Talk:Paper - The development of the lobule of the pig's liver (1919)
THE DEVELOPMENT OF THE LOBULE OF THE PIG's LIVER
FRANKLIN PARADISE JOHNSON
Department of Anatomy, University of Missouri
Development of the connective tissue septa 301
Capsula fibrosa 308
Formation of new lobules 308
Role of the connective tissue septa 318
Growth of the liver 319
The development of the Uver lobules offers a difficult problem owing to the fact that in most animals the lobules have no definite boundaries, one running directly into the other without demarcation. In section, therefore, the livers appear to consist of solid masses of parenchyma, pierced at more or less regular and alternating intervals by various-sized branches of the portal and hepatic veins. This lack of lobule definition is so great that one readily appreciates why Weber, in 1842, denied the presence of true lobules in the human liver, and one may himself doubt the appropriateness of the term 'lobule' for either the portal or hepatic units.
Early recognizing this difficulty, I chose for material the liver of the pig, for I hoped that by using an animal in which the liver lobules are definitely marked out, this difficulty Avould be greatly overcome. I soon found that the pig's liver does not show indications of dividing septa until about birth, and that the connective tissue septa are not definite until about the second month of postnatal life. Contrary to the state 299
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ment of Mall ('06), lobule formation is not complete at this time, for I find with Lewis ('12) that the multiplication of lobules continues long after birth." The few late stages of the pig which I have been able to procure, consequently, have been of great value in furnishing evidence concerning the development of the hepatic lobules.
Because of the importance of the connective tissue septa, I have found it advantageous to divide the development of the pig's liver into two definite periods — one before and one after the septa are indicated. I shall speak of the former simply as the 'early stages,' of the latter as the 'late stages.' The early stages include those up to but exclusive of an embryo of 254-mm. in length; the later stages include the 254-mm. embryo and extend to the adult.
In attempting to determine the manner in which the units of the liver multiply, I first gathered together a series of selected stages of developing livers. But so far as lobule formation is concerned, this was unnecessary. I agree with Mall, ('06) when he says, "the great difficulty is to recognize the same thing from step to step," but I find the greatest difficulty is to recognize the limits of a lobule in its three dimensions in any single early stage. In the later stages, however, because of the presence of the connective tissue septa, the lobules are definitely bounded. Since in any developing liver the lobules present numerous instances of every stage of development, it is possible by a little study to arrange the various stages in their proper sequences. In this manner the development of the liver lobules may be easily and most satisfactorily determined from any single late stage.
Inasmuch as I have worked out the development of the liver lobules from stages in which the connective tissue septa are present and continually growing and delimiting new lobules, I found it necessary to study first the origin of the septa. This was the more essential, for an understanding of the different developmental stages of the septa makes it possible not only to recognize dividing lobules, but to distinguish between the newly formed and the old lobules.
DEVELOPMENT OF LOBULE OF PIG's LIVER 301
DEVELOPMENT OF THE CONNECTIVE TISSUE SEPTA
In stages until nearly birth the liver of the pig shows no indications of connective tissue septa. The parenchyma is made up of cells not greatly different from those of the adult. The sinusoids, which appear proportionately large, are lined with endothelial cells. The 'Gitterfasern' or 'reticulum of Mall' ('96) is demonstratable following Bielschowsky's silver-impregnation method, and is also slightly discernible after staining with Mallory's triple connective tissue stain. The branches of the hepatic and portal veins interdigitate with one another and furnish a means by which hepatic and portal lobules may in certain areas be roughly outlined. The large branches of the portal veins are readily distinguished from the hepatic, for they are accompanied, as in the adult, by branches of the hepatic artery and of the bile duct; some of the smaller veins are determinable only with the aid of serial sections.
In figure 1 is shown a longitudinal section through a branch of the portal vein, taken from the liver of a pig 229-mm. in length. The interdigitation of its branches with those of the hepatic vein is clearly shown. Surrounding the larger branches of the portal veins, bile ducts, and hepatic arteries is connective tissue. Staining with Mallory's triple connective tissue stain clearly demostrates in it the presence of collagen fibrils. Where the collagen fibrils are in contact with the parenchyma they often send short fibrils into its reticulum. Surrounding the hepatic veins there is but a thin layer of connective tissue, which, moreover, does not extend out to as small branches as does that surrounding the portal veins. The hepatic cells are polyhedral in shape, with slightly granular protoplasm and distinct chromatic nuclei. They are grouped in strands, but, as stated by Theopold, a radial arrangement about the central vein is not yet to be found. Here and there are to be seen small clusters of nucleated round cells. These cells, which we know to be developing blood-cells, were first described by Kolliker ('79), but were thought by Toldt and Zuckerkandl ('75) to be young hepatic cells. That Kolliker's interpretation is correct,
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Fig. 1 Section of liver of pig 229 mm. in length. Mallory's triple connectivetissue stain. H, hepatic veins; P, portal veins; c. /., collagen fibrils; h. d., bile duct; h.a., hepatic artery; b.c, group of blood cells; g.c, giant-cells. X 50.
Fig. 2 Section of liver of pig 254 mm. in length, showing first indications of interlobular septa. X 50.
DEVELOPMENT OF LOBULE OF PIG's LIVER 303
however, has been abundantly proved by the researches of Van der Stricht ('91); Kostanecki ('92), Engel ('99), Lobenhoffer ('08), and others. Large mononuclear giant-cells, also described by KoUiker, are to be found sparsely scattered through sections of the liver. In the later fetal stages both the group of embryonic blood-cells and the giant-cells become fewer and fewer, as is agreed by all investigators. I find with Theopold, however, that they have not all disappeared at birth and that a few may be found for several weeks after birth.
