were three groups of veins in the long bone. (1) The central medullary veins could be seen accompanying the central artery (fig. 4). From one to four parallel veins accompanied the artery and traversed the shaft from each end to unite near the center in a single efferent vein which occupied the nutrient foramen, together with the entering artery. (2) Several large veins emerged near the vascular area of red marrow, always more prominent toward the epiphyses. (3) There were numerous small veins along the diaphysis (fig. 6) which drained the compact tissue and the peripheral area of the marrow and, with the small nutrient arterioles of the shaft, formed the abundant vascular network of the periosteum (not shown in the diagram). This general vascular pattern held for both the radius and the ulna of the pigeon, the individual bones differing only in the number of their central vessels, in direct relation to their relative size, and in the extent of bone-marrow to be supplied. In relatively complete injections, the central vessels could not be seen from the surface, even in the most perfectly cleared specimens, so dense was the network of carbon-filled vessels, as will be shown later.
In figure 6, which shows the next stage of a partially complete injection, the gross picture observed in figure 4 is again illustrated in the cleared specimen with the marrow in situ. The central vessels are still visible and smaller branches may be seen coming off at an angle from the main artery and extending toward the circumference. These begin almost at the center of the shaft but become more numerous and dense toward the ends. At each epiphysis there is a veritable spray-like shower of fine vessels which ramify to every part of the marrow and supply the epiphysis as well, but which stop abruptly at the line of cartilage forming the articulation of the joint (fig. 1). The characteristic vessels of embryonic cartilage have disappeared in the mature state.
Fig. 1. A detail drawing of a part of the epiphysial end of specimen shown in figure 4 (plate 1). There is a most extensive ramification of the vessels at the epiphysis, radiation stopping abruptly, however, at the line of cartilage. X 140.
The artery and its branches were easily distinguished from the veins by
virtue of their smaller caliber, firmer walls, and less tortuous course; also by the
fact that the lumen was more closely packed with particles of carbon. The divisions
of the artery were characteristic, the branches came off at an acute angle, and the
subdivisions were much less numerous than those of the corresponding veins.
The arterioles at the periphery were characteristic in their delicacy, scarcity,
and apparently limited distribution.
Figure 6 illustrates very graphically the "tuft-like" character of the venous branchings. Coming off from the central vessel, almost at right angles, are the large distended veins which at once branch outward toward the circumference in an ever-widening balloon-shaped bed, to anastomose eventually with branches from tufts on either side. The large caliber of the vessels is strikingly maintained ; and though there is some decrease in the lumen toward the periphery, it is not commensurate with the extent of the branching. The most apparent and striking thing about the entire vascular system of the bone-marrow, both in gross and in microscopic view, is this extensive venous ramification and its very evident capacity for large quantities of blood.
A still better comprehension of these venous and arterial tufts and the means by which they become continuous with each other is obtained from a study of a third more complete injection (sections 100 to 150 micra thick). Figure 5 gives such a picture. In this preparation can be plainly seen what I have termed the "transitional capillaries " leading directly from the arterioles to the venous sinusoids and with apparently very little true arterial capillary bed. This patent capillary link connecting arterioles and venules is extremely circumscribed, and it is not until the venous sinusoidal anastomoses are reached that the blood spreads out in lacing and interlacing vessel tufts, thence to be directed from the tuft-like branchings into larger and larger vessels, eventually to enter the central longitudinal vein almost at right angles or to find egress by way of one of the other venous outlets. It will be seen that the marrow assumes almost the appearance of a segmentally or lobularly divided organ, dependent upon the structural circulatory distribution of these venous tufts, so completely do they ramify in definite areas, yet anastomosing on all sides with the ramifications of bordering tufts. The relationship of the arterial tree to the venous tufts on either side and the capillary transitions from one to the other, even though not extensive, were easily distinguished and were very characteristic in sections of injected marrow. There is little doubt, however, that the extensively distributed, spacious, thin-walled venous sinusoids form normally the principal functioning vascular bed for the actively circulating blood in the marrow; i. e., they correspond largely to the capillaries of other organs. These are the vessels that have been seen and described as the fundamental units of the bone-marrow by those who have worked in this field; and, while being the most outstanding structures in injected marrow, by virtue of their caliber they are quite as easily seen and followed in the uninjected state. By most writers they are termed the venous capillaries. It would seem that venous sinus or venous sinusoid might be more appropriate and desirable terminology, inasmuch as there are already two types of true capillaries in the marrow, as recognized and interpreted in these observations.
