Paper - The histology of the spleen and suprarenals of echidna (1932)

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Basir MA. The histology of the spleen and suprarenals of echidna. (1932) J Anat. 66: 628-649. PMID 17104400

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This historic 1932 paper by Basir the echidna spleen and adrenal gland.



Modern Notes: echidna | spleen | adrenal


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The Histology of the Spleen and Suprarenals of Echidna

By M. A. Basir M.B., B.S. (Mapras), Px.D. (Lonp.),

Professor of Physiology, Medical College, Vizagapatam

From the Department of Anatomy and Embryology, University College, London

  • Part of thesis approved for the degree of Ph.D, in the University of London.

I. The Spleen

The spleen of the Monotreme, remarkable on account of its form, was first described by Meckel in his classical monograph on Ornithorhynchus. The Echidna spleen has very much the same shape as that of Ornithorhynchus. In both species it differs very materially from the typical mammalian form, being tri-radiate or Y-shaped (text-fig. 1) and possessing a large pear-shaped swelling at the end of one of its limbs. The surface is indistinctly divided into irregular lobules by slight constrictions, whilst the hilum extends along its entire dorsal surface.

The spleen consists of three distinct parts, as can be seen in text-fig. 1. Of these, the body (b.) lies in the great omentum, along the greater curvature of the stomach, being directed towards the oesophagus. The process (d.pr.) bearing the large pear-shaped body (d.enl.) at its end, also lies in the great omentum and is directed dorso-laterally, extending as far as the distal colon, whilst the other process (v.pr.), also embedded in the great omentum, is directed ventrally. It is longer, thicker and more freely movable than the other two limbs of the organ.

The dimensions of the specimen examined were as follows: The body (b.) measured 4-5cm. long and 0-5 cm. in diameter. The dorsal process (d.pr.) was about 3-5 em. in length (excluding the bulbous portion) with a diameter of 0-5 cm. The pear-shaped portion (d.enl.) itself measured 3 cm. long, 2 cm. in the transverse plane and 1-5 cm. antero-posteriorly. These two parts of the dorsal process were connected together by a slender bridge of lymphoid tissue (l.st.). The ventral process (v.pr.) measured 7 cm. in length and 1 x 0-5 cm. in diameter.

The spleen was fixed in corrosive sublimate and was kept in alcohol for a good many years. It nevertheless proved to be extremely well preserved, and showed many interesting histological details. Serial sections of pieces from different regions of the organ were cut and stained by the following methods: Ehrlich’s haematoxylin and eosin, Heidenhain’s iron-haematoxylin


and Van Gieson, Dominici, Pasini, Mann’s methyl-blue eosin, Mallory’s connective-tissue stain and Weigert’s resorcin fuchsin. Special methods of staining, such as silver impregnation methods and the Prussian blue reaction for iron, could not be used. In order to supplement my observations on the Echidna spleen, I have examined the spleens of hedgehog, mouse, rabbit, Hapale, baboon, alligator, Python, the lizard, Trachysaurus and the Gymnophionan, Siphonops annulatus. These were fixed in a variety of fixatives (Zenker, Bouin, bichromate-formol, alcohol and formol) and stained by the methods mentioned above. My observations on these spleens are incorporated in the text.


Text-fig. 1. Photograph of the spleen of Echidna. Mag. 7 times natural size. Note the triradiate appearance of the organ and the slight constrictions on its surface, giving rise to indistinctly divided irregular lobules. It consists of a body (b.), a ventral process (v.pr.) and a dorso-lateral process (d.pr.) which is connected with a pear-shaped enlargement (d.enl.) by means of a delicate stalk of lymphoid tissue (J.st.).

The Supporting Framework

(i) The capsule and trabeculae

The capsule which completely enclosed the organ consists of dense fibrous tissue, some elastic fibres and numerous smooth muscle cells, Arising from the capsule are strong trabeculae (Plate II, fig. 3, T.), similar in structure to the capsule, which form an anastomosing network in the interior of the organ. These trabeculae carry with them the blood vessels which run in them for some distance and eventually enter the splenic pulp, covered in a connective tissue sheath, derived from the trabeculae—the so-called “hilar sheath” of Whitting (13).