The first indication of a segmentation of the liver parenchyma into hepatic lobules which I have found has been in an embryo of 254-mm. in length. According to Engel ('99), this pig would be one at about full term, since he states that a pig at birth measures 25 cm. Theopold ('10), however, shows that pigs at full term vary considerably in length; that they even attain a length of 35 cm. before birth. In the 254-mm. specimen, the liver cells appear more vesicular, their protoplasm stains but faintly. The boundaries of the hepatic lobules as seen in figure 2 are in many places definitely marked. A close analysis of the dividing medium, however, shows that it contains no collagen fibrils, but rather its appearance is due to a change in the parenchyma. A higher-power drawing of this stage is shown in figure 3. To the right and left of the drawing are shown terminal hepatic on central veins; above and below are terminal portal veins. Extending from portal vein to portal vein is shown the first evidence of an interlobular septum. It consists of hepatic cells which are more coarsely granular and stain more deeply than the remaining cells; similar cells are found surrounding the portal veins. However, the thing which makes the septum most apparent is the arrangement of the cells. As seen in sections, they form two more or less regular parallel rows, an arrangement which becomes greatly accentuated in slightly older stages. Another factor which in places intensifies the distinctness of the septa is the presence of small branches of the portal veins. Nucleated blood-cells found in the sinusoids along the septa also aid in making it more distinct. I have studied carefully the retic
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Fig. 3 Higher magnifications of portion of figure 2, sliowing interlobular septum. H, hepatic vein; P, portal vein. X 260.
Fig. 4 Section of liver of a pig four days old, showing arrangment of lobule border cells in parallel layers. H, hepatic veins; P, portal veins. X 50.
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ulum of the septum stained by the Bielschowsky method and beUeve that a thickening of it at this stage is very doubtful.
In a slightly longer pig measuring 265-mm. (also unborn) a somewhat similar condition is found, although it is not quite so far advanced as the 254-mm. specimen. This variation in the degree of development I have found to be especially marked in stages around birth. Thus, in specimens of two hours and twenty-four hours after birth, the septa appear no more distinct than those of the 265-mm. stage, while a specimen of three days is no further advanced than a 254-mm, embryo. A similar varation in the degree of development is noted by Theopold ('10). Again a radial arrangement of the hepatic cells is absent.
In a pig of four days the liver shows a more advanced condition (fig. 4). The cells are coarsely granular and stain deeply. The lobules are in most places definitely marked out by the arrangement of their border cells. This consists in their forming continuous sheets of cells about each lobule; thus, when seen in cross-section, they form two parallel rows. Between these rows of cells is found reticulum which is slightly thickened, but contains no collagen fibrils. A Berlin-blue-gelatin injection of the liver of a second pig of four days also shows definite evidence of the lobular nature of the liver. As shown in figure 5, the injection mass has passed through the sinusoids into the central veins. The sinusoids between the lobules, although present and forming free anastomoses between one lobule and another, are smaller and somewhat less numerous than those within the middle of the lobules. This condition results in the appearance shown in the figure, the lobules being separated from one another by a clear translucent zone — an appearance which is more strikingly brought out in the thick sections.
For the first time can a radial arrangement of the liver cells be seen. This point is shown in the injected specimen, since the direction of the trabeculae of liver cells corresponds with the direction of the sinusoids. Although the radial arrangement is not well marked at this stage, it becomes increasingly
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more definite in livers of pigs of one, two, and three weeks. At two months it is as strongly marked as in the adult. These observations are in accord with those of Theopold ('10), who states that the radial arrangement of the liver cells is first seen in pigs in the second half of the first week of postnatal life, and that the arrangement gradually increases in definition, although at the sixth week it is not strongly marked in all lobules.
In pigs of three and four weeks old the hepatic lobules are still more strongly marked. The parallel rows of cells with the intervening layer of reticulum are still in evidence. In addition, however, the reticulum in many places shows the addi
Fig. 5 Portal injection of liver of a pig four days old, showing fewer and smaller sinusoids along interlobular septa. X 40.
tion of delicate strands of collagen fibers (fig. 6), as demonstrated by staining by both Mallory's and Bielschowsky's methods. In certain places these fibers extend from portal vein to portal vein; in other places they extend from one portal vein toward another, leaving, however, the nodal-point region devoid of them. From various degrees of this condition it is safe to conclude that the connective tissue of the interlobular septa has its orgin in the connective tissue of the portal canals and grows outward from them, pushing its way into the reticulum already separating the lobules. According to Theopold ('10), complete septa arising from connective tissue already present are in evidence, in certain places in the liver of a pig eight days
DEVELOPMENT OF LOBULE OF PIG S LIVER
old. He does not state, however, whether or not this connective tissue contains true collagen fibrils.
Fig. 6 Interlobular septa of liver of a pig three weeks old. Mallory's triple connective-tissue stain. X 250. Fig. 7 Same, two months old pig.
The exact manner in which the connective tissue of the septa is laid down, that is, whether the collagen fibrils have their origin from fibroblasts situated in the portal canals and push their way out into the interlobular reticulum or whether the fibroblasts first migrate and give rise to the collagen fibrils
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in situ, I have not determined. This point, it seems to me, is of little moment, inasmuch as in either case the origin is from the connective tissue of the portal canals. It is interesting to note that the connective tissue surrounding the sublobular veins does not take part in the formation of the septa to nearly so great an extent as does that of the portal canals. Around some of the larger sublobular veins a few out-pushing collagen fibrils may be found, but it is evident that the bulk of the connective tissue forming the septa springs from the portal canals.
