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=The Angiology, Angiogenesis, and Organogenesis of the Submaxillary Gland=

By

Joseph Marshall Flint, M.D.

From the Hearst Anatomical Laboratory of the University of California.

With14 Figures 


In a preliminary note<ref>Journal of Medical Research, Vol. VII, No. 4. 29</ref> on the blood-vessels of the submaxillary gland and their development, the writer outlined brieﬂy the angiogenesis of the circulation, together with its arrangement and distribution in the adult organ. At the same time, attention was called to certain researches of Thoma concerning the principles involved. in the development of bloodvessels and their application to the evolution of the circulation in highly organized glands. The following extracts from this paper emphasize these points again brieﬂy.

:“ The well-known researches of Thoma on the histogenesis of the vascular system offer an explanation of some of the phenomena of vascular development, particularly to the relation between the velocity of the blood current and the size of the vessel that conducts it. The question of the ancestry of arteries and veins was solved by Thoma, who showed in chick embryos that originally they were always simple capillaries. Their subsequent transformation, according to this author, was due to their fortuitous location with reference to the primitive aortae, and the venous ostia of the heart. It has been shown that these facts apply to the vascular development in mammals as well as the chick, and for vascular systems developing in three dimensions, as well as those found in the area vasculosa where the vessels grow in two directions only. In considering the problems of angiogenesis in mammals, it is apparent that Thoma’s histo-mechanical principles do not suﬂice to explain all the facts, nor do they even entirely accord with them. The statement that a new growth of vessels follows a rise of blood pressure in the capillary area must be considered only as an hypothesis and not a demonstrated fact, for this would make the vascular system the stimulus to the development of new cells, while there is considerable probability that it is the new cells which give the stimulus for the growth of new capillaries. It must be obvious that the principal factors that govern organic growth are resident in the cells rather than in the blood-vessels, as is indicated by their behavior in the embryo before the vascular system is laid down. We have still much to learn concerning the factors that arrest the growth of organs when they reach the adult type, but there is little doubt that these phenomena are expressions of cellular rather than vascular activity, since the vascular system maintains. far beyond the usual period of growth, its power of progressive development. It is, so to speak, always in a state of unstable equilibrium, in which both progressive and regressive changes are possible. Certain facts in the development of the bloodvessels of organs have already been demonstrated, the most important of which is that the intrinsic blood supply of organs usually marks out the paths along which the units of structure that compose it have developed.‘ And by following the gradual increase in complexity through a series of injected embryos, the succeeding changes from the simple embryonic to the adult form can be easily demonstrated. It is important to trace these changes not only for the light they shed on the development of the vascular system, but because many obscure features in the structure of organs are elucidated when the mechanics of their development are known.


Excepting certain radical modiﬁcations that take place at the time when the embryo ceases to receive its nourishment from the maternal blood sinuses and independently undertakes the aeration of its own blood, it has been shown that the conditions of the systemic circulation in embryos at term approximate very closely that in adult life. At the same time considerably less differentiation occurs in the intrinsic circulation of the individual organs than in the larger vessels of the general circulation. At the time of birth the structure of most organs is well developed, and the changes which take place are usually quantitative rather than qualitative.- Accordingly, the material for this research was obtained from the submaxillary gland of injected embryo pigs, and the results were subsequently shown to conform to the conditions found in the glands of dogs and human beings.

==Methods==

The technique for the injection of embryos used in the study of the angiogenesis of the submaxillary has been described in detail in another place.’

9 Flint, Welch, Festschrift, 1900, and Reports of the Johns Hopkins Hospital, Vol. Ix., 1900.  

In working up the blood supply of the submaxillary two of the simpler methods have been found especially serviceable, namely, the use of silver nitrate and a saturated aqueous solution of Prussian blue. The chief value of the silver nitrate method lies in the fact that by far a greater proportion of the silver is precipitated in the arteries, giving by a single injection a natural differentiation between them and the veins. Moreover, the endothelial lining of the blood-vessels is beautifully demonstrated by this method. A =Z to 1 per cent solution gives good results. The only disadvantage lies in a tendency for this solution to extravasate somewhat more than the Prussian blue, but there is compensation in the fact that silver injections are usually incomplete. These give clearer pictures of the blood supply in the developing organ, because the details are not obscured by a general ﬁlling of the entire capillary system. Double injections are possible and serviceable in the embryos where it is necessary to diiferentiate arteries and veins, but in the submaxillary, at least, most of the differences between the arterial and venous systems can be clearly shown in the silver nitrate specimens. (Figures 2 to 8.) For the blood supply of the adult organ Prussian blue and lamp black gelatin or a carmine mass followed by a suspension of lamp black in gelatin gives very sharp pictures. The circulation in the adult organ, however, differs in no marked feature from that of the pig at birth, except quantitatively. For a study of the relationship between blood—vessels and cells, sections made by the routine methods demonstrate clearly these points. And, in following the development of the cellular elements of the ducts and alveoli, Mallory’s aniline blue connective tissue stain is of great value: This stain seems to be peculiarly adapted to the use of the submaxillary because it brings out in sharp relief differences between mucous and parietal cells, and stains as well the duct epithelium. Moreover, it differentiates clearly between connective tissue ﬁbers and cell elements.