All of the vessels thus far described were plainly apparent, either grossly or with the aid of the binocular microscope. The analysis of the circulation up to this point had been comparatively simple through the study of injected material; when an attempt was made, however, to study, under an oil-immersion lens, the detailed ramifications of the smaller vessels and the extent and continuity of the individual endothelial cell distribution, difficulties were at once encountered. It was found that analysis of tTiese finer points in normal marrow is extremely unsatisfactory, if not quite impracticable. In order to analyze with any certainty the finer ramifications of the vascular pattern, i. e., the cytological relationships, it is essential, in the first instance at least, to have a marrow depleted as far as possible of all the free cells. An attempt was therefore made to produce experimentally a hypoplastic bone-marrow in the pigeon. The desired condition was secured through simple starvation for periods varying from 10 to 18 days.
Protocol, Pigeon 19 A.
January 29. Pigeon in excellent condition, weight 475 grams. Diet restricted to fresh water every morning. Condition remained excellent up to February 7. February 10, condition good.
February 15, pigeon in fair condition but emaciated; weight 340 grams. Operation sanv date.
3.15 p.m., ether anesthetization; posterior incision, cannula inserted. 3.25 p.m., warm physiological salt solution started at 130 mm. Hg. 3.31 p.m., salt stopped. 3.32 p.m., warm india-ink (1-4) at 85 mm. Hg. 3.39 p.m., ink stopped.
One radius and ulna fixed in 10 per cent formalin and cleared. Marrow from opposite radius and ulna fixed in Helly's fluid (Zenker-formol). Imbedded and cut in serial sections.
In such an experimentally produced hypoplastic marrow (fig. 2) three types of cells were observed, fat-cells, reticular cells, and endothelial cells. In order to analyze the relations of these three cell-types the vessels of the marrow were washed out with physiological salt-solution and then injected with india ink. The fat-cells, together with their nuclei, were readily distinguishable and quite characteristic. They were more numerous in the hypoplastic marrow, having apparently replaced to a large extent the depleted cellular areas. In the fixed tissue these cells appeared as empty spaces, limited by a thin but distinct membrane. Each contained a more or less flattened oval nucleus, eccentrically placed and but faintly stained, owing to the small amount of chromatin. Such cells made an easily discernible network. Frozen sections of the fresh tissue, stained with Sudan III, indicated the increased extent of these deposits of fat in the cytologically depleted marrow.
Reticular elements which conformed to all of the known criteria were to be seen. They were large pentagonal or hexagonal cells with large, round, vesicular nuclei; the cytoplasm took a faint eosin stain, the nuclei showed moderate chromatin content.
The endothelial cells, in the main, conformed to certain standards and were recognized through various characteristics. In the areas where the endothelium could be seen lining the venules and the capillaries connecting them with arterioles there was no difficulty in its identification; but there were capillaries in the bonemarrow where, even after taking all the histological characteristics of endothelium into consideration, certain cells could not be definitely classified. This was especially true in normal uninjected marrow. Unfortunately, a specific stain for identifying endothelium in sections has not been developed up to the present time, and such characteristics as size, morphology and peculiarities of the nuclei are not always adequate criteria. The methods developed by McJunkin (1919), Foot (1921), Wislocki (1921), and others, dependent on the specific phagocytic function of endothelium for various colloidal suspensions and vital dyes, were all tried in the bone-marrow with indifferent success, but it is possible that additional experiments, now being carried out, will give us at least some valuable leads in further finer differential data applicable to the problem. It must not be forgotten, however, that such methods depend upon direct contact between the phagocytizable particles and the endothelial cell; therefore, assuming that the capillaries described below are probably normally non-patent to the circulating blood, we still have left the need for further means of differentiating endothelium. Realizing fully, then, the limitations of our present methods and the difficulties for final determination in the case of a certain few individual cells, I have tried to analyze the picture presented by these injections on the basis of data available at this time for their interpretation.