Ca-psula fibrosa (Glissoni). Collagen fibrils are differentiated in the pig's liver as early as the 80-mm. stage. They are found principally in the portal canals, and to a less extent around the larger sublobular veins, being especially abundant in the region of the porta hepatis. Extending out in either direction from this region, as seen in the cross-section, there are thin strands of collagen fibrils which spread around, but do not entirely encircle the liver. These strands represent the forming external capsule of Glisson. A similar section of the liver of a pig 111 mm. in length, shows a complete capsule. It consists, however, of a very delicate layer of collagen fibrils which lies close to the liver parenchyma. In all stages up to birth the capsule has this appearance, the collagen fibrils showing no appreciable thickening; on the other hand, in certain stages it appears to be less distinct than in the younger ones; in some its presence as a distinct layer is extremely doubtful. After birth it rapidly becomes thicker and continues to augment in strength and density until the adult condition is reached.
FORMATION OF NEW LOBULES
Mall ('06), in his study of the structural unit of the liver, concluded that Thoma's laws concerning the formation of new blood-vessels explain the formation of new lobules. He states:
As the vessels grow the liver tissue increases in quantity, but the liver lobules do not increase in size indefinitely, because Thoma's first law is constantly at work and will soon break up the larger lobules into a number of smaller ones. In all cases the length of the capillaries remains constant, and when they appear to be too long and too
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numerous, it is always found that some of them have ah'eady turned into small veins and thus mark the beginning of new lobules or of new portal units.
While in general the idea expressed in the above statement may be correct, nevertheless it should be emphasized that the sinusoids of the liver are not all of equal length. Mall, himself, shows that within a single lobule they are of various lengths, that there are short paths and longer ones between the portal and hepatic veins. Yet he believes that all the capillaries of the lobule and of the liver as a whole are equally favored by the circulation. In a former paper (; 18 a) I have shown that in the adult pig the lobules vary greatly in diameter; the lengths and numbers of their capillaries, consequently, must also vary. Again, in my study of the development of the lobules, I find that the lobules do not reach a certain constant size before they begin to divide into new ones; a large lobule and a smaller one placed side by side may be in a similar stage of division.
In his recent studies on the growth of the blood-vessels in the tail of the frog larva, Clark ('18) has shown that as certain areas of tissue increase in size, the surrounding capillaries send into the area endothelial sprouts which develop into additional capillaries. When these new capillaries become functional, that is, when a flow of blood through them is inaugurated, there results an increased flow through and an increase in the size of certain ones of them which are favorably located. According to Clark, it is the increased amount of blood flowing through these vessels w^hich causes them to enlarge. Conversely, Clark has shown that when the flow through capillaries is diminished, the capillaries become smaller, w^hen the flow" stops altogether, they close, break in two, and are gradually retracted into the vessels with which they were originally connected.
The application of these observations to the growing liver may be made as follows : As the hepatic cells increase in number, new- capillaries are formed, bringing about an increased flow" of blood in, and a subsequent increase in size of, certain capillaries leading to and from the new ones. These enlarged capillaries become new" branches of the portal and hepatic veins.
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As the portal and hepatic veins increase in length, the blood flow through certain capillaries along their sides is diminished and finally stopped, with the result that these capillaries disappear through retraction. Thus it is that capillaries arise from only the terminal branches of the hepatic and portal veins.
It was Mall's idea ('06) that new lobules form by the splitting up of old ones, yet he does not make clear the exact manner in which the splitting takes place. Apparently, he believed that the lobules split and become fragmented into a number of parts, each of which is capable of giving rise to a new lobule. Furthermore, he believed that the fragmented parts of several different lobules are capable of uniting into a single lobule, for on page 278 he describes a lobule as arising from three adjoining ones. Yet his diagrams illustrating the formation of new lobules (figs. 41 to 43) are not suggestive of any such 'shattering' and reuniting of parts of lobules, but rather of an accumulation of orderly binary fissions; if we should add other figures to his diagrams to fill in the wide gaps which he has left, we should have similar pictures to those which I have shown in figures 11 to 15.
As stated above, the manner in which the hepatic lobules of the pig develop may be understood from the study of any single stage of development. However, it is only in the later stages where lobule boundaries are made definite through the ingrowth of connective tissue that the process can be determined with any degree of accuracy. I have accordingly used for the following description a liver from a pig two months old. The observations recorded have been checked with other stages, both before and after the connective tissue septa have made their appearance; so far as I can determine, the formation of lobules is the same in all.
The development of new hepatic lobules can be best understood by watching the changes which take place in the growing hepatic veins. Each of their new branches marks the beginning of a new hepatic lobule. The portal veins spread over the surfaces of or between the lobules, dividing repeatedly, their terminal branches being more numerous than those of the
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hepatic vein, as pointed out by Mall, In their growth they bear a certain constant relation to the growing hepatic veins, through which relation a constant flow of blood is maintained.
In figures 8 to 10 are shown hepatic lobules in successive stages of development. Figure 8 is a section of a slightly elongated lobule with a bifurcating vein. Although in all other respects this lobule is similar to other single lobules, it can be definitely stated, because of the branching of its hepatic vein, that it has begun to divide. In figure 9 the new branches of the central vein are longer, having kept pace with the lengthening lobule. The new veins grow, as described by ]Mall, by the enlargement of certain sinusoids. In addition to the new veins is to be seen the beginning of a cleavage of the lobule. This is represented by the arrangement of certain hepatic cells into parallel rows, between which collagen fibrils later appear. It is to be noted that the septum occupies the plane of the original central vein and bisects approximately the angle formed by its two new branches. The free edges of the septum lie just above the fork of the central vein, which position (fig. 9) it maintains for some time.