==Angiology==

In man, the A. submaxillaris is derived from the A.'maxillaris externa, which runs in a small sulcus on the surface of the glandula submaxillaris or directly through its substance as it mounts up over the lower border of the ramus of the mandible. Occasionally the A. submentali contributes submaxillary branches that enter the organ. The Vv. submaxillares sometimes empty into the V. submentalis, but usually are tributaries of the V. facialis communis. In man the arteries do not enter the hilus of the organ with the duct, but join the latter a short distance after it penetrates the gland and take up immediately the close relations which are observed throughout the developmental and adult periods of life. The veins follow the arteries throughout their course. In the pig, however, the duct is joined by the blood-vessels almost immediately after their passage through the hilus, even before branches of the ﬁrst order are given off. The relationship between the duct and vascular systems is absolutely constant and dates from the earliest embryonic history of the organ.

3 Flint, Welch, Festschrift, and Johns Hopkins Hospital Reports, Vol. IX.



Inasmuch as the'nomenclature of the blood-vessels is taken from that of the ducts, a short account of the latter may simplify the description of the vascular system. In pigs, the distribution of the ducts is not unlike that in man, although in the former it is somewhat more regular. In man the duetus submaxillaris enters the hilus of the gland and almost immediately breaks up into branches of the ﬁrst order. These run a very short distance and then terminate in a secondary system, forming interlobular ducts that ramify extensively throughout the gland. From the latter are derived a set of sublobular ducts. These divide once or twice and then terminate in lobular ducts which, entering the hilus of the ultimate lobules, at once exhaust themselves in the extensive ramiﬁcations that form the intralobular ducts and radiate from the hilus to the periphery of the lobule. They terminate in a short duct of slightly smaller caliber termed the intercalary portion connecting them directly with the secreting alveoli of the gland. The intercalary ducts are usually considered as having a much smaller caliber than the ducts of the next lower order. This, however, is not the case, as their lumen, as shown by corrosion preparations, is only slightly smaller than those just preceding. This mistaken impression probably arose from the consideration of the ducts when clothed with their epithelium. The total diameter of the intermediate ducts is much smaller, because the intralobular ducts on one side are lined with columnar epithelium and the alveoli have either high mucous or serous cells on the other, while the intercalated portions of the duct between them have only ﬂattened epithelial cells for their walls. Running in the thick processes of fasciculated connective tissue that form the interlobular spaces, are the blood-vessels that always accompany the ducts. In one way, the interlobular spaces of the salivary glands have a certain resemblance to those of the liver except that the former have two veins instead of the single branch of the portal system found in the hepatic interspaces.

In describing the blood-vessels of the adult organ, it is convenient to divide them into three systems: 

# The glandular circulation from the A. submaxillaris to the alveolar plexus and back to the Vv. submaxillaris. 
# The circulation about the ducts. 
# Circulation in the capsule and septa.

==The Glandular Circulation==

The submaxillary artery in pigs at the hilus or point of entrance is separated by a short distance from the ductus submaxillaris. The two rapidly converge, however, and, as a rule, meet just before the point where ducts of the primary order are found. Before joining the duct, the artery may give ofE_one or two short branches that enter adjacent lobules, as well as a few branches to the large mass of connective tissue which enters the organ at the hilus. The ducts of the ﬁrst order are accompanied by the Aa. principales. These now run in the connective tissue of the larger interspaces at a short distance from the lumen of the duct. There is no marked tortuosity of the main artery or its principal branches, but, at the different angles where the chief ducts are given off, the arteries sometimes wind partly around them or may take a slightly spiral course about the duct. As a rule, however, while there is a slight wavy irregularity in the course of these vessels, a complete spiral arrangement about the ducts is rarely observed. From the principal branches of the main artery the Aa. interlobulares are derived, accompanying ducts of the same order in the interlobular interspaces. From these branches the blood is distributed in radiating directions throughout the gland. They are longer than blood-vessels of any other order. In their course they may divide once or twice and ﬁnally exhaust themselves in lateral branching in the sublobular arteries which run with the sublobular ducts in the center of the primitive lobules. Arteries of this order may in thick injected specimens be easily recognized running in the center of the groups of ultimate lobules bounded by the secondary septa. They divide three or four times with the sublobular ducts and then exhaust themselves in the Aa. lobulares, each one of which enters a primitive lobule at its hilus together with the lobular duct. Once within the limiting membrane of the lobule, the ramiﬁcation is more abrupt and more marked. The intralobular branches of the A. lobularis radiate from the hilus toward the periphery of the lobule, divide with and accompany the intralobular ducts. The terminal divisions of the intralobular arteries run with the intercalary ducts and divide under the clusters of alveoli into the alveolar capillary plexus. The capillaries mount up over the alveoli external to the re ticulated basement membrane and anastomose with the capillary plexus 293. FIG. 1. Diagrammatic reconstruction of the submaxillary gland. Magniﬁed 12 diameters. The reconstruction shows the distribution of the ducts and their termination in the alveoli, together with the arraigementof the accompanying blood~vessels. For the sake of clearness in drawing, there is some discrepancy between the relative size of theblood-vessels and ducts. The blood supply is taken from a pig at birth where the type of circulation is so developed that it diﬂE‘ers in no way from that of the adult. The histological portion and the digested section are from the human submaxillary, while the piece digestion is from the submaxillary of the dog. The limiting membranes of these two tissues have simply been forced to meet in order to show their relationship by the dltlerent methods. Otherwise no violence has been done to the original specimens. (J. M. Flint, A7z_qz'ology of subrnazillary gland.) of adjacent alveolar groups. The meshes are small and regular, and occupy three dimensions. This plexus has anastomoses extending throughout the lobule, but terminates on all sides at the membrana limitans. Each lobule has, therefore, with certain exceptions that will be considered later, an independent circulation.