The amount of smooth muscle, connective tissue and elastic fibres in the capsule and trabeculae varies considerably in the different spleens examined, as does the development of the trabecular system. For example, the spleen of the alligator possesses an extremely thick fibrous capsule, which is. poor in muscle, and gives off no trabeculae, whilst that of Siphonops possesses neither capsule nor trabeculae. It is interesting to note that in these two forms, the reticular network is much more strongly developed, so that the reticular cells would appear to have taken on the supporting function of the trabeculae.

(ii) The reticulo-endothelial framework

The supporting framework of the splenic pulp, which occupies the meshes of the capsule-trabecular system is formed of a uniform spongy framework, composed of branching reticular cells (Plate II, fig. 3, ret.c.) which are closely applied to the reticular fibres (ret.f.). This loose network is infiltrated with the blood cells (7.61.) which give rise to the characteristic red colour of the splenic pulp. The delicate reticular fibres (ret.f.), as can be seen in Plate II, fig. 8, become directly continuous with the collagen fibres (c.t.) forming the capsule, trabeculae and tunica adventitia of the blood vessels, whilst some of the reticular fibres appear to be attached to the smooth muscle fibres (s.m.) present in these regions.

(iii) The vascular system

The arteries and the veins enter and leave the spleen at the hilum. This lies along the whole length of the organ on its dorsal surface. The arteries at first run in the trabeculae, but they soon pass into the splenic pulp itself, enclosed in a connective tissue sheath—the “‘hilar sheath” derived from the fibrous trabeculae. In the pulp, the “hilar sheath” becomes infiltrated with small lymphocytes, forming a lymphoid coat round the vessel, the white pulp or Malpighian corpuscle. The Malpighian corpuscles vary in size, but they are frequently large, running for more than 5 mm. along the arteriole, whilst they are not of uniform thickness throughout. Several such corpuscles may occur at varying intervals along the length of an arteriole.

As is well known, the form of this lymphoid sheath varies in different spleens. In the alligator and Trachysaurus, the arterioles are enclosed in a uniform lymphoid sheath throughout their entire extent, whilst in Siphonops— such lymphoid sheaths are also found along the veins. In Python, on the contrary, the vessels are completely devoid of a lymphoid covering.

In Echidna, the Malpighian corpuscles (M.c.), as can be seen in Plate I, fig. 1, are surrounded by a thin capsule of condensed reticular fibres, in which also occur smooth muscle fibres, and collagen fibres, derived from the “hilar sheath.” The artery (text-fig. 2, c.a.), after running a short distance through the corpuscle, may divide dichotomously, and some of the branches (a.) so formed, may immediately leave the corpuscle and run into the red pulp, where they develop fresh corpuscles (M.c.), whilst other branches, the smaller, run right through the corpuscles and open into the reticulum of the red pulp. The majority of these branches, however, do not reach the reticulum, for their endothelial walls break down and the blood is poured into the lymphoid tissue, forming a blood sinus (Plate I, fig. 1, bl.s.) round the periphery of the corpuscle and enclosed by the reticular capsule (Plate I, fig. 1, cap.M.). The blood sinus (Plate I, fig. 1, bl.s.) is not an endothelially lined space, but the blood actually lies free in the reticulum, from which it is eventually drained into the surrounding pulp (sp.p.) by means of endothelially lined capillaries (c.). This peripheral blood sinus is absent in the spleen of the young animal, and it may be that the sinus is concerned with the increased blood destruction in the older animal. In the latter there is present chiefly in the sinuses, but also in the pulp, a considerable number of red blood cells undergoing haemolysis which were not found in such quantity in the younger animal.

The main arteriole eventually leaves the Malpighian corpuscle and runs into the red pulp, where it divides into two or more penicillate arterioles (text-fig. 2, p.a.) which acquire dense, though ill-defined sheaths or ellipsoids (el.) around them.