That the hew central veins lengthen in many instances at the expense of the old one, that is, by cleavage of the latter, there is little doubt. In those lobules in which the central vein is just beginning to bifurcate it is relatively long; in those in which the branches are long the old central vein is usually shorter. This apparent splitting of the old central vein seems to be caused by the growth of the septum into the fork of the central vein. In many instances where the cleavage of a lobule is nearly complete the old central vein is split down almost to the point where it enters the lobule.
The further separation of the lobule into two new ones takes place slowly by the continued growth of the new septum from the sides of the lobule toward the old central vein. "\Mien the septum is complete, the remaining portion of the old central vein is found to lie between the two new lobules; it may then be spoken of as a small sublobular vein.
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Fig. 8 Dividing lobule from liver of pig two months old. //, hepatic vein; P, portal vein. X 50.
Fig. 9 Dividing lobule from liver of pig two months old. C\ and C-i, branches of central vein; 8, new interlobular septum. X 50.
Fig. 10 Dividing lobule from liver of pig two months old. Ci, old central vein; Ci, new central vein; S, new interlobular septum. X 50.
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Usually, however, before the time the connective tissue septum is complete, the new lobules themselves have begun to divide, thus giving rise to what Kiernan ('33) has described as compound lobules. That the compound lobules of the adult are, as I have stated before ('18 a), incompletely divided lobules due to incompletely developed connective tissue septa is quite apparent; they are similar in every detail to those found in the developmental stages.
Compound lobules have also been described and modeled by Debeyre ('10) who states that in the pig as well as in other mammals they are more numerous than the single lobules.
"Le petit lobule classique, isolable, existe, mais il est presque exceptionnel."
While I am able to confirm Debeyre in that the compound lobules of the pig's liver are numerous, I have not found the single lobule to be exceptional. If blocks of pig's liver are treated with 50 per cent to 75 per cent hydrochloric acid (Johnson,' 18), the connective-tissue septa are destroyed and the lobules fall apart. The compound lobules, joined together by liver parenchyma, are preserved intact. Examination of the lobules under the binocular microscope shows that many of the lobules are single. It is true that the compound lobules may be torn into their component parts, but this does not happen if they are handled carefully. If the maceration is allowed to proceed to the proper degree, the lobules separate from one another on a very slight amount of shaking. The compound lobules can be divided only upon rough handling. From a number of such preparations I have observed that the compound lobules vary greatly in number, size, and number of component parts in the livers of different adult pigs.
In addition to the above-described process of division, there is another which frequently takes place. Instead of the distal end of the hepatic vein bifurcating, a new branch is given off from its side. The new branch, as shown in figure 10, enters a portion of the lobule. As it gradually becomes larger and longer a cleavage takes place in the lobule in a similar manner to that described above. The cleavage divides the original
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lobule into two new ones, one of which is supplied by the old central vein, the other by the new one. That new branches of both the hepatic and portal veins arise as outgrowths from the main trunks is stated by Mall.
Surface lobules often divide by the combination of the two processes just described. The long axes of such lobules are placed at right angles to the surface of the liver. When a surface lobule divides its central vein bifurcates, the new branches spread apart and then bend to become perpendicular to the surface (fig. 23). A septum then starts from the surface, midway between the two new branches, and grows down to the fork of the central vein. Thus two elongated lobules are marked off. These then become cut up transversely, new branches from either the new or old central veins extending in to the lower ends of the lobules. In some instances, however, the new hepatic veins which supply the lower ends of the lobules are formed earlier by a bifurcation of the new central vein.
Slight variations in development are often met with in certain groups of new lobules. Figure 24 is from a wax reconstruction of three almost completely formed lobules which have developed from a single one. The original central vein H has bifurcated into the two branches h and h. The original septum is shown between A and H; it is almost complete. A new cleavage, B, however, has grown in from the side and has joined the original septum, thus completely cutting off the lobule N. A new vein, h' , has grown out from the original central vein into the new lobule 0, which is not yet entirely separated from the lohule M. In the process of splitting, the original central vein H has become interlobular in position.
Very seldom in the pig's liver is found a lobule through which the central vein extends to become the central vein of another lobule. One of the few which I have found in looking over a large number of lobules is shown in figure 25. Judging by the stage of development of its connective tissue septa, I have explained its existence in the following manner: The three lobules. A, B, and C, were originally one: its central vein H bifurcated into h and h, and the longitudinal septum grew
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down from the surface, bisecting the angle formed by the new branches of the central vein. This septmii instead of following the central vein, either turned off to one side or was met by another septum from the side, thus leaving the old central vein completely surrounded by liver cells. That such central veins do not long remain within the lobules is evident, since none of the larger sublobular veins are found passing through single lobules. The manner in which such a vein leaves its lobule, however, I have not fully determined. It may be that they later become interlobular in position by the ingrowth of additional septa. It seems more probable, however, that they gradually become shifted to one side, since in several other similar lobules, I have found them eccentrically placed. In these lobules new central veins were found arising at right angles from the eccentrically placed ones, seemingly marking the path along which the old veins must have shifted.