As stated in a preliminary communication (Doan, 1922), it is not until sections
as thin as 5 micra (fig. 2), from a relatively complete injection of a hypoplastic
marrow, are seen under an oil-immersion lens that the full import of the nature
and extent of the bone-marrow circulation begins to be realized and perhaps partially understood. First of all, the gross structures — the main longitudinal vessels,
transverse smaller branches, arterioles, a few transition capillaries, and the venous
sinusoids described above — were easily verified in the serial sections. But in
addition to these I have found, appearing between the fat spaces in well-outlined and clearly defined channels, a most extensive system of capillaries, hitherto
unsuspected. Many of these capillaries appeared to have been non-patent and
functionally dormant so far as the active blood circulation is concerned. This was
borne out by the difficulty and infrequency of then- demonstration in the ordinary
marrow injections, where they were totally collapsed and could be seen only as
septa surrounding the fat-cell spaces.
Fig 2. Drawing of a hypoplastic marrow, injected with india ink, showing venous sinusoid and intersinusoidal capillaries. From the radius of an adult pigeon (19 A), e. c, endothelial cells lining capillaries, r. c, reticular cells of the marrow; n. /. c, nuclei of fat-cells; r. 6. c, red blood-cells; v. s., venous sinusoids; cap., intersinusoidal capillaries surrounding the fat-cells, with the granules of carbon of the injection fluid scattered throughout the extent of their channels. These capillaries are seen to be in direct communication with the large venous sinusoids via the characteristic conical openings. Hematoxylin and eosin; 5/jX700.
Figure 2 shows these extensively ramifying channels to be semi-collapsed.
Only a trace of fine ink-granules reveals the presence of a potential lumen, the
caliber of which appears insufficient for the passage of even a single blood-cell
without difficulty. Toward the epiphyses there is this complete encircling of each fat-space by these minute vessels. They are seen to lead directly from the large
venous sinusoids by way of typical conical openings and appear to be continuous
with them. This is illustrated in figure 3, which is an enlarged drawing of the
portion of figure 2 indicated by the square. These vessels are not capillaries, in
the sense of an arterio-venous transition, but extend from venous channel to
venous channel; they are intersinusoidal. There is no break in the continuity of
the endothelium which forms these slender channels from sinusoid to sinusoid.
There was no extravasation at any point and the material injected followed these
vessels everywhere. It was quite evident that these channels were closed, in the
sense that there was no extravasation or diffuse permeation of the tissues by the
injected ink.
The attempt to differentiate an extravasation from a true circumscribed distribution of perfused particles within definite channels was not made without a full appreciation of the marked tendency of such granules to follow a reticular framework closely in any injection into diffuse connective tissue. This characteristic of reticular tissues to be outlined by extravasated particles, thus simulating, more or less closely, definite channels, is recognized and acknowledged, and it is obvious that the possibility of error of interpretation in injections of mesenchymatous tissue requires a corresponding amount of attention and care in analysis. There were, however, five points apparent in the interpretation of these studies which emphasize strongly the non-fenestrated character of the vascular bed of the bone-marrow. (1) In injections showing a diffuse permeation of the medullary parenchyma there have been demonstrable ruptures in vessel walls. (2) In extravasation it was clear that the extruded granules were neither phagocytized nor regularly distributed along one side, but adhered promiscuously and heterogeneously to the surface of the parenchymal cells, thus more or less concealing their outline. In contrast to this, the particles within a definite lumen were scattered here and there along the sides of the lining cells on the inside of the channel only. (3) In an analysis of comparatively complete injections, showing this extensive, inter-sinusoidal capillary bed, not only could these channels be distinctly followed by the granules of ink, but the reticular network or framework of the medullary parenchyma could be seen in the same areas without any attached granules of ink. (4) The walls of the veins and venules appeared as continuous endothelium-lined channels, similar in appearance to the vascular bed elsewhere in the body, but with conical openings into the tiny capillary network. (5) Finally, I have obtained relatively complete injections of these very fine, extensive, lacelike vessels without the slightest evidence of any of the injected particles outside the closed channels in the parenchyma. In other words, in the adult bone-marrow here studied, there was no evidence of any fenestrated vessel-wall, similar to that described by Mollier (1909) for the spleen. One need only contrast a true extravasation with one of these injections to recognize the difference at once. It is very possible, however, that in an injection of normal bone-marrow that filled only arterioles, transition capillaries, and venous sinusoids these conical capillary projections might be interpreted as fenestrated openings.
