On the Reticular Tissue and Lattice=fibers occurring in the Milk=spots of Omentum.

By

Dr. Yukio Hamazaki.

From the Pathological Department of Okayama Medical College (Director: Prof. Oto Tam ura).

2 Figures (Plate III) and 3 Text Figures.

Ranvier and Weide n reic h regarded the omentum as a flattened- out lymph gland and the abdominal cavity as its lymph sinus. The latter, furthermore, theoretically emphasized that the omentum is nothing but a sheet of reticular tissue, the "taches laiteuses" correspond- ing to the secondary nodules. Lately, Kiy ono agreed with the above view, though lie pointed out that the histiocytic cells in the milk-spots do not form reticular tissue, unlike those in the lymph glands. At the Fifteenth Pathological Congress of Japan (1925) I reported that the milk-spots in the rat, cattle, and pig are provided with a certain kind of reticular tissue. The purpose of the present paper is to settle this problem using specific stainings for the reticular fibers and for the lattice-fibers ("Gitterfasern" of v. Kupffer), which may be in an intiniate relation with them.

Material and methods.

The material was obtained from the cattle, pig , dog, cat, rabbit, guinea-pig, rat, mouse, chicken and human subject. As the control the organs containing reticular tissue, i. e., lymph glands , and thymus gland were also examined. The material fixed with 10% solution of lormalin was studied as stretched specimens and as sections . For reticulum-staining the eosin-methyl blue method modified by the author was used: 1. Sections are stained for 30 minutes in 1% solution of eosin (a few drops of glacial acetic acid is added to 100 cc of the solution) . Then they are washed with distilled water . 34 Yukio Hamazaki, 2. They are rinsed in 1% solution of phosphomolybdic acid for a few seconds and washed with distilled water. 3. They are stained with 0.1% solution of methylblue for five minute's and then washed with distilled water. 4. They are differentiated and dehydrated in 95% alcohol, keeping them in constant motion, so as to obtain uniform decolorization. 5. Finally they are mounted in balsam, passing through absolute alcohol and xylol. In sections thus treated, reticular fibers are stained green, the nuc- lei of the reticular cells violet-red, those of the wandering histiocytes violet and cytoplasm blue. The nuclei of lymphocytes are stained red, the erythrocytes orange-red. The lattice-fibers were studied with the Bielschow sk y-Maresch 's impregration method. In some cases of rat material vital staining with lithium-carmine was performed prior to eosin-methylblue method and the Bielscho w sky's method to make clear the relations of the histiocytes and reticulum.

Experiments.

As is well known, the "Wiles laiteuses," are best developed in the omentum of rat. In this paper the reticular and lattice-fibers of this form, especially in its follicular milk-spots, will he dealt with.