From the above description, it is evident that lobule formation takes place by the binary fission of an elongated lobule. This is preceded by either a bifurcation of the central vein or by the sprouting out of a new vein from it. In either case, the two veins extend into the halves of the old lobule. By the time the cleavage is complete, each half of the old lobule represents a fully formed new lobule. The self-explanatory figures 11 to 15 show diagrammatically the manner in which a single lobule may give rise to a larger group.
In figure 28 is shown a wax reconstruction of a group of lobules, all of which are supplied by a single sublobular vein. This vein and its accompanying branches are shown in figure 27. From the above description, it follows that all the lobules of this group must have arisen from a single lobule, and, conversely, all the lobules which have arisen from this original lobule belong to this group. Each branch of the hepatic tree goes to an individual lobule and represents an independent cleavage of the hepatic parenchyma. With the exception of a few of the terminal branches, it is impossible to tell which have arisen through a dichotomous bifurcation of terminal central veins and which as new outgrowths from existing stalks.
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It is interesting to note, however, that all the branchings of the hepatic vein shown, with possibly one exception, are dichotomous. This condition which has been mentioned by Roux ('95) obtains in the adult and is easily verified by the examination of celloidin corrosions of either the hepatic or portal trees.
Figs. 11 to 15 Diagrams representing manner of formation of hepatic lobules. The numbers represent the number of times the hepatic vein has bifurcated. Vessels marked 1', 2', etc., indicate new growths, not terminal bifurcations.
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Thus far I have shown that the terminal hepatic veins always keep within the lobules ; that each of its new branches is indicative of the formation of a new lobule. The portal veins in growing spread between the lobules, tending always to keep within a certain distance of the central veins. It should not be inferred, however, that the growth of the portal veins follows in point of time that of the hepatic veins. Both undoubtably grow hand in hand, increasing in length and diameter, and branching as the increasing parenchyma demands. With the increase in the size of lobules, there is ever the tendency to spread the two sets of veins further apart and this tendency is at all times being counteracted by the formation of new veins.
The manner in which the new^ veins develop, that is by a widening out of certain sinusoids, has already been described. These new veins, according to Mall, grow into those regions on the surface of the lobules which are most distally situated from the branches of the portal veins, areas which Mall has termed 'nodal points.' The whole matter of nodal points can only be clearly understood from a study of the lobule in its three dimensions; they are uncircumscribed areas on the surfaces of the lobules which lie between the terminal branches of the portal veins and between the central vein of one lobule and that of an adjacent lobule. In the dividing lobule shown in figure 26 as many as ten nodal points may be counted, one of which is just forming coincidentally with the formation of the new dividing septum. The statement of Mall's ('06) that the portal and hepatic veins alternately grow toward the nodal points and break them into fragments to form new nodal points holds good, according to my observations on the pig's liver, for only the portal veins. In figure 26 the whole upper surface of the lobule was originally represented by a single nodal point; the newbranch of the portal vein has broken it up into two new ones. But the terminal hepatic veins always lie within the lobules, they grow toward the nodal points only as the parechyma of the lobule increases, but they never reach them. In the lobule represented in figure 26 the single central vein H was originally directed toward the single nodal point of the top surface, but
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with the sphtting of the lobule two new nodal points are formed, and the two new central veins are again found to be directed toward these new nodal points. That the hepatic veins do not grow into the nodal points and break them into fragments is still more evident when it is considered that the terminal central veins are always found within the lobules, while the nodal points are always interlobular in position.
The role of the connective tissue septa in lobule formation. In order to understand clearly the role played by the connectivetissue septum in the splitting of one hepatic lobule into two, it will be necessary to review in their proper sequence the events which take place in the formation of new lobules. The first unmistakable evidence of splitting in a lobule is the formation of an additional branch of the hepatic vein, either by the bifurcation of a terminal vein or by its outward growth from the side of an existing vein. At the same time, however, new branches of the portal vein are similarly forming, although these latter branches are not indicative of the formation of new lobules. Next follows the beginning of the cleavage, which consists at first of the parallel arrangement of liver cells and is afterward followed by an ingrowth of connective tissue. The growth of the new branches of the portal vein takes place along the fissures of the new septum, if they have not preceded it (fig. 26). The new septum comes to possess in this way a new nodal point. Branches of the portal vein, however, do not actually enter the new septum to break up its nodal point until both new lobules are fully formed, and not until the new lobules have increased sufficiently in size to warrant this addition to the vascular system.
It is evident, therefore, that the real and essential phase of lobule formation is that concerned with the vascular system. New lobules are actually in existence when certain relations between the new branches of the portal veins and those of the hepatic veins become established. The new branches of the veins determine the position of the new septa, just as they do shortly before birth when the first evidences of dividing septa appear. In the early stages, before traces of the septa are at all apparent, similar branches of both svstems of veins are formed, establish
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ing similar relations to one another. Thus they form undefined lobules, which are only different from those which are separated from one another by connective tissue septa, in that their vascular systems are in more open communication with each other.
We may unhesitatingly conclude, therefore, that the connective tissue septa play no active part in the formation of lobules in the pig's liver. The boundaries of the lobules are determined by the vascular system; these in turn become apparent by the parallel arrangement of the border cells; finally the connective tissue fibers invade the reticulum between the parallel layers of cells and the septum becomes established. Just what may be the stimulus which causes the growth of the connective-tissue fibers, I have not determined; it may or it may not be due to certain influences of the circulatory system. When this problem is solved we may understand why it is that in certain animals the septa are complete, in others partially, and in still others entirely absent; and further, why in the seal (Mall, '06) the connective tissue septa bound the portal rather than the hepatic lobules.