Beginning in small radicals around the base of the alveoli, venules are formed through the coalescence of capillaries. These rapidly converge to a. point near the terminal arterioles about the intercalary duct to form venae terminales. The veins are short and there is but a single one accompanying each terminal arteriole. At the point of junction of several intercalary ducts, the accompanying terminal veins through their conﬂuence form the intralobular veins which collect the blood from different portions of the lobule and terminate in the lobular vein. This vessel makes its exit from the lobule at the hilus, side by side with the lobular artery. There is no reduplication of the veins of the intralobular system. As soon as the veins leave the lobules, however, and enter the sublobular interspaces, the venous system is doubled, yielding venae comites to each successive division of the A. submaxillaris. The sublobular veins parallel the course of the artery, giving oﬁ numerous anastomotic branches which run over and under the artery forming oblong meshes. It is not uncommon, however, to have the veins separate and pass around the duct and then join on the opposite side, or else have anastomotic branches pass from the venae comites, embrace the duct, and ﬁnally join the main veins lower down. The venae comites with their blood current ﬂowing in the reverse direction follow in a convergent manner the previous divergence of the arteries. Thus the sublobular veins unite to form those of the interlobular system, while these, in turn, join to make the venae principales. The principal veins part with the ducts at the same point Where the artery joins them and leave the gland along with the A. submaxillaris as the Venae submaxillares. These usually empty into the vena facialis communis. In the general glandular circulation We ﬁnd two vascular units, one, a complete terminal system, forming, in Ludwig’s sense, the true unit of circulation, that is to say, blood-vessels supplying a group of structures which have deﬁnite relations throughout the organ with the general framework. These form, in this way, the ultimate indivisible complete unit of structure of which the organ is built up. Such a unit in the submaxillary gland would be represented by the lobule and the whole organ is simply formed of a series of these units. Another vascular unit could be formed of the terminal arteries. These break up in the alveolar capillary plexus which reunite into small venule radicals and empty into the terminal vein. Each lobule is— composed of a. series of such units. Even another larger unit might be constructed from the circulation embraced between the sublobular artery and the sublobular vein, supplying groups of lobules that have deﬁnite relations with the secondary septa. The ultimate lobular unit might Well be considered the least, While the_primitive lobule would be the greatest common structural divisor of the submaxillary gland. The smaller terminal units are comparable to the conditions of the circulation in such glands as the stomach and adrenal where cell complexes having deﬁnite- relations with larger bands of connective tissue do not exist.

==Circulation about the Ducts==

The ductus submaxillaris is embraced by a rich supply of arteries derived from the Aa. principales, branches of which run in a recurrent directionalong the walls of the ducts as far as the hilus. These arteries may anastomose with some frequency (Fig. 2), although the plexus formed by them is not a rich one. They are distributed around the surface of the duct and give off terminal branches which sink to the region just around the base of the duct epithelium and terminate in a capillary plexus formed of small, irregular, polygonal meshes. Through the conﬂuence of the capillaries, numerous venules are derived which empty into large veins forming an irregular venous plexus about the duct. The caliber of these veins is larger than the arteries, and the meshes formed by them are irregular and polygonal. The venous plexus lies below the arterial and from a convergence of its elements larger veins are formed that terminate in the venae principales. In ducts of the interlobular order there is the same arrangement of the blood-vessels around the ducts, except that the elements of the venous plexus are not so large nor is the connection between them so frequent. As one progresses upWard from the main ductus submaxillaris to the lobular ducts, there is a gradual simplifying of the duct circulation, until, in the ducts of the sublobular order, we have afferent arteries derived from the Aa. sublobulares running part way around the duct, breaking up into capillaries which reunite in small anastomosing veins, that pass around the walls of the ducts to terminate in the venae comites. Alternations of artery and veins in these instances are not uncommon and arterioles and veins are in all cases above the capillary plexus. Fig. 3, which shows the circulation in a duct of this order, gives the relations beautifully. It should be noted, however, that one of the venae comites has been removed for the sake of clearness. The lobular and intralobular ducts show a still simpler form of duct circulation. Small arterioles almost immediately break up into capillaries when they are derived from the lobular or intralobular arteries. These quickly reunite again into veins that terminate into venules which empty directly into intralobular veins. This gradual‘ simpliﬁcation of the circulation about the ducts gives a characteristic demonstration of their age, and, in the angiogenesis of the submaxillary circulation, parallels can be drawn between the circulation about the ducts at diiferent periods of embryonic life and the conditions found in the vascular supply of ducts of the different orders. In the very earliest stages the circulation of the main ductns submaxillaris is much like that about the adult lobular duct. At a some— what later stage is approximates that of the duct of the sublobular order, while still later there is a similarity to that of the interlobular system, and ﬁnally the highest development is attained with the complete forma— tion of the arterial and venous plexuses which embrace the main ductus submaxillaris in the adult. It is very easy, moreover, to follow in a single, well-injected gland the evolution of the duct circulation from that of the lobular type to that which embraces the main duct. The small arterioles break up into capillaries and reunite into small venules that ﬁnally terminate in the accompanying veins. These vascular elements enlarge so that the arteries branch and come to lie upon the capillary plexus and the isolated veins receive a few anastomoses from each other. With the growth of the duct and the lengthening of the artery and its ramiﬁcations, the venules which are last formed from FIG. 4. Circulation about an intralobular -capillaries mme to he under the The artery is the upper vessel of the arterial but over the capillary sys tem, and thus we have the three superimposed series of vessels noted about the ducts of the larger order. Throughout the course of the successive divisions of the submaxillary arteries, branches are given off which supply the framework of the interspace. These vessels are extremely irregular and after breaking up into a capillary system that ramiﬁes around the fascicles of connective tissue, venules are formed emptying into the venae comites at irregular intervals. The circulation to the sympathetic ganglia buried in the interspaces also has a similar derivation.