Reticular fibers. Rat. Inthe stretched specimens one can clearly see that many fibers supporting the net-trabecules of the omentum go into the milk-spots. The fibers ramify into several branches, thus dividing the milk-spots into many lobales. Since the milk-spots of rat are crowded with many cell-elements, it is very hard to make out the relations of the collagenous fibers to the reticular ones or to the cells. In horizontal sections, one can distinguish three layers in the milk- spots. The outer layer is composed of many wandering histiocytes, mast- cells and some lymphocytes, polymorphonuclear leucocytes. In this region the reticular cells are scattered over in a small number and the fiber-elements are poorly developed. The reticular cells are either spindle- shaped or stellate. The meshes are relatively large, measuring about 17p in diameter and accommodating many wandering cells mentioned above. The cell-body of the wandering histiocyte in the meshes contains a On the Reticular Tissueand Lattice-fibersoccurring in the Milk-spotsof Omentum. 35 delicate net-apparatus consisting of very fine fibrils. Each cell is con- nected with the neighboring ones by means of fibril-bridges which arise from the net-apparatus. The mast-cells also have fine net-apparatus in the cytoplasm, though they produce no fibril-bridges. In the intermediate layer the reticular cells with round or oval nuclei (about 62) are present in large numbers. They are triangular or stellate in shape. The cytoplasmic processes with fiber-element are gentrally short and form small reticular meshes (about 15/2 in diameter) and accordingly they can accommodate only few wandering cells. The wandering cells mainly consist of lymphocytes and histiocytes. In this area the fibril-bridges between the histiocytes are not so well developed as compared with those of the outer layer. The reticulum in this region resemble very much that of the splenic pulp (red pulp) or of the lymph- sinus. In the inner layer the reticular cells with flattened or triangular nuclei (about 6p) are present in a relatively small number, but time fibers are numerous, long and tortuous forming reticular meshes of somewhat polygonal shape (about 30p in diameter). The meshes are filled with many lymphocytes. The reticular fibers are connected with the adventitial fibers of blood-vessels, which are chiefly distributed in the intermediate layer. The reticulum in this layer closely resemble that of the splenic nodule (white pulp). The vital staining with lithium-carmine was followed after fixation by eosin-methylblue to investigate the relations of the histiocytes and the reticulum. Lately, Fahr studied on the lymph glands at the hilus of the and he recognized very many epithelioid cells are developing from the reticular cells. He inferred that these epithelioid cells belong to the histiocytes of Ascho ff and K iy o o. In my material the change process of the reticular cells into wandering histiocytes could be clearly brought out. The stellate cells of the reticulum shorten their processes, become more and more spherical and finally set themselves free from .the reticulum. The color of carmine-granules turn into blue-violet by methylblue. A small number of such granules are present in the reticular cells. The granules increase in number as the cells become more spherical and detatched from the reticulum. (Fig. 1, Plate III) . Guinea-pig and mouse. The milk-spots of these two forms are similarly constructed as those of the rat but the cell-elements, especially the lymphocytes , are less than in the rat. Cattle and pig. 36 Yukio Hamazaki,

In the milk-spots of the cattle the reticular cells are mostly stellate in shape. The cytoplasmic processes of the superficially disposed cells are relatively short, but in the deeper part of the milk-spots such processes become gradually long and slender, forming a medium-sized meshes. Iii the cytoplasmic processes many tortuous and anastomosing fibrils can be detected, but they show little tendency to form the true reticular fibers, unlike those of the rat. In the reticular meshes some large and small mononuclear wandering cells, lymphocytes and eosi- nophils are situated. These pectures resemble very much the sinus- reticulum of lymph glands. In the milk-spots of pig, however, the fiber- elements are somewhat well developed. Chicken. The milk-spots of chicken are chiefly developed in the superficial part of the mesentery. The subserous connective tissue buried under the milk-spots') send off many collagenous fibers into them and the fibers are divided into several branches anastomosing with the reticular fibers. The reticular fibers reach the outer-layer of the milk-spots where they branch into fine fibrils and become connected with the net-apparatus in the cytoplasm of the large mononuclear wandering cells. The reticular meshes are relatively large sized and lodge various kinds of wandering cells. Man and (log. The milk-spots of man are divided into several lobules by sinusoid spaces whose branches go into the central part of the lobules. The spaces conmunicate with the perivascular lymph-spaces. The wall of the sinusoid space is composed of endothelioid cells, the appearances of which are quite similar to the reticular cells. Some of their processes are wedged into the cell-elements of the milk-spots. In the parenchyma the reticular cells are scattered over in a small number and their fine fibers surrounding each wandering cells are connected with the - ous fibers which come from the subserous connective tissue. The reticulum in the milk-spots of dog resemble that of man, but the sinusoid space can not so clearly be made out. The solitary milk-spots developed on the peritoneal surface are composed of large mononuclear wandering cells as in the outer layer of the milk-spots of rat. Each sends out fine fibrils which anastomo,se with those of neighboring cells. Some of these fibrils are fused to form reticular fibers. Rabbit and cat. The milk-spots are mainly composed of large mononuclear cells. Many connective tissue fibers go into the milk-spots from the net On the ReticularTissue and Lattice-fibersoccurring in the Milk-spotsof Omentum. 37 trabeoules of the omentum and so the reticular . fibers are hardly differentiated from the collagenous fibers. Lattice•fibers. In the milk-spots of rat impregnated with the Bi el s c ho wsk y- Mar es ch's method the wandering histiocytes of the outer layer stain themselves in a light brownish color and their nuclei in a deep brown. The cell-boundaries of the histiocytes are somewhat clearly made out. In the deeper area, however, cell-bodies are generally stained diffusely in a light gray and their boundaries being blurred. The nucleus of the (histiocytic cell) is colored deep gray, while that of lympho- cyte black. The latter usually contains in it a large nucleolus. The above two kinds of nucleus can therefore be readily distinguished. The lattice and collagenous fibers are stained black and deep brown respec- tively, as M a resell firstly pointed out, but the transformation from one kind to the other is not clear. • Rat . In the stretched spechnens many collagenous fibers go into the milk- spots from the net-trabecules and along the blood-vessels of the omentum