The endothelial cells of the inter-sinusoidal capillaries were thinned out, in contrast to their number and arrangement in a larger vessel, and in many instances had been forced apparently into the interstices between encroaching fat-cells and looked more nearly like primitive embryonic endothelium. They could, nevertheless, be seen to line these spaces through which granules of the injected fluid had been forced. The picture then was that of a very extensive capillary bed which simulated, in the appearance, distribution, and arrangement of its vessels and cell elements, an embryonic plexus rather than the ordinary mature capillary plexuses elsewhere recognized in the adult. This plexus was lined everywhere by intact endothelium.
It may now be possible to bring out, clearly and definitely, the really striking contrast between the type of circulation to be found in the spleen and that inherent in the bone-marrow. There has been, in the past, a tendency to draw analogies between the two circulations. This, we feel, is quite unjustified, both from the standpoint of the function and from the very different nature of the two structures. Mall (1902, 1903) showed, in a final and crucial experiment, that the spleen was adapted to an easy, rapid, and complete emptying of its blood-content at any given moment. He tied all of the splenic veins in a dog, under ether, and let the arteries fill the spleen with blood to its maximum distention; he then cut the ligatures from the veins and watched the speedy contraction of the organ, and proved by frozen sections that the pulp, which had been engorged with red cells, became totally empty in a few seconds. This could be possible only in case the entire splenic pulp were to be regarded as a peculiar capillary bed in very free communication with its efferent veins. The demonstration of the fenestrated endothelial lining of the veins of the splenic pulp by Mollier (1909) completed the understanding of this special type of circulation. The well-known bands of smooth muscle in the trabecular are accessory structures peculiar to this system. The spleen is therefore a contractile organ, capable of emptying itself at intervals, and thus providing a means of propelling the whole blood, which has free access to the interstitial tissues, back into and through the general circulation. In contrast to this, the venous sinuses of the bone-marrow have an intact endothelial wall; the intersinusoidal capillaries are discrete and are perhaps never, or almost never, in the direct line of the circulation. Furthermore, the organ is inclosed within rigid bony confines, frequently with bony trabecule subdividing the marrow-substance, a condition as far as possible from that found in the contractile spleen. The spleen and the bone-marrow are unlike both structurally and physiologically, and without any real basis for analogical comparison. C. K. Drinker, in association with K. R. Drinker and C. C. Lund, to whose work reference has already been made, attempts to explain the circulation of the bone-marrow in relation to its physiological function. He has found that no experimentally induced increase of pressure will cause an increased discharge of cellular elements from the marrow into the general circulation. He has been unable by any physiological method to "wash out" the developing cells of the marrow. The red cells are delivered into the circulation in cycles at varying intervals, independent of circulatory influences. The areas of developing red cells, as seen in the bone-marrow, show all the cells in a given area to be in the same phase. Drinker hypothesizes a "growth pressure" delivery of these blood elements into the general circulation after first having "grown through" the extremely delicate walls of the sinusoids. This process occurs periodically and without any definitely demonstrable relation to the blood-pressure or circulation and obviously without the possibility of any inherent expansilecontractile mechanism.
In injections of the white rat, the marrow (of the ribs particularly) showed the same gross vascular arrangement as that described for the long bones of the pigeon. There were two central vessels with transverse branchings giving rise to an extensive plexus toward the circumference. In a few experiments on the rabbit, cat, and dog, the normal marrow of both the radius and ulna showed the same general characteristics, though in an apparently less extensive degree throughout the shaft. An occasional section from the mammalian tissues presented here and there the typical inter-sinusoidal, semi-collapsed type of channel, with a few fine ink granules marking its existence. Drinker and his coworkers find these same indications in their most carefully controlled mammalian injections. One of their figures shows a single inter-sinusoidal lumen, as identified by a perceptible line of fine ink granules, identical in appearance with the channels we have seen so much more extensively distributed in the pigeon. While the primary purpose for inducing a hypoplasia of the pigeon's marrow was that more accurate cytological relationships might be determined, it may be, as Dr. Drinker has suggested, that the hypoplastic marrow, through an increased fluidity supplanting the depleted cellular areas, has provided the optimum conditions for demonstration by injection of* this otherwise non-demonstrably patent or occult system. In other words, the normal incompressibility of the marrow-tissue within its bony cavity may be altered. If such be the case, a similar condition of induced hypoplasia in the mammal must precede the demonstration of the completeness of the analogy between the vascularization of avian and that of mammalian marrow. This is a problem in itself, inasmuch as simple starvation of the mammal will not produce the hypoplasia desired.