THE GROWTH OF THE LIVER
I have thus far considered the formation of new lobules without regard to the changes which thej' produce in the liver as a whole. The growth of the liver is accomplished by two means: first, by an actual increase in size of the lobules; second, by a multiplication of lobules. I have shown in a previous paper the approximate rate of growth of the hepatic lobule of the pig. In the table below, it will be seen that the size of the larger lobules remains constant between the 80-mm. and the 154-mm. stages. From the 229-mm. stage on they show a gradual increase. The growth of the lobule is shown graphically in figures 16 to 19, all of which are camera-lucida drawings of equal magnification.
It was believed by lUing ('05) and Theopold ('10) that after the connective-tissue septa are laid down the growth of the liver takes place entirely by an enlargement of its lobules. Theopold, however, states that the septa are not completely formed until
FRANKLIN PARADISE JOHNSON
the first few weeks of postnatal life and that during this time innumerable new lobules are forming. He further states that in the early stages of the liver the hepatic branches outnumber the portal, and that lobule formation ceases when a single portal
Figs. 16 to 19 Outline camera-lucicla drawings of lobules of four different stages to show increase in size of hepatic lobules. Fig. 16. 254 mm.; fig. 17, four weeks old; fig. 18, two months old; fig. 19, adult. X 25.
DEVELOPMENT OF LOBULE OF PIG's LIVER
Table showing the average diameters of some of the larger lobules of the pig's liver at various stages of the development
0.33 0.33 0.33 0.35 0.43
1 The diameter of the lobules of the adult pig as given here (1.2 mm.) is the same as that given previously (Johnson, '18 a). Mall ('06) gives it as 1.4 mm. There can be no question that Mall obtained his figures as I did, by averaging only the diameters of large lobules as seen in sections of the liver. The actual 'average diameter' of the liver lobule is an altogether different thing, one difficult to calculate. It must be pointed out, as I have recently shown ('18 a), that the hepatic lobules of the pig vary greatly in size within a single liver, and also that the average size varies for different adult livers. Reference to the table given in my former paper will show how I obtained the 'average weights per lobule' given in the table. From an average of these 'average weights per lobule,' I have attempted to calculate the average diameter, assuming a spherical form for the lobule. This gives 0.8 mm. Since, however, the lobules are not spherical, the diameter would in reality be less than 0.8 mm. With regard to the relatively average diameter of the lobules in the young stages, I am confident that there is less variation in size than in the adult, and that the average diameter of the lobules of the stages 80 to 154 mm. is only slightly less than 0.33 mm.
branch has formed for each hepatic branch present. These observations, however, I have been unable to confirm.
The growth of the lobules likewise takes place in two ways: first, by an increase in the numbers of the hepatic cells and sinusoids ; second, as has been pointed out by Toldt and Zuckerkandl (75) andby Illing ('05), by an actual increase in the size of the hepatic cells. The latter, however, is counterbalanced to a certain degree by a decrease in the diameter of the sinusoids. The rate of increase in size of the cells and sinusoids, and numbers of the cells, is shown in table 2.
The formation of lobules begins at an early stage (Mall's onelobule stage), and, I believe, continues throughout all stages to the adult. This is in agreement with the statement of Lewis ('12),
FRANKLIN PARADISE JOHNSON
that "the multipHcation of lobules continues long after birth, and partly divided, compound forms were recognized in the adult by Kiernan." At just what time lobule formation is fully completed I have not determined ; there are some evidences that a few lobules are undergoing division in the so-called 'adult' stages I have studied.
In studying the growth of a solid mass of tissue such as the liver, we must consider the possibility of both peripheral and central growth; in the former the increase takes place by a laying down of successive layers on the surface ; in the latter the growth takes place by an increase in the size and number of the units within the organ. The growth of the liver may be described as
TABLE 2 I
OF SINUSOIDS IN
CELLS IN AVER AGBD-SIZED
13.9 12.5 16.01 11.7 13.1 19.8
4 weeks old
2 months old
1 The cells in this stage were unusually large and vesicular.
central, that is, taking place more or less evenly throughout. After a study of sections of developing livers, I was at first led to believe that mitosis proceeded somewhat more rapidly in the surface lobules than in the deeper seated ones ('18 b) but the further study of this point does not confirm this view. Mitotic j&gures even in favorable sections of well-preserved livers are not abundant,^ making it extremely difficult to note any actual variation in their distribution. I am inclined to believe that in general all parts of the liver are growing at the same rate, since the liver
1 lUing ('05), who has studied the liver cells of growing and adult animals, states: ' 'In hiesigen Institute (Tierarztlichen Hochschule zu Dresden) sind im Laufe der Jahre Tausende von Leberpraparaten von alien Haustieren untersucht, aber niemals mitotische Kernfiguren gefunden worden."
DEVELOPMENT OF LOBULE OF PIG's LIVER 323
tissue is everywhere of the same type, since all its parts are equally favored by the circulation, and since, so far as can be determined, dividing cells and lobules are uniformly distributed in sections. Concerning the growth of the liver. Mall ('06) states :
In development the liver structure shifts distalwards, successively tearing off its capillary connections with the main veins, gradually rearranging the architecture of the lobules, often fracturing and scattering them.
Although I agree with Mall that the liver parenchyma shifts distalward as the liver grows, I do not believe that the lobules become fractured and scattered or that capillary connections are torn away. I find no instances of fractured lobules other than those undergoing normal binary fission and no instances of the scattering of lobules since the lobules maintain their connections with the hepatic veins throughout. As for the tearing away of capillary connections along the beginning of sublobular veins, it seems more probable that such capillaries retract (Clark, '18), because of the decreased flow of blood through them. I believe that the shifting of the liver substance peripherally produces no destructive changes in the arrangement of its units other than that the form of the lobules is altered by the pressure of one lobule against another due to their manner of growth.