FIG. 2. Blood-vessels about the ductus submaxillaris in a. pig 26 centimeters long showing the arterial, venous and capillary plexuses: arteries light, veins and capillaries dark.




FIG. 3. Blood-vessels about a. sublobular duct. The artery is the upper of the two vessels. One of the venae comites has been left out for the sake of clearness.



==Circulation of the Capsule and Septa==

Blood-vessels that run to the primary and secondary septa are derived from the As. intralobulares. Branches from vessels of the latter type perforate the limiting membrane and almost immediately break up into a loose irregular plexus, the venules from which unite into a single vein that accompanies the septal artery and ﬂows back into the lobular circulation. The blood-vessels in the capsule are derived from two sources, in part from the arteries and veins in the periglandular connective tissue which run on the surface of the connective tissue envelope of the organ in the form of a very irregular plexus of polygonal spaces. These vessels are very small. Frequently capsular branches from the lobular arteries perforate the capsule and join the capsular plexus. These capsular arteries are accompanied by capsular veins. When septal arteries arise from lobules at a point near the capsule, they may pass from the septa and ﬁnally unite with the capsular plexus. With these two exceptions, the circulation in each lobule seems to be complete and independent, save in those cases where a lobule of the first order is not dompletely subdivided into ultimate lobules, so that at their bases anastomoses may occur between the alveolar capillaries of the peripheral portions of adjacent ultimate lobules.

==Angiogensis==

Chievitz<ref>Chievitz, Arch. f. Anat. u. Phys. Anat. Abth., 1885.</ref> has shown that the submaxillary gland develops as a bud from the buccal epithelium. This grows backward toward the angle of the mandible and there begins a dendronal branching that marks the anlage of the submaxillary gland. Owing to the difficulty in injecting very early embryos, a study of the relationship between vessels and cells can best be made in stained sections. The submaxillary gland is shown in a simple form in pigs represented by about 8-3,; centimeters, nape-breech measurement. At this time the branching column of cells is accompanied by an artery that enters with the duct at the simple hilus and lies close to the developing basement membranes. Capillaries forming an irregular plexus can be seen here and there about the ramiﬁcations of the simple tube forming the ductus submaxillaris. The arterioles, venules and capillaries at this stage are usually separated from the reticulated basement membranes by a short intervening mass of syncytium, but as the ducts increase in size this distance is diminished. These relations can be found in stained sections from the glands of pigs 4-3; to 6 centimeters in length. When, however, the pig reaches an age represented by 8% centimeters, the injection of the gland becomes a simple matter and the description can be taken up from injected organs that are cut in half and studied under the stereoscopic microscope. This gives not only the relations of the vessels throughout their course, but also enables the observer to follow them in three dimensions. At this time the gland is reniform, and the vascular supply is comparatively rich. Since the relationship between arteries, veins and ducts established in pigs of earlier age always persists, the course of the ducts accordingly may be determined by the course of the blood-vessels. Indeed, in a pig of this age the duct and its branches are represented negatively, making it possible to follow them nearly as well as though they were either stained or injected. At this period the artery enters the primitive organ at the hilus and branches with the duct. This is the simple stage of the arteria submaxillaris. In its course the artery gives off small branches which almost immediately break up into capillaries. These run around the column of cells and empty at once into the vein accompanying the afferent vessel. The meshwork formed by this capillary plexus is small and irregular and the vessels composing it are often unequal in caliber. Following the branching ducts, the A. submaxillaris divides with each successive ramiﬁcation, giving off still the collateral arterioles that embrace the walls of the ducts. The ramiﬁcation at this time extends to about four orders and the arterioles terminate in a capillary plexus embracing the terminal buds or swellings that form the apices of the growing ducts. These vary in number from four to seven and are distinctly seen as clear spaces sharply demarcated by the embracing capillary plexus. At this 

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no anastomoses with each other. The "mini bud“ may be blood, derived from the terminal arteri oles, passes through the capillary plexus and is gathered again into venules which accompany the terminal artery along the walls of the duct from which the buds have grown, but the alveolar plexus is ﬁner than that around the ducts. Already at this stage, it is possible to see that the blood supply of the entire gland now looks like the vascular distribution in a single lobule of the adult organ. Attention has already been called to the analogy between the two, so the four ramiﬁcations of the A. submaxillaris; Aa. principales, Aa. interlobulares, Aa. sublobulares, Aa. lobulares are like the divisions of the intralobular arteries in the adult. Fig. 5 represents half of an injected gland at this stage, photographed so as to obtain a stereoscopic effect. The branching of the ducts is shown by the course of the blood-vessels and the irregular plexus formed by the latter upon the former is clearly visible. The alveolar plexus, however, does not stand out clearly in this specimen.