In Echidna, the ellipsoids (Plate II, fig. 4, el.) consist mainly of longitudinally arranged condensed reticular fibres (ret.f.) intermixed with large reticular cells (ret.c.). The penicillate artery (p.a.) is lined by an irregular layer of endothelial cells (end.c.) which possess scanty protoplasm and large nuclei ‘bulging into the lumen (J.). Peripherally the dense reticulum of the ellipsoid. gradually merges into the reticulum of the pulp. The penicillate artery does not branch in the ellipsoid and no canaliculi, such as are described by Bannwarth (1) and more recently by McNee(8) can be seen. In the matrix of the ellipsoid, however, there are visible isolated red blood corpuscles which suggest that such canaliculi may exist.

The ellipsoids are better developed in the spleen of the alligator and their structure can be more easily studied. The penicillate artery here divides into four or five smaller branches within the fibrous sheath, producing a quadrior penta-cornuate ellipsoid, whilst outside this, enclosing the penicillate artery and its ellipsoids, is a lymphoid sheath. This lymphoid covering to the ellipsoid appears to be peculiar to the spleen of the alligator, and I have found nothing comparable to it in any of the spleens I have examined.

In the spleens of the baboon, Hapale, rabbit and mouse, which have welldeveloped Malpighian corpuscles, the ellipsoids are poorly developed, so that I can confirm the statement of McNee(8) that as a general rule, in animals with well-developed Malpighian corpuscles, the ellipsoids are poorly developed. Further, in this connection, Bannwarth (1) observed that during development the ellipsoids appear before the Malpighian corpuscles and that they become indistinct later in embryonic life, when the Malpighian corpuscles make their definite appearance.


The mode of termination of the penicillate arteries is still a matter of controversy. Helly 3) and Thoma(11) believe that there is a closed circulation in the human spleen and that the penicillate arteries open directly into the venous sinuses, but that some of the blood passes into the pulp through openings in dilated terminations of the arteries—the so-called “ampulla.” These openings, according to Thoma(1l), are very minute, whilst Helly @), on the other hand, believes that they are of considerable size. Many other workers, Kultschitsky (4), Mall (6) and Sobotta (10), believe that the circulation is an open one, whilst Wiedenreich (12) states that both forms of circulation exist in the same spleen.

Mollier (7) more recently has made a further study of the structure of the venous capillaries and sinuses in sheep and Man, and he showed that they are provided with innumerable stomata, through which a considerable amount of blood can pass from the pulp during the passage of blood from the arterial to the venous side. Neubert (9) has further shown that the same holds good also in dog, cat and pig.

In Echidna, the penicillate artery (Plate I, fig. 4, pa.) after leaving the ellipsoid becomes funnel-like, its endothelial lining (end.c.) flattens out and gradually becomes indistinguishable from the surrounding reticular cells (ret.c.). The blood thus passes directly into the red pulp, so that the circulation appears to be of the open variety. In addition, however, some arterioles (text-fig. 2, p.a.1) run directly through the pulp (sp.p.) to open into the venous sinuses (v.s.), but this is not of frequent occurrence.

Certain spleens, those of the alligator, Siphonops, hedgehog and Hapale, which I have examined, possess an open circulation, whilst in others, those of Python and Trachysaurus, the circulation is closed, as McNee(8) has stated.

The blood is eventually collected from the pulp by small capillaries, which open into the venous sinuses (Plate IT, fig. 5, v.s.). The origin of these venous capillaries has been much discussed. Thoma (11) described the venous capillaries as being continuous with the distal part of the ampulla, whilst Kultschitsky (4) thought that they arise directly from the pulp spaces. Mollier(7), on the other hand, describes the blood cells passing to the venous sinuses, through stomata in the walls of the sinuses.

In Echidna the venous capillaries originate in the reticulum of the red pulp and possess an incomplete endothelial lining, like that of the venous sinuses, so that blood drains into them from the surrounding tissue. These capillaries, after a longer or shorter course in the pulp, run into the venous sinuses.

The general scheme of the circulation of blood in the Echidna spleen is given in the accompanying text-fig. 2.