Fig. L Stretched specimen of small milk-spot of rat. Showing the spongy appearance of network of lattice-fibers and its relation to collagenous fibers of trabecules of omentum. 38 Yukio Hamazaki, to anastomose with the network of lattice-fibers. However, the aiwunt of the lattice fibers is quite superior to that of collagenous ones and so the former cannot be regarded as the branches of the latter, but is the production of the milk-spots in themselves. The net-work of the lattice- fibers appears spongy and frequently individual fibers are not discernible owing to their extreme density (Fig. 1). Iii the sections the lattice-fibers are numerous in the deeper area of the milk-spots, while in the outer layer they are scarce. Thick fibers usually originate from the neighboring region of the blood-ves.,,nis and after repeated divisions and anastomosing form a delicately close-meshed network. They on the other hand come into connection with the tine lattice-fibers which encircle the blood-capillaries, so called adventiti a ca pillar i s of Mar es c h. The lattice-fibers thread between the cell- elements taking a wavy course which is motsly influenced by the nuclei and in the least by the cell-bodies. In its course the fibers frequently enibrace a nucleus more than half of its circumference and in such cases they send out several branches to surround the nucleus. The lattie- fibers, as is well known, do not show perceptible change in their thickness by either ramification or anastomosing. In the central region of the milk-spots they become fine forming small meshes. Here I shall describe some observations on the relations of the lattice- fibers and the nucleus. For this purpose lightly impregnated specimens were studied to best advantage. If otherwise, the fibers surrounding the nuclei cannot be clearly differentiated from the nuclear membranes. Light staining is preferable for the reason that carmine-granules become brownish which bring out the boundary of cell-body. In such specimens one can see some fine fibers arising iiitracellularly around the nucleus. Sometimes one fiber comes to the nucleus, runs along over half of its circumference and goes out of the cell-body (Fig. 2, a.). Sometimes two fibers arise from the opposite poles of a nucleus, and after circling each pole, leave the cell-body to anastomose with thick fibers in the neigh- boring region (Fig. 2, b.). In some cases the fibers originated from near a nucleus are cut by their fibers anastomosing with a thick one (Fig. 2, e.). Still in other cases many line fibers make a fibril-basket surrounding a nucleus, just like that of the Purk iuj e cells (Fig. 2, d and e.). In the sections from the vitally stained material most the cells around whose nuclei there are fine lattice-fibers, as mentioned above, possess carmine-granules which have been decolorized into brown. So they may belong to the histiocyte. On the Reticular Tissue and Lattice-fibers occurring in the Milk•spots of Omentum. 39

Fig. 2. Specimen vitally stained with lithium-carmine preceding Bielschowsky 's method, showing the relations of lattice-fibers and nuclei in milk-spots of rat. Cell-bodies, excepting (d), have carmine granules in varying quantities.

Rabbit, dog, etc. The collagenous fibers which come out of the trabecules of the omentum into the milk-spots do not change their original until they reach a thick cell-group where they communicate with the network of lattice-fibers. In these forms the lattice-fibers are generally poorly developed. 41) Yukio Hamazaki,

Fig. 3. Horizontal section through milk-spot of rat. Biel s- c ho w sky 's method. a outer layer of milk-spot consisting mainly of large mononuclear wandering cells.