In a {{pig}} 12 centimeters in length the ramiﬁcation has proceeded to higher orders, the ducts as shown by the embracing meshwork have increased in size and the plexus embracing them is more highly diﬂerentiated. Small lateral arterioles are now given off from the main arteries which divide into capillaries. These reunite into little venules emptying into the main venae eomites. In a pig 8 centimeters in length, the veins are duplicated as high as the third order, but have not yet taken up their course in such close relation to the artery. In the next stage, however, the veins follow more closely the course of the artery and the capillary plexus extends around the duct from the point where they are situated. Each successive ramiﬁcation remains practically a terminal system, although occasional anastomoses are found rimning from the vessels accompanying one branch of the duct to those about another. These communications, however, are usually venous and not arterial, although connections between vessels of the latter type are occasionally found. The capillary plexus embracing the terminal buds is now much ﬁner and more deﬁnitely organized than in the earlier stages. Arterioles run along with the terminal ducts and suddenly break up into an irregular, ﬁne plexus composed of small polygonal meshes which mount up over the summit of the alveoli and are collected into venules which return to the point of junction of the ducts and alveoli, and there unite into little terminal veins that accompany the ultimate arterioles. At this period an occasional branch from one of the terminal arteries passes out from between the~buds and joins the small plexus that is being formed on the capsule of the gland. As yet septal arteries are not found. Fig. 6 shows an injected silver preparation of a pig at this stage. Except in certain points the mass has not passed over into the veins, for the most part leaving the arteries and capillary plexus around the alveoli the single visible portion of the vascular system. The greater regularity of the alveolar plexus is now clearly shown. 


FIG. 6. Submaxillary gland of pig’s embryo 12 centimeters long. Same method of preparation as Fig. 5. x 10 diameters. In this specimen only the arteries accompanying the ducts and the terminal alveolar plexus are visible.



In a {{pig}} 18 centimeters long the main submaxillary vessels and submaxillary duct are now somewhat separated. Arteries and veins are distinctly seen in the plexus embracing the ductus submaxillaris. These are derived from its vasa comites. The ramiﬁcation has not increased so much in number of orders as in the number of branches. The arteries still preserve their individuality but a duplication of veins is observed as ducts in


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At this period vessels of the lobular type have just been formed and the capillary plexus around the alveoli is very sharp and distinct. Anastomoses have been established between the capillary systems of adjacent alveolar groups. This forms for the ﬁrst time in the history of the organ the ﬁnal arrangement of the circulation in the lobule, although representing it in a very simple form. Here and there the connections between the alveolar circulation and the circulation of the capsule are seen, while, for the ﬁrst time, perforating vessels leave the lobules and ramify in septa of the ﬁrst and second orders. These appear best in Prussian blue specimens where the injection is complete. Compared with other organs, the amount of syncytium from which the ﬁbrillar and cellular parts of the submaxillary framework are derived is proportionately excessive to the quantity of cells forming the primitive ducts which are destined to yield all of the epithelial elements of the gland. In all of the early stages, the blood-vessels are conﬁned to the immediate neighborhood of the ducts, while the syncytium has none. As this syncytium is transformed into strands and septa, they receive a vascular supply of their own, which is not well shown until the pig reaches an age indicated by a nape-breech measurement of 18 centimeters. The interspaces have now attained a deﬁnite size and blood-vessels derived from the vasa comites of the main ducts leave the plexus and ramify in the substance of the connective tissue, here and there giving off arteries to supply the sympathetic ganglia that are embedded in the ﬁbrous tissue forming the main interspaces. Even at this period there is no connection between the circulation in one lobule and that of its neighbor, save occasionally at the lobular hilus where lobules of the ﬁrst order have been incompletely separated by membranae limitantes into ultimate lobules. Fig. 7 shows an incomplete silver injection of the circulation at this stage. The arteries are seen as deﬁnite round tubes while the veins in the photograph appear collapsed. Owing to the lack of depth, many of the ﬁner relationships of the blood-vessels are lost. The alveolar plexuses are clearly shown, together with the beginning of the demarcation of the ultimate lobules by the branches of the lobular arteries.


In a pig 22 centimeters long the arrangement of the blood-vessels has not been altered in any marked degree. Each of the six divisions of the branches of the arteria submaxillaris has been formed. The growth subsequent to this stage, therefore, lies more in the differentiation and complexity of the lobule rather than in the general plan of the organ, a fact which is conﬁrmed by the study of stained specimens. The plexus about the main submaxillary duct as it enters the gland becomes more complex. Branches derived from the main artery anastomose and ramify a short distance, and then break up into a dense capillary plexus formed by irregular polygonal spaces which entirely embrace the duct, lying just external to the basement membrane. Venous radicals formed in this plexus unite and ﬂow into larger units which form an irregular venous plexus lying on top of the capillary plexus. Emissary veins from this plexus empty into the venae comites. The double plexus can now be followed as far as the interlobular ducts, Where the two layers of vessels are replaced by one. In the development of the circulation the growth of the vessels like the growth of the ducts has been entirely centrifugal.‘ If one takes a thick tangential section of the gland and views it with the stereoscopic microscope from above, the outlines of the lobules are clearly shown and the branching and radiating vessels are always found in the center looking much like the branches of a small tree viewed from above. At birth the relations of the last stage remain


5In another communication, Archiv fiir Anat. u. Phys. Anat. Abth., 1903, the writer has called attention to the centrifugal growth of the ducts of the submaxillary with reference to the formation of the lobes and lobules of the ﬁrst and second orders. These structures always occupy the center of the lobes and the center of the lobules, from which point they radiate out towards the capsule or the limiting membranes. The entire arrangement of the frame-work of the submaxillary depends, in fact, upon physical factors involved in successive action of this principle of centrifugal growth upon the ﬁbrils that compose the supporting tissue of the organ. Joseph Marshall Flint 433 practically unchanged, save in the enlargement and diiferentiation of the lobules brought about by an increase in the ramiﬁcations of the intralobular ducts and consequent increase in the number of alveoli. Pam passu with these changes the Aa. intralobulares have undergone a numerical increase corresponding to the division of the intralobular ducts. The capsular plexus is now very Well marked. Arteries in the connective tissue surrounding the gland, run to the capsule, forming there an irregular polygonal plexus which breaks up into capillaries that pass irregularly in the outer layer of ﬁbrous tissue that composes it, 



FIG. 8. Submaxillary gland of pig‘s embryo 24 centimeters long. X 10 diameters. Aa. prinpipales, inter

These caplﬂanes reunfce lobulares, sublobulares, lobulares, and in ralobulares are now distinctly seen. The lobules have increased into venules which ﬂow into venae comites that accompany the arteries forming a capillary plexus. As in the earlier stages, perforating branches from the lobules and septal arteries run up and join this plexus.

somewhat in size and the lobular veins and the venae sublobulares, interlobulares, prlncipales are Well formed. The plexus about the duct is not injected, arteries appear tubular and glistening while the veins are brownish and collapsed. The photographs from Figs. 5 to 8 are given their stereosco 1c et_1ect by painting the back of the slides with ndia ink and focusing a strong light on the cut surface of the glands. They were then photographed with a Zeiss apparatus with the above result.