Discussion
The question that immediately presents itself is that of the function of this vast bed of endothelium extending throughout the bone-marrow, which, as far as can be determined, does not function as a channel for the active circulating bloodstream, at least not normally and regularly. In the absence of full experimental evidence, it is natural and helpful for one to reason by analogy in an attempt to secure working hypotheses in explanation of the phenomena not at present fully understood. This is not without full comprehension of the very great difficulty of following such a line of reasoning without the possibility of grave error.
Richards (1922) has recently reported observations on the glomerular activity in the frog's kidney. He believes that the majority of the glomerular capillaries are not continuously functioning actively, but that there are intervals during which the individual glomerular capillary is closed to the main blood-current. It is possible that could the hemopoietic tissues be examined directly and as satisfactorily as has been done in the case of the frog's kidney by Richards, a similar phenomenon in the marrow capillaries would be found.
Krogh (1918, 1919) has published some most illuminating observations on the capillary circulation in the muscle of the frog and guinea-pig. He finds that in resting muscle most of the capillaries are in a state of contraction and closed to the passage of blood. It was impossible to inject, even under high pressures, any but the few functioning capillaries that were patent at the moment; but by tetanic stimulation, with gentle massage, or in spontaneously contracting muscles a large number of capillaries were opened up and were subsequently observed to contract again. He found the average diameter of open capillaries in resting muscle to be much less than the dimensions of the red corpuscles which become greatly deformed during their passage. Finally, he has shown and called attention to the important fact that clinical hypersemia and anaemia are due mainly to changes in the caliber and number of open capillaries, and that the capillaries are not merely passively dilated by blood pressure but are controlled by a " capillario-motor system" independent of the " arteriomotor system."
It was only through ingenious pressure injections that capillary channels, long suspected but often denied, were finally demonstrated in the valves of the heart by Bayne-Jones (1917). It is conceivable that under certain physiological conditions they may be more obviously patent. Rich (1921), in experiments on the omentum, has shown, both by induced inflammation and by histamin injection, a capillary bed much increased over that seen in the normal omentum, demonstrating the large number of ordinarily non-patent, occult vessels capable of responding and functioning protectively when occasion demands. Lee (1922), in some investigations on lymphatic circulation following the ligation of the thoracic duct, described a most interesting phenomenon. Within 10 days after the careful complete ligation of the thoracic duct he found a most extensive anastamotic distribution of fine lymphatic vessels spreading out along the wall of the aorta, and eventually (within two weeks) a completely compensated, equilibrated lymphatic circulation was established. It seems probable that these may be preexisting collapsed channels which become functionally patent under the stimulus of the new conditions. In view of what we know of the capillaries elsewhere, may it not be that, under excessive demand for blood-cells, when we recognize grossly an increased activity and vascularity of the marrow (red marrow versus yellow marrow), these otherwise collapsed capillary channels become patent and function to help meet the crisis? They may thus be a very important unit of the defensive mechanism of the body. Drinker and his associates were unable, however, to demonstrate satisfactorily these accessory channels in the mammal following the return of the blood-volume to normal after a large hemorrhage, when it might be expected that all possible avenues of delivery for cellular elements would be functioning.
On the other hand, in view of Sabin's (1920, 1921) derivation of red bloodcells, clasmatocytes, and monocytes in the chick embryo from endothelium, there remains still another possible function for the marrow-capillaries, or rather the endothelium of the marrow-capillary. It will be remembered that the endothelium of these capillaries is embryonic in appearance. In hyperplastic marrow injections, Drinker has described the disappearance of a detectable endothelial lining to the vessels and ascribes the lack of extravasation of injection granules, even with these apparently open vessel walls, to the close packing together of the developing cells, which he believes grow into and through the .yielding endothelium. If the red cells were formed intravascularly in an extra-circulatory capillary bed with embryonic endothelium as their source, the apparent cellular border described by Drinker might be these developing cells inside a greatly distended capillary, with wall so stretched and endothelial cells so altered by rapid proliferation as to be unrecognizable as such. The fact that there is no parenchymal diffusion with injections, even though the wall appears to be patent, would seem to suggest this. After saponin injection, Drinker and his collaborators noted the appearance of nucleated red cells in the peripheral circulation prior to an increase in the leucocyte count. They ascribe this to the fact that the developing red cells are in "closer proximity to the circulating blood." This would be literally true were their development assuredly intravascular. Even though the extravascular origin of the erythrocytes in the adult mammal is practically universally accepted to-day, Drinker, it would seem, has more nearly sensed the only justifiable attitude tenable at the present time when he states: "Red cells are apparently formed outside the blood-stream and enter the moving current as a result of growth pressure. It will be noticed that we have not declared for the extravascular origin of the erythrocytes, but have simply said that they arise outside the blood stream." In our present state of knowledge this is all that can be said.