That the shifting of the liver tissue from the center towards the periphery is taking place constantly in the growing liver, there can be no doubt. This does not mean, however, that certain lobules or groups of lobules are shifting or slipping past one another or that they shift along the walls of the blood-vessels. In fact, the shifting takes place in such a way that the general relations are not at all disturbed. The blood-vessels, ducts, connective tissue, etc., grow and shift correspondingly with the lobules, and while the liver tissue is shifting from the center toward the periphery, it is not to be inferred that the deeper-seated lobules will eventually come to lie on the surface or even nearer to it. On the contrary, they are continuously getting further and further away from the periphery for the simple reason that the more peripheral lobules are likewise constantly increasing in number.
FRANKLIN PARADISE JOHNSON
These points concerning the growth of the hver can best be exemphfied by the following diagrams. In figure 20, let the circle A represent a circumscribed mass of liver tissue surrounding a large vein of either the hepatic or portal systems. The zones B and C represent concentric zones of liver substance about A. When A increases in size (fig. 21), it might at first seem that it would press against the zone B and this in turn against C, tending to produce changes in them. But the zones B and C are likewise growing at the same rate as A. In their growth they expand just as metal rings expand when they are heated. The
Figs. 20 and 21 Diagrams to show manner of growth of liver as a whole.
fact that the center is filled by A alters in no way the shape the zone B will assume. The expansion of C similarly makes room for B. The process is exactly similar to that which takes place in the expansion of any solid ball of metal when heated evenly. A lobule in figure 21 at the point m would be further away from both center and periphery than at its former point ?n in figure 20. Similarly, the vein increases in length as the liver expands. Its increase is due in part to the growth at the tips of its branches and in part to an increase in length of its trunk.
The above hypothesis explains in a general way the growth of the liver and is based on the assumption that the liver tissue
DEVELOPMENT OF LOBULE OF PIG's LIVER 325
is everywhere growing at an equal rate. While this assumption is probably true for groups of lobules taken collectively, it is not always true within individual lobules. I have shown above that when a lobule grows, it may increase more in one diameter than in another. It is probable that this type of unequal growth gives rise to local pressures here and there and that these in turn determine to a certain degree the shapes of the lobules.
1. The first evidence of connective tissue septa found was in an embryo of 254 mm. in length. The growth of the septa is gradual and they are not fully formed until about two months after birth. Their origin is first indicated by the arrangement of the border cells of the lobules in parallel layers. The connective tissue of the portal canals sends sprouts into the reticulum between the layers of border cells. These sprouts, coming from opposite directions, meet in the region of the nodal points thus completing the septa. Additional septa are formed with the development of new lobules. Their paths are similarly marked out by the parallel arrangement of border cells. The collagen fibrils of these septa sprout out from the connective tissue about the portal veins and from that of those septa already present,
2. The collagen fibrils of the capsula fibrosa (Glissoni) appear first in the region of the porta hepatis and spread over the surface of the liver. They completely cover the liver in a pig of 1 11 mm. The capsule remains as an extremely thin and delicate layer until birth, after which time it gradually thickens.
3. The formation of new lobules may be described as a necessary readjustment on the part of the parenchyma to circulatory difficulties. It is accomplished by a binary fission of lobules already present, the cleavages taking place only in lobules which have developed two central veins. The new central veins may arise, 1) by a bifurcation -of the tip of a growing central vein, or 2) by a sprout from the side of an existing vein. The plane of cleavage bisects the angle formed by two new veins. The cleavage is evidenced by the arrangement of the hepatic cells into parallel
326 FRANKLIN PARADISE JOHNSON
layers, into the reticulum between which collagen fibrils push from the surface of the lobule. In the case of 1) the septum completes itself gradually in the plane of the old central vein, which, when the septum is complete, becomes a sublobular vein. Usually before the septum is fully completed the newly formed lobules show evidences of further segmentation.
4. The growth of the portal veins takes place by an increase in length and by the formation of new branches which spread themselves between the lobules. They grow into the nodal points and split them up into additional ones.
5. The central veins grow by an increase in length and by the formation of new branches which are always intralobular in position. They are directed and grow^ toward certain nodal points. but never reach them.
6. In their growth both the terminal hepatic and portal veins never approach nearer one another than one-half the diameter of the lobules, as pointed out by Mall.
7. The connective tissue septa of the pig's liver play only a passive role in the formation of new lobules. They grow in between the new lobules after they have really formed.
8. The growth of the liver takes place throughout all stages, 1) by an increase in the number of its lobules; 2) by an increase in size of its lobules.
9. The growth of the lobules likewise takes place, 1) by an increase in the numbers of its hepatic cells and sinusoids; 2) by an increase in the size of the hepatic cells. The latter is counterbalanced to a certain degree by a decrease in the diameters of the sinusoids.
10. In general, all parts of the liver grow simultaneously and equally. There is a constant shifting of lobules peripherally, but this takes place in such a way as to produce a minimum amount of change in the relation of one lobule to another.
11. It is probable that the lengthening of lobules in their growth (not an equal swelling in all directions) produces local disturbances which manifest themselves by giving rise to the great variety in shapes of the lobules.
DEVELOPMENT OF LOBULE OF PIG's LIVER 327
Clark, E. R. 1918 Studies on the growth of blood-vessels in the tail of the
frog larva — by observation and experiment on the living animal. Am.