==Literature==

The only investigations on the blood supply of the submaxillary are those of Kowalewsky,“ who described the blood supply and drew from the arrangement of the vessels certain conclusions concerning the secretion of the gland, depending upon the anatomical relations of the vascular system to the cells and the lymphatics. Kowalewsky worked upon the parotid and submaxillary of the cat and dog. He believed many instructive ideas could be gained from the diﬁerent resistances of the vascular stream in different parts of the blood system as shown by complete and partial injections. His description is taken from the submaxillary gland of the cat. Both the arteria carotis communis and the vena jugularis externa were used for injection. An incomplete injection from the arterial side filled the capillary plexus in the walls of the ducts, while the interalveolar capillary plexus remained completely empty as the mass reached only the arterioles running in between them. He concludes that these facts indicate “the presence in the salivary glands of two vascular systems with unequal resistances; a system of diminished resistance with capillaries in the walls of the ducts, and the system of greater resistance with capillaries in the lymph spaces between the alveoli.” The arteries of the salivary glands, according to Kowalewsky, as well as the ducts and veins, run in the connective tissue between the lobules. They give, in their course, small twigs that divide twice or three times and run mostly in recurrent direction in the connective tissue wall that surrounds the ducts. Here the arteries pass over into capillaries which sink quickly to reach the neighborhood of the epithelium under which they form a comparatively thick plexus. The veins from these capillaries appear in a more superﬁcial position, change their direction so that it is parallel to the surface of the ducts and unite to form larger trunks which, at a certain distance from the afferent arteries, terminate in the accompanying veins. Small branches are also given off from the interlobular arteries which supply the sympathetic ganglia situated in the interspaces. As the ramiﬁcation proceeds there is a thinning of the connective tissue Wall of the salivary ducts and the capillary plexus at these points is not so well developed. The arteries and veins accompany the interlobular connective tissue until they ﬁnally penetrate into the interior of the lobules. The intralobular ducts, like those of the extralobular system, have a capillary plexus derived from the intralobular arteries. This empties into the vein that accompanies the duct. The intralobular system divides with the ducts until they reach the surface of the lobules. Arriving at this point, the terminal arteries are some what thicker than capillaries, but in the absence of muscle elements they can scarcely be distinguished from them. As soon as the lobular surface is reached, the arteries customarily divide into several divergent branches which form recurrent arched loops and immediately break up into capillaries around the alveoli. These vessels or vascular arcades, according to Kowalewsky, are already in the lymph spaces and send from their concave portion capillary twigs into the interalveolar lymph spaces which surround the single alveoli. From these capillary plexuses between the alveoli appear numerous short venous radicals which run in the neighborhood of the salivary tubes and soon terminate in large veins which accompany the ducts in their subsequent course. In this short description Kowalewsky does not trace the exact transition from the arterial to the venous system in various parts of the gland, nor does he discuss the important relations of the vascular and anatomical units. Moreover, this author does not describe the vascular units in the diﬁerent circulatory systems as such, nor does he consider the capsular and septal circulation and their relations to that within the lobules.

6 Kowalewsky, Arch. f. Anat. u. Phys. Anat. Abth., 1885.


The alveolar capillaries are not situated in the lymph spaces, as Kowalewsky believes, but in the interalveolar spaces between the basement membranes of adjacent acini. Here they are limited by the reticulum ﬁbrils that bind together the alveoli which they supply.

==Development of the Alveolar Epithelium and the Demilunes of Gianuzzi==

In a pig 3 centimeters long (Fig. 9) the terminal buds of the branching column of cells forming the ductus submaxillaris indicate the primitive alveolus in its simplest form. It is composed of a group of irregular polygonal cells embraced and enclosed in a reticulated basement membrane.


FIG. 9. Sectionof primitive O“-tslde of 3 alveoms from Subm mar _ , FIG. 10. Alveolus_from glandula of pig’s embryo Bcentlgieterh Slightly deﬁnlte S“b".1“Xﬂ1a“5 °f P13,“ emb "Y0 . 8 long Hardened in zeukmds centimeters long. Hardened in ﬂuid stamad by Mauoryas amangement of Zenker s and stained by Mallor-y’s method. X 650 diameters. method. )< 650 diameters.’ The arTms drawing Shows the h,_ the outer layer rangement of the cells in two or regular columns of the cans three layers about the lumenis now forming terminalbudm. apex of cells , the re- apparent, together with the beginof the rowing column of _ mug of the f_01'r_nat10n of the mucous cells in ﬁle developing sub_ m 3, 1 n d e 1- a 1' e globules within the cells of the

maxmarx V packed’ 

m inner layer‘.