Finally, there is the possibility that these strands of endothelium are never opened up to the circulation as such in the bone-marrow, but represent filaments of cells, like the angioblastic chains described by Sabin (1920) in the embryo, which, in repeated cycles, multiply and make new generations of red corpuscles, the preexisting cone-shaped openings into the sinusoids marking the avenue of entrance for the cells into the blood-stream. Such an interpretation would explain the discrepancies in connection with the relation of endothelium to the formation of the red corpuscles and thus harmonize the two divergent ideas of the intravascular versus the extra-vascular origin of erythrocytes. Under such a view the red cells could be considered as coming from endothelium, but endothelium so placed that the new red cells would not be in the active current of the blood as actually within the sinuses. The cells would, nevertheless, be so placed with reference to the sinuses as to gain a ready access to the functioning lumen without calling for any special destruction of the wall of the sinusoid.
Cunningham (1922), in his study of the cellular reactions during the production of exudates in the peritoneal cavity, obtains no evidence either for or against the participation of the endothelium of the neighboring capillaries of mesentery and omentum in the formation of exudative cells. He points out the difficulty of differentiating reticulum and endothelium in spleen and lymph-glands of the adult mammal when attempting to determine which of these cells is progenitor of the circulating mononuclear. However, certain observations have led him to "suggest the hypothesis that if the circulation be cut off from a group of capillaries, the endothelial cells of which still obtain sufficient nourishment to prevent cell death [the condition that probably exists in the bone-marrow normally], these cells may undergo a cataplastic reversion to the syncytial angioblastic or embryonic endothelial type, with subsequent differentiation into clasmatocytes." Sabin (1921) has proved conclusively the endothelial origin "of the clasmatocyte in the embryo. Furthermore, the work of Macklin and Macklin (1920), who found that areas of endothelium in new-formed capillaries appear to become transformed into clasmatocytes, and the work of others along similar lines, make it practically certain that the protean possibilities of endothelial differentiation in various parts of the functioning mature organism are only beginning to be appreciated.
The mere knowledge and recognition of the presence of this extensive distribution of endothelium in bone-marrow not regularly functioning as a blood-channel is a step in the direction of the determination of its relation to the blood-cell production of the marrow and at least a presumptive indication for further studies, with this possible specific relationship as an objective hypothesis.
This investigation is the direct outcome of Dr. F. R. Sabin's work on the origin of blood-cells in the chick embryo and was undertaken at her instigation. Throughout the development and interpretation of the reported findings, her constant help and criticism have been indispensable. It is a pleasure to express also my gratitude to Dr. R. S. Cunningham for his advice and many helpful suggestions, and to Mr. James F. Didusch for the excellent illustrations accompanying the text.
Summary
- The arterial supply of the bone-marrow is secured via the medullary artery, the periosteal vessels along the shaft, and some vessels near the articular extremities which supply the epiphyses as well. The arterioles are relatively few in number.
- Normally there are a few "transition capillaries" functioning as the intermediary communication between arterioles and venous sinusoids.
- The very extensive distribution of large-lumened, thin-walled venous sinusoids, probably forming the real, active, functioning vascular bed of the marrow, is the most characteristic thing about the gross circulation in bone-marrow. The venous drainage is threefold, corresponding to that of the arterial supply.
- A hypoplastic marrow is essential for the analysis of the finer distribution of the blood-channels. In such a marrow can be seen a very extensive intersinusoidal capillary plexus, hitherto unsuspected, its normal state being possibly one of collapse.
- The vascular system of the bone-marrow is a closed system, no fenestrated vessel- walls being demonstrable in this series of experiments.
- Endothelium apparently forms a continuous lining throughout the vascular ramifications in the marrow, being therefore much more extensively distributed through the medium of the widespread capillary plexus than has been indicated in the usual marrow injections heretofore described.