(iv) Cytology of the spleen The cellular elements forming the red pulp, apart from those of the supporting framework, comprise the cells of the blood, macrophages and lymphoid elements. In some animals, e.g. mouse and hedgehog, the red pulp contains myelocytes, megakaryocytes and plasma cells in addition.


Text-fig. 2. As can be seen, the artery (A.), on leaving the trabecula (7.) enters a Malpighian corpuscle (Df.c.), wherein it gives off numerous branches. Some of these enter the peripheral blood space (6l.8.) of the corpuscle (M.c.), the blood from which eventually drains into pulp (sp.p.), whilst others leave the corpuscle to form penicillate arteries (p.a.) and these either open directly into the pulp by funnel-like openings (f.) or pass (p.a.1) to open directly into the venous sinuses (v.s.). From the pulp (sp.p.) the blood passes.into the venous capillaries (v.c.) or through the stomata (st.) in the walls of the venous sinuses into the latter, and thence into the veins (v.).

(a) Cells of the supporting framework, including the histiocytes (the so-called reticulo-endothelial system).

The connective tissue cells, plain muscle cells and plasma cells, which are found in the capsule and the trabeculae, are so well known, that it is not necessary for me to do more than mention them here. The reticular cells (histiocytes) on the other hand, present a more complex problem.

These cells, as I have already mentioned, form the reticular framework of the splenic pulp, in which reticular fibres can be seen when stained by appropriate methods, such as Mallory’s or Pasini’s stains. By means of Foot 684 . -... ... . - M. As. Basir.

and Ménard’s silver impregnation method (), still clearer pictures of this reticulum can be obtained, but unfortunately this method could not be used on the Echidna spleen.

The reticular cells are large and are provided with irregular protoplasmic processes, which unite with similar processes of the neighbouring cells to form a syncytial network, in which the reticular fibres are embedded. These cells possess a granular acidophil cytoplasm and contain one or two oval or spherical nuclei, with well-defined nuclear membranes, rather scanty chromatin, clumped into one or two large granules and one or two nucleoli.

Such reticular cells also line the venous sinuses. They differ from the ordinary endothelial cells by the fact that they project into the lumen of the sinuses and are actively phagocytic, whilst their processes are often in continuity with the syncytial network.

In the spleens of the alligator, Siphonops, hedgehog and Echidna (although to a less extent in the Echidna spleen than among the others I have mentioned), macrophages are formed from the reticular cells. These macrophages separate off from the reticulum and become actively phagocytic, ingesting pigment or other cell débris. They measure up to 10 in greatest diameter and possess oval nuclei with rather scanty chromatin and a prominent nuclear membrane, whilst their cytoplasm is abundant and is stained faintly with basic stains.

Pigment may also be seen in the reticular cells lining the venous sinuses, and is very noticeable in those of Siphonops, whilst extracellular reddish brown pigment, exhibiting a well-marked iron reaction with Prussian blue, was noticed round the ellipsoids in the spleens of the alligator and the hedgehog. In the spleen of Echidna, however, neither extracellular nor intracellular pigment was observed.

(b) Cells of the lymphoid series. In Echidna, the Malpighian corpuscles are formed entirely of small lymphocytes and no germ centres are present. 1

(c) Blood constituents.

Blood cells are found scattered in large numbers throughout the red pulp as well as in the blood sinus round the periphery of the white pulp. Red blood cells were observed in process of fragmentation and were in course of ingestion by macrophages, whilst others had undergone haemolysis and their débris was seen lying in the venous sinuses. No erythropoiesis was taking place in the spleen.

(d) Special cells.

In the pear-shaped enlargement of the dorsal limb, certain large cells are found scattered in the reticular network of the red pulp (Plate II, fig. 6, gr.c.). They measure up to 0-016 x 0-012 mm. in diameter, and are remarkable in that their cytoplasm is filled with coarse acidophil granules (gr.), spherical in shape and in such abundance that the nucleus is displaced to one side of the cell and presents a spherical pyenotic appearance (p.nuc.). Similar cells are also found in the spleen of the alligator, Siphonops and mouse, although in these animals the cells are smaller, whilst the granules are less abundant and the nucleus is round and vesicular. Such cells were also seen in the spleens of the rabbit, Hapale and baboon, but they are extremely rare. I can find no reference to such cells in the literature.