Summary and discussion.

Regarding the origin of reticular tissue many theories have hitherto been put forth. Kiilliker emphasized that the reticular tissue is composed of connective tissue cells which form net-works with their cytoplasmic processes and he named it " cy toge nes Bindegewebe." Ile and Billroth pointed out that in the follicle of lymph gland the nucleus disappear,, while in the sinus it is present. The views of Bizozze ro and Ranvier in which G Oland and S ta r agreed is that the reticulum consists of tine connective tissue bundles to which stellate reticular cells attached. Using vital staining method, Aschoff. f and Kivono lately claimed that the reticular cell as well as the Kupffer's stellate cell of time liver, splenic sinus-cell, etc. belong to the histiocytic cell which they called " Re tic ulo en dot heli e n ." Whether the exists intracellularly or intercellularly is the problem which has been discussed by many authors. Flemming and Golowinski stated that in the embryonal period the connective tissue-tiber is produced from the surface of the cytoplasm. On the lattice- fiber Riissle and Yoshida, and on the ring fiber of splenic sinus Monier stated that the fibers are produced from the surface of both the undifferentiated endothelial and reticular cells. Judging from its chemical nature ( wi n ski and Matsui considered that the lattice- fiber is nothing but a pre-collagenous one. Kon has claimed that in the embryonal period the lattice-fibers of liver take their origin in black particles or elongated rods in the cytoplasmic processes of the endothelial cells of capillaries. On the ReticularTissue and Lattice-fibersoccurring in the Milk-spotsof Omentum. 41