- The splenic and marrow circulations are contrasted, with a view to showing the fallacy of an analogous comparison of the two.
- The possible significance of the endothelial distribution and occult capillary system of the marrow is discussed.
Bibliography
The blood-vessels of the Jour. Anat., vol. 21, pp.
Bayne-Jones, Stanhope, 1917.
heart valves. Amer.
449-462. Bizzozero, G., 1891. Nouvelle recherches sur la structure
de la moelles des os chez les oiseaux. Arch.
Ital. de Biol., vol. 14, p. 294. Brinckerhoff, W. R., and E. E. Tyzzer, 1902. On
amphophile leucoeytogenesis in the rabbit.
Jour. Med. Res., vol. 8, pp. 449-495. Bunting, C. H., 1906. Experimental amemias in the rabbit.
Jour. Exp. Med., vol. 8, pp. 625-646.
1919. The regulation of the red blood-cell supply.
Contributions to Medical and Biological Research, dedicated to Sir William Osier. Paul B. Hoeber, vol. 2, pp. 824-828.
Cunningham, R. S., 1922. On the origin of free cells of serous exudates. Amer. Jour. Physiol., vol. 59.
Danchakoff, W., 1908. Untersuchungen iiber die Entwicklung des Blutes und Bindegewebes bei den Vogeln. Anat. Hefte., vol. 37, pp. 473-589.
1908. Zur Herstellung der Celloidinserien. Ztschr.
f. wissensch. Mikr., vol. 25, pp. 32-37.
1909. Untersuchungen iiber die Entwicklung von
Blut und Bindegewebe bei Vogeln. Das lockere
Bindegewebe des Hunhchens im fetalen leben.
Arch. f. mikr. Anat., vol. 73, pp. 117-181. Dents, J., 1888. La structure de la moelle des os et la
genese du sang chez les oiseaux. La Cellule,
Lierre and Louvain, vol. 2, p. 241. Dickson, W. E. C, 1908. The Bone Marrow. Longmans,
Green and Co. Doan, C. A., 1922. The capillaries of the bone-marrow of
the adult pigeon. Johns Hopkins Hosp. Bull.,
vol. 33, pp. 222-226. Drinker, C. K., K. R. Drinker, and C. C. Lund, 1922.
The circulation in the mammalian bone-marrow,
etc. Amer. Jour. Physiol., vol. 62, pp. 1-92. Ehrlich, Paul, 1891. Farbenanalytische Untersuchungen
zur Histologic und Klinik des Blutes. August
Hirschwald.
Ferrata, Adolfo, 1918. Le Emopatie, vol. 1. Milano.
Foot, N. C, 1921. Studies on endothelial reactions. Jour. Exper. Med., vol. 33, p. 271.
Foote, J. S., 1921. The circulatory system in the bone. Smithsonian Misc. Coll., vol. 72, No. 10.
Hoyer, H, 1869. Zur Histologie der capillaren Venen in der Milz. Anat. Anz., vol. 17, pp. 490-497.
Krogh, August, 1918-1919. The supply of oxygen to the tissues and the regulation of the capillary circulation. Jour. Physiol., vol. 52, p. 457.
Langer, K., 1S77. Ueber die Blutgefasse der Knochen der Schiideldaches und der harten Hirnhaut. Denkschr. d. k. Akad. d. Wissensch. Math, naturw. Klasse, Wien, vol. 37, pp. 217-240.
Lee, F. C, 1922. The establishment of collateral circulation following ligation of the thoracic duct. Johns Hopkins Hosp. Bull., vol. 33, pp. 21-31.
Macklin, C. C, and M. T. Macklin, 1920. A study of brain repair in the rat by the use of trypan blue. Arch. Neurol, and Psychiatry, vol. 3, p. 353.
Malt., F. P., 1902-03. On the circulation through the pulp of the dog's spleen. Amer. Jour. Anat., vol. 2, pp. 315-332.
Maximow, A., 1909. Untersuchungen iiber Blut und Bindegewebe. I. Die friihesten Entwicklungsstadien der Blut- und Bindegewebszellen beim Saugetierembryo, bis zum Anfang der Blutbildung in der Leber. Arch. f. mikr. Anat., vol. 73, pp. 444-561.