irreglarly within the alveolus. The cells are polygonal in shape, have a deﬁnite outline and granular cytoplasm, staining a golden brown by Mallory’s method. The nuclei are vesicular or ovoid, have a distinct nucleolus and considerable chromatin deposited on the linen threads. Exclusive of the regularity of the arrangement, there is, however, no morphological difference at this stage between the cells of the central and peripheral portions of the acinus. In a pig 8 centimeters long (Fig. 10) a lumen has appeared in the center of the developing alveolus and the cells are arranging themselves in two or three _fairly deﬁnite layers. No marked changes are observed in the character of the parietal group of cells, but in those of the inner layer of the lumen of the alveolus, one notes the formation of mucous globules within the cells which stain a deep blue with one of the elements of the Mallory stain. The mucous globules appear to be formed in the part of the cell next to the lumen, as the nucleus is pushed off to the edge nearest the basement membrane where it begins to show some signs of intracellular pressure, evidenced by its semilunar


f V J : ,) " form. At the same time the . r - 2 ». 1 I. . fiw A ,;"_,~._-E)’ V .l nucleus takes a red stain, 2" ~13‘ , ‘ 2,5 5‘: .3 " while the nuclei of the paris ,-L 1 .} . A.‘ ‘ . _ . A _V . _ —- ' etal cells remain brown in color. F1G.11. Terminal cluster of alveoli from the sub— - ~ Iliiaxéllaryir ;:la;1do1;fpig‘§er3‘ory(p 1EaCe]Il3i;]et1\EI']S1lDI1g- At thls tlme l3h€ mucous ‘ ’ ' ' or ‘s ‘ . mglhgdfa l<n6f?0nd§Imsete]1'1s. alibi nigiinitg ailrangya Cells are not much larger

th 11 f th (1 t (1 1 ll ' §3}$2.tr§§t. 3132013 cell: fo“r%u%3"iro%1V§§e S5133 than those of the parletal

- 1:. Al 1 ll 1 ~ pggﬁrslpgpvnapparen veo at ampu as are 2180 group and the” cytoplasln

looks like a reticulum of even blue lines or else as a mass of minute globules closely pressed togcther within the limits of the cell. At a somewhat later stage (Fig. 11) the arrangement of the cells is more deﬁnite and they are placed in two distinct layers. Mucous cells have increased in size but otherwise show the same characteristics of the cells at an earlier period. In an alveolus of a pig 18 centimeters long (Fig. 12) the mucous cells have begun to approximate laterally and, in some instances, have reached and impinged upon the reticulated membrana propria of the alveolus. The parietal cells now show evidences of pressure and are grouped 011:’ towards the reticulated membrane between adjacent mucous cells. In these instances, however, the parietal cells are not as a rule single, but occur in clumps of three or four. In many places the mucous cells are still separated from the basement membrane by an entire layer of parietal cells.


The alveoli in a pig two days old (Fig. 13) show, in general, a considerable advance in the arrangement of the mucous cells. They become larger and, as a rule, either rest upon the basement membrane or are separated from it by a very slight distance. The distended mucous cells have increased in size, but otherwise their character is not altered. The parietal cells, however, are wedge—shaped and already begin to have some of the typical appearances of the demilune. Throughout the series of embryos there is a considerable variation in the alveoli of the same gland, but the series of illustrations represents, however, the average condition found in the alveoli at the ages mentioned. In the adult submaxillary of a pig (Fig. 14) stained by Mallory’s method, there is a marked difference in the microchemical reactions of the two classes of alveolar cells. The mucous cells have increased greatly in size, the mucous globules are slightly larger, and give the body of the cell a clearer, more glairy appearance, while the nucleus still retains its crescentic shape and is pushed off to the pole of the cell nearest the membrana propria. Much more marked changes have occurred in the parietal cells; the cytoplasm has taken on a distinct bluish tinge and is now very granular, while the nuclei are less vesicular but are still brightly stained with the orange element of the dye.



FIG. 12. Terminal alveolus from the submaxillary gland of pig’s embryo 18 centimeters long. Hardened in Zenker’s ﬂuid and stained by Mallory’s method. x 650 diameters. Mucous cells have now in places reached the basement membrane. Parietal cells are pushed off into the spaces left between the cells of the former group.

FIG. 13. Terminal alveolus from submaxillary gland of pig two days old. Hardened in Zenker's ﬂuid and stained by Mallory’s method. x 650 diameters. The mucous cells have in most cases reached the basement membrane. Parietal cells are now found here and there between them, already giving the ﬁnal and deﬁnite relations of the demilunes of Giannzzl.

FIG. 14. Terminal alveolus of submaxillary gland of adult pig, showing the ﬂnal form and arrangement of the demilunes. Hardened in Zenker’s ﬂuid and stained by MalIory’s method. X 650 diameters. The diﬂerences in the staining reactions between the cells of the central and parietal groups are indicated.


Ever since the demilunes were ﬁrst described by Gianuzzi they and their functions and relations have been the subject of an extensive dis cussion in the literature. Stiihr, who believed that the demilunes are mucous cells in various stages of activity, speaks of the false or pseudolunulae, classifying among the latter Pfl1'iger’s demilunes, which are the peripheral protoplastic portions of unﬁlled mucous cells and membrana propria demilunes consisting of sections of thickened basement membranes. Another point which has given rise to considerable discussion is the Stratiﬁcation of the alveolar epithelium, many authors holding that the cells are arranged in two layers while others maintain that only a single layer exists. The conditions probably diﬁer in diﬁerent animals. In man, apparently, both groups of cells, central and parietal, touch the basement membrane, while in pigs it is not uncommon to ﬁnd even in the adult many mucous cells separated from the basement membranes by demilunes. The chief point of interest in this connection, however, is the fact that the two groups develop from a double layer of epithelium.