According to my own observations the fibers are very fine and are in the cytoplasmic processes of the reticular cells both in the outer and intermediate layers of the milk-spots, while in the inner layer they are situated intercellularly. Both the kinds of reticular fibers are presu- mably produced from the reticular cells, but the latter may be a higherly differentiated portion which can persist for a long time after the detach- ment of the reticular cells or even after the death of the cells. Such an inference was made by ROssl e and Yoshida as to the lattice- fibers of lymph nodules. Oppel, ROssle and Yoshida, Matsui, et al. described that the reticular fibers are nothing but lattice-fibers. However, in the light from the studies on the milk-spots the two kinds of fibers exhibit many morphological differences. The reticular fiber looks as though it were a membrane consisting of many fibrils (Fig. 2. Plate III), while the lattioe- fiber gives an impression of a single string. The former takes rather straight course forming a network of polygonal meshes, while the latter runs tortuously forming a network with rounded meshes. As a whole the latter fibers exceed the former in number. It is noteworthy that the lattice-fibers are usually situated intercellularly, but their fine branches frequently originate from around the nuclei of the histiocytes. Taking these facts into consideration, it may safely be said that these two kinds of fibers are produced by the same kind of cells, but they differ from each other in their degree of differentiation. The presence of the reticular tissue has been confirmed in the lymph glands and spleen and as to its presence in the thymus opinions are still divided. Using the eosin-methylblue method I could scarcely make out the reticular tissue in the thymus. The milk-spots of the omentum are taken by W ei de n r eich and Kiyono to be in the category of lymph glands. But K iy on o insists that the histiocytes of the milk-spots never form reticular tissue. He, therefore, calls them " Gewebs histio cy ten " together with the histiocytes in the subcutaneous connective tissue in opposition to "Retie ulo end othelien ." He thinks that, there is no fundamental difference between the tissues of milk-spots and the omentum excepting the numerical difference of the histiocytes. It should be mentioned that my results are at variance with Kiy on o's. As has been dealt with at some length, I reached the conclu- sion that the histiocytic cell in the milk-spots form reticular tissue, and that these cells ingest actively various foreign bodies. Freeing themselves from the reticulum they become wandering histiocytes. Sometimes they 42 YukioHamazaki, turn into giant cells, each with two nuclei." The more the histiocytes acquire vitally staining capacity towards carmine the more they show tendency to free themselves from the reticulum (Fig. 1, Plate III). It is interesting to note that I came across in the material obtained at autopsy of a case of cardiac defect many histiocytes of the milk-spots loaded in a marked degree with blood-pigment. The reticular tissue of the rabbit is not clearly brought out to view by the eosin-menthylblue method due to a large quantity of collagenous fibers, whereas it is clearly made out by employing glycogen-staining from the arrangement of Best's positive granules." It may be added that in the milk-spots of the rabbit and rat the reticulum-formation begins at two days after birth.° In the parenchyma of the milk-spots relatively many lymphocytes are present, but the lymph vessels or sinus can hardly be found (except man and the dog). The milk-spots are provided with many tortuous blood-capillaries and frequently they are dilated to form the so-called "sinusoid" of Minot .° The endothelial cells of the sinusoid can hardly be stained vitally with the carmine method, contrary to those of the venous sinus of spleen. In the sinusoid many large and small wander- ing cells and lymphocytes can often be met with as described in one of my previous papersP From this fact, it will be seen that the endo- thelium of the sinusoid permits a ready passage of the wandering cells like that of the splenic sinus. In the case of inflammation of the omentum of the guinea-pig Marchard observed that many wandering cells existed in the blood-capillaries and he expressed his view that the remarkable production of the cell-elements of the milk-spots may result in the immigration of some of these cells into the capillaries. Furthermore, Do min i ci reported that the appearance of the myelo- cytes in the omentum of rabbits in the cases of experimental typhus peritonitis and of post-hemorrhagic anemia. Later, Schwarz made a detailed study on the myelocytes with ,8-granules and the production of polymorphonuclear leucocytes found in the milk-spots of the omentum of normal rabbits. In my studies it was observed that in the inner layer of the milk-spots of rat there exist some lymphocytoblasts with many Altmann 's granules. The functional correlation between the milk-spots and spleen has been investigated by me in extirpating the spleen. My results will be described in detail in a forthcoming paper, but here it should be mentioned that a month after the operation the milk-spots enomously enlarge as compared with the normal ones. It is of much interest to observe that intraperitoneal injection of normal saline solution or Indian-ink easily causes hemorrhage which is On the Reticular Tissue and Lattice-fibers occurring in the Milk-spots of Ome.ntum. 43 mainly limited to the milk-spots. Such a phenomenon may be due to the nature of the capillary walls which has a decided tendency towards diapedesis and in addition, to the obstruction of capillaries resulting from the insufficiency in the collateral circulation. Such conditions are well known in the splenic vein. In the rat, cattle, pig, chicken, etc., the mesothelial cell-layer of the milk-spots does not make a continuous membrane, but at places meso- therial cells are substituted by mononuclear wandering cells!) Therefore the peritoneal fluid may freely pass into the reticular tissue and the wandering cells of the milk-spots can readily emigrate into the peritoneal cavity. Thus the milk-spots of omentum supply the peritoneal cavity and also the blood-vessels with the large and small mononuclear wandering cells and lymphocytes. Conclusion. The milk-spot of omentum is a lymphoid organ distinctly differen- tiated from other omental tissues. There is no room for doubt that it possesses both the reticular tissue and the lattice-fibers. In nature it resembles more closely the spleen than any other structure.

In closing this paper I wish to acknowledge my indebtedness to Profs. 0. Tamura and H. Tanabe. December 29, 1925.

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Explanation of the Plate (III). Fig. 1. Horizontal section through milk-spot of rat. Vital staining with lithium- carmine preceding cosin-methylblue method. Showing the developmental process of wandering histiocytes from reticular cells in intermediate layer. Carmine-,2-,ranuleshave become blue-violet by methylblue. The more histioeytes detach from the reticulum, the more carmine-granules. Note also that many fine fibrils in reticular fibrils go into cell-bodies. Fig. 2. Horizontal section through milk-spot of rat. Eosin-methylbluemethod. a Reticular cells with violet-red nuclei of inner layer. Meshes are filled with many lymphocytes. b Stellate reticular cell of intermediate layer. c Mono- nuclear wandering cell connected with reticulum by means of fibril-bridges.

, FOLIAANATOMICAJAPONICA,BD.IV. TAF。1口.

Y.Hamazaki.