Jour. Anat., vol. 23, pp. 37-88. Debeyre, a. 1910 Morphologic du lobule hepatique. Bibl. Anat., T. 19, pp.
249-263. Engel, C. S. 1899 Die Blutkorperchen des Schweines in der ersten Halfte
des embryonalen Lebens. Arch. f. mikros. Anat., Bd. 54, S. 24-59. Felix, W. 1892 Zur Leber- und Pankreasentwickelung. Arch. f. Anat. und
Entw., S. 281-328. Illing, G. 1905 Vergleichende histologische Untersuchungen iiber die Leber
der Haussaugetiere. Anat. Anz., Bd. 26, S. 179-193. Johnson, E. . P. 1917 The later development of the lobule of the pig's liver.
Anat. Rec, vol. 11, pp. 371-372.
1918 a The isolation, shape, size, and number of the lobules in the
pig's liver. Amer. Jour. Anat., vol. 23, pp. 273-283.
1918 b Additional notes concerning the development of the pig's
liver. Anat. Rec, vol. 14, p. 40. KiERNAN, F. 1833 The anatomy and physiology of the liver. Trans. Roy.
Soc. of London, pp. 711-770. KoLLiKER, A. 1879 Entwickelungsgeschichte des Menschen und der hoheren
Wirbeltiere. II Aufl., Leipzig. KosTANECKi, K. V. 1892 Die embryonale Leber in ihrer Beziehung zur
Blutbildung. Anat. Heft., Bd. 1, S. 303-322. Lewis, F. T. 1912 The development of the liver. In Keibel and Mall's
Human Embryology, vol 2, pp. 403-428. Philadelphia and London. Lobenhoffer, W. 1908 tJber extravaskulare Erythropoese in der Leber
unter pathologischen und normalen Yerhaltnissen. Beitr. zur. path.
Anat. und zur AUg. Path., Bd. 43, S. 124-146. Mall, F. P. 1896 Reticulated tissue and its relation to connective tissue
fibrils. Johns Hopkins Hospital Reports, vol. 1, pp. 171-208.
1906 A study of the structural unit of the liver. Am. Jour. Anat.,
vol. 5. pp. 227-308. MiNOT, C. S. 1900 On a hitherto unrecognized foi-m of blood circulation
without capillaries in the organs of vertebrata. Proc. of the Boston Soc. of Nat. Hist., vol. 29, pp. 185-215. Roux, W. 1895 Gesammelte Abhandlungen iiber Entwickelungsmechanik der
Organismus. I. Functionelle Anpassung. Leipzig. Theopold, J. 1910 Untersuchungen iiber die Entwickelung der Leberlapp chen des Schweines. Bern. 32 pp. ToLDT, C, UND Zuckerkandl, E. 1875 tJber die form und Texturveranderun gen der menschlichen Leber wahrend des Wachstums. Sitz.-Ber.
der Kais. Akad. der Wiss. Wien, Bd. 72, Abt. 3, S. 241-295. Van der Stricht, O. 1892 Nouvelles recherches sur la genese des globules
rouges et des globules blancs du sang. Arch, di Biol., T. 12, pp.
EXPLANATION OF FIGURES
22 Dividing lobule of the liver of a two-months-old pig. X 55.
23 Dividing surface lobule of same liver. X 55.
24 Dividing lobule of same liver; for description see page 314. X 55.
25 Dividing surface lobule cff same; for description see page 314. X 55.
26 Dividing lobule of same, showing both portal and hepatic veins; for description see page 315. X 55..
DEVELOPMENT OF LOBULE OF PIG'S LIVER
FRANKLIN PARADISE JOHNSON
EXPLANATION OF FIGURES
27 and 28 Wax reconstructions of hepatic tree with the group of lobules which it drains. The central veins and the lobules which they drain have corresponding letters. Those central veins not lettered belong to lobules not shown in figure 28.
DEVELOPMENT OF LOBUI.E OF PIG'S LIVER
FRANKLIN PARADISE JOHNSON
Resumido por la autora, Lucile Witte.
Histogenesis del musculo cardiaco del cerdo con relacion a la aparicion y desarrollo de los discos intercalados.
1. El tejido cardiaco del cerdo tiene al principio estructura celular y esta compuesto de celulas fusiformes que mas tarde se anastomosan terminal y lateralmente para formar una red de fibras. 2. La estriacion aparece mas temprano en los discos que en las otras partes, pero en focos diseminados en toda la extension del tejido, 3. Los discos aparecen en el estado de embrion de 76 mm. de longitud, mucho mas pronto que en cualquiera de los animales estudiados hasta el presente, con la excepcion del embrion del gato de cuatro dias. 4. Los discos aparecen primero como puntos o bandas iricompletas que comienzan en la periferia de la fibra y crecen hacia su interior. Avanzando el desarrollo se transforman en discos pianos que atraviesan toda la fibra extendiendose despues a dos o mas fibras y finalmente adoptan el tipo mas complejo de discos. 5. Los discos no son mas numerosos en las areas contraidas que en las relajadas. 6. Los discos no se presentan en los extremes de una serie de micleos, constituyendo de este modo una celula, porque se presentan invariablemente en mtimo contacto unos con otros, apareciendo en manchas, y raras veces existe un nucleo entre ellos. 7. La autora propone la teoria que supone a los discos como bandas encargadas de reforzar las fibras musculares, puesto que aparecen proximamente durante la transformacion de las celulas en fibras y aumentan en numero y complejidad con el crecimiento y actividad del corazon.
Translation by Jose F. Nonidez Columbia University