The signiﬁcance of the demilunes has been the point of several theories that have given rise to more or less discussion. Among the prominent ones are the “Ersatztheorie ” of I-Iaidenhain, “ Phasentheorie” of Stohr and the doctrine of the speciﬁcity of the demilunes suggested by Von Ebner.


The “Ersatz” theory is based on the idea that there are two groups of cells, a central group with the characteristic arrangement which consists for the most part of mucous cells and a peripheral group corresponding to the demilunes which through mitosis increase and undergo a mucous metamorphosis to replace the destroyed cells of the central group. Against this theory, however, the following objections are made by Oppel:

# That the mucous cells are not destroyed or disintegrated during the secretion.
# Pure mucous glands without demilunes are known to exist.
# The demilunes never show mitotic ﬁgures.

The “ Phasentheorie ” is based on the assumption that the mucous and parietal cells are merely different functional stages of the same elements. Stohr and others believe that the mucous cells are not destroyed during the secretion of the gland but persist like the cells of the stomach epithelium and that the parietal cells represent the peripheral protoplasmic part of the mucous cells which are not transformed into mucous globules. Supporting this fact are the pictures obtained during the various stages of activity of the gland, which seem to suggest, according to these authors, that the cells at ﬁrst have a round nucleus with only the central portion of the cell next to the lumen transformed into mucous. As the production of this substance increases, the nucleus is Joseph Marshall Flint 439

pushed more and more toward the periphery of the cell, where, with the remainder of the untransformed protoplasm, it rests against the basement membrane. These with similar neighboring cells form the parietal group. In extreme cases the nuclei may be pressed ﬂat against the base of the cell. Miiller raies the following objections to St6hr’s theory:

# The demilunes are not empty of secretion but contain secretion granules.
# Intermediate stages between ﬁlled mucous cells and demilunes do not exist. I
# The secretion capillaries described by Stiihr with the Golgi method are simply intercellular spaces, while true demilunes have distinct intracellular secretion canaliculi.

The doctrine of the speciﬁcity of the demilunes suggested by v. Ebner is based upon the following conclusions:

# The cells of this group contain secretion granules.
# They contain secretion canaliculi which may be demonstrated by the Golgi method.
# Certain substances injected into the circulation of living animals are demonstrable in the demilunes or their secretion canaliculi.
# They react distinctly to certain microchemical tests.

From the study of the development of the demilunes in the submaxillary gland in a series of embryo pigs, it becomes patent that the doctrine of the speciﬁcity’ of the demilunes is the only one which is at all tenable. Aside from the various conclusions given by Miiller supporting this fact, the indisputable evidence of their ancestry shows that, throughout the period of embryonic life, they are cells of an individual speciﬁc group, derived from the peripheral layer of epithelium in the embryonic alveolus. They maintain their identity throughout life and represent neither young cells which undergo, at a later period, transformation into the mucous cells of the alveolus, nor one of the functional stages of the mucous group. Embryologically and anatomically they are deﬁnite units and must be considered as cellular complexes which form deﬁnite structural elements of the alveoli of certain of the salivary glands.

==Changes in the Duct Epithelium==

In a pig 42} centimeters long the main ductus submaxillaris is already composed of two layers of cells which are cubical in shape and not distinctly columnar. These can be followed in the main divisions of the ducts and terminate in the solid buds that form the apices of the growing ducts, which we have termed the embryonic alveoli. The cells are polygonal in shape and have deeply staining nuclei, while the cytoplasm is granular.

In a pig 8% centimeters long the epithelial lining of the ducts of all orders from the ductus submaxillaris to the alveolus is in the form of a double row. The character of the epithelium is not changed from the simple form shown in the earliest embryos, where a similar condition also exists, but only in the larger ducts.

In a pig 1232- centimeters long, the rami principales, interlobulares and sublobulares are already formed. At this time the epithelium of the inner layer of the ducts of the lower orders shows a tendency to become columnar, the nuclei ovoid, with their long axes parallel to the long axis of the cell. The epithelium of the outer layer is more irregular and conical in shape, while the nuclei are rounded and vesicular. The portion corresponding to the intercalary ducts in the adult now joins the alveolus directly to the sublobular duct. This indicates accordingly that ducts of the lobular order have not yet been formed, but in these intercalated portions, the cells appear quite like the parietal cells of the alveoli, and are arranged in two deﬁnite strata.

At 19 centimeters there is no change in the ducts of the higher order, but, with the deﬁnite appearance of the secondary lobules, We note the formation of the lobular and intralobular ducts for the ﬁrst time. The epithelium of'both orders is in two layers, an inner cubical and an outer more irregular layer. The ducts connecting them with the acini show a considerable ﬂattening with a tendency for the inner stratum to take the stain more intensely than those of the outer layer.

At 22 centimeters the ducts of the higher order show an increase in the columnar arrangement of the inner layer of cells; the central portions now stain a deep blue with a clearer peripheral portion on the side of the nucleus near the basement membrane. The ducts of the sublobular, lobular and intralbbular order remain unchanged. Deﬁnite intercalary duets have now been formed and appear to have but a single layer of epithelium with the long axis of the nuclei parallel to the axis of the duct.

In a pig 261} centimeters long the main ducts have now a distinct columnar epithelium adjacent to the lumen. This has a bluish staining pole and a clearer peripheral pole. As the ramiﬁcation proceeds, particularly in ducts of the sublobular order, there is a tendency for the epithelium to become more columnar. There is no striation in the cytoplasm of the cells of the intralobular ducts. These now appear in many


(edited text to be added)


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