1910. L T ntersuchungen liber Blut und Bindegewebe.
III. Die embryonale Histogenese des Knochenmarks der Saugetiere. Arch. f. mikr. Anat., vol. 76, pp. 1-113.
McJunkin, F. A., 1919. The origin of the phagocytic mononuclear cells of the peripheral blood. Amer. Jour. Anat., vol. 25, p. 27.
Minot, C. S., 1912. Development of the blood. Manual of Human Embryology (Keibel and Mall), 1912, vol. 2, Philadelphia. (Handbuch d. Entwicklungsmesch. d. Menschen (Keibel and Mall), vol. 2, 1911).
Mollier, S., 190'J. Ueber den Bau der Milz. Sitzngsb. d. Gesellsch. f Morphol. u. Phsyiol. in Milnchen. 1911. Die Blutbildung in der embryonaten
Leber dea Mensehen und der Saugetiere. Arch. I raikr. Anat.. vol. 74, pp. 474-524.
Pappenheim, Artstdb, 1919. Morphologisehe Hematologic. Hand 1. Included in vol. 23 of Folia hsematol.
Rich, A. R., 1921. Condition of the capillaries in histamine shock. Jour. Exper. Med., vol. 33, pp. 287-298.
Richards, A. N., 1922. Kidney function. Araer. Jour. Med. Sci., vol. 103, pp. 1-19.
Rindfleisch, G. E., 1880. Ueber Knockenmark und Blutbildung. Arch. f. mikr. Anat., vol. 17, pp. 1-11: also pp. 21-42.
Sabin, F. R , 1920. Studies on the origin of blood-vessels and of red blood-corpuscles as seen in the living blastoderm of chicks during the second day of incubation. Contributions to Embryology, vol. 9. pp. 213-202, A Memorial to Franklin Paine Mall, Carnegie Inst. Wash. Pub. 272.
1921. Studies on blood. The vitally stainable
granules as a specific criteria for erythroblasts and the differentiation of the thiee strains of the white
blood-cells as seen in the living chick's yolk sac.
Johns Hopkins Hosp. Bull., vol. 32, pp. 314-321. Schafer, E. A., 1912. Quain's Anatomy. Vol. 2, pt. 1, p. 154. Schriode, H., 1907. Die Entstehung der ersten embry onalen Blutzellen des Mensehen. Verhandl. d.
deutseh. path. Gesellsch., Dresden, 1907; Jena,
1908, vol. 12, pp. 360-306. SpaI/TEHOLZ, W., 1914. Ueber das Duichsichtigmachen von
menschlichen und tierischen Praparaten. 2nd
edition. S. Hirzel, Leipzig. Van der Stricht, O., 1892. Nouvelles recherches sur la
genese des globules rouges et des globules blancs
du sang. Arch, de Biol., vol. 12, p. 199-344. Venzlaff, W., 1911. tjber Genesis und Morphologic der
roten Blutkoruerchen der Vogel. Arch. f. mikr. Anat., vol. 77, pp. 377-431. Weidenreich, F., 1903-1901. Die rotem Blutkorperrhen.
Ergebn. der Anat. u. Entwicklungsgesch., vols.
13 u. 14. Wisi.ocki, G. B., 1921. Experimental observations on bonemarrow. Johns Hopkins Hosp. Bull., vol. 32.
pp. 132-134.
Description of Plate
Fig. 4. Distal half of radius, pigeon 16 As, injected with india-ink (dilution 1-4). Marrow cleared in situ by the Spalteholz method. Injection very incomplete. The nutrient artery and efferent vein are seen occupying the nutrient, foramen. The longitudinal distribution of the main vessels is seen. Near the epiphysis one small artery enters the marrow cavity while several small veins emerge. There is extensive anastomosis between the medullary vessels and these extra-diaphaseal vessels. There is an indication of the venous-tuft distribution seen more distinctly in the other figures. X 10.
Fig. 5. Radius from pigeon 35 A, the marrow having been embedded and sectioned serially, 150 p. The central longitudinal vein is shown with two main venous tufts anastomosing. A branch of the longitudinal artery connects with the venous tufts by way of the "transition-capillary" link. These vessels function normally and, though few in number, appear to be the regular avenues for the passage of blood from the arterial to the venous side. XI 10.
Fig. 6. Portion of radius of pigeon 36 A, cleared with marrow in situ, showing a more extensive injection than figure 4. The venous and arterial tufts suggest a segmental distribution. The nutrient vessels of the bony cortex are seen extending into and anastomosing with the medullary vessels. X26.
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