Megakaryocytes, such as occur in spleens of the hedgehog, rabbit and mouse were not fqund in Echidna.

Summary

  1. The spleen of the Echidna is tri-radiate in form, with a pear-shaped enlargement at the end of one of its limbs.
  2. It is invested by a well-developed fibrous capsule, which contains a considerable quantity of smooth muscle and which sends trabeculae into the splenic pulp.
  3. The stroma of the gland consists of a branching syncytium formed by the reticular cells, in which run reticular fibres. These reticular fibres are continuous with the collagen fibres of the capsule and trabeculae, and are also attached to the smooth muscle cells.
  4. The Malpighian corpuscles which are developed round the arterioles are characterised by the presence of a large blood sinus round their periphery.
  5. The penicillate arterioles possess ellipsoids, each of which consists of a condensation of reticular cells and fibres. On leaving the ellipsoid, the penicillate arteriole becomes funnel-shaped and opens into the splenic pulp or into a venous sinus.
  6. Venous capillaries and sinuses are lined by an incomplete layer of reticular cells, so that there are numerous openings in their walls, through which the red blood cells can pass directly into their lumina.
  7. The cellular elements of the pulp comprise reticular cells, macrophages derived from the reticular cells and blood cells. In the bulb-like enlargement of the dorsal limb there were also present large coarsely granular acidophil cells, the origin of which is unknown. Giant cells were not observed.


II. The Suprarenal

The Monotreme suprarenal has been described by Elliott and Tuckett (14). According to their account in both Ornithorhynchus and Echidna, it consists of a cortex and medulla, distinct from each other and separated by a layer of connective tissue and a sinus-like “blood space.’’ The cortex forms the major portion of the gland and surrounds the medullary tissue except at the caudal end; in Ornithorhynchus it is divided into lobules by the blood vessels passing into it from the capsule. The cells on the edge of these lobules are small and closely packed together, but those in the centre of the lobules are larger and coarsely vacuolated. This lobulation is not found in the narrow zone immediately adjoining the medulla. In Echidna the cortex is not lobulated and the cells are arranged in anastomosing columns, in the meshes of which are found large sinus-like capillaries. At the caudal end of the gland prolongations of the cortical tissue are found intermixed with the medullary cells, whilst small ganglionic masses are found lying near the medullary tissue or actually embedded in it. The material used by these workers was not well preserved, so that they do not give any detailed account of the structure of the cortical or medullary cells.

Material

The suprarenals were fixed in formalin and kept in alcohol for a number of years. After embedding in paraffin, serial sections were cut along the long axis of the gland and sections were stained with Ehrlich’s haematoxylin and counterstained with eosin, Pasini, Dominici, with Masson’s triple stain and Dustin’s silver impregnation method. In order to supplement my observations I have examined the suprarenals of pigeon, rabbit, guinea-pig, dog, cat, ox and Man, which were lent to me by Miss R. Deanesly.

Observations

The suprarenal in Echidna lies on the ventro-lateral aspect of the corresponding kidney, enclosed with this organ in a fold of the visceral peritoneum. It is elongated or oval in form, measuring in cross-diameter approximately 1-2 x 0-65 cm. and is invested with a thick fibrous capsule.

As can be seen in text-fig. 38, the medulla (chr.c.) forms an elongated mass, surrounded by the cortical cells (z.7.2, z.g.) except over the caudal pole of the gland where the medulla comes to the surface (hk). The blood vessels enter and leave the gland in this region and a small ganglion (g.n.) is also often found embedded in the medullary tissue (chr.c.). The cortex and medulla are more or less distinctly separated from each other by a layer of connective tissue and blood sinuses (text-fig. 4, c.¢., bl.s.). In the caudal region prolongations of the cortical tissue are found among the chromaffin cells.

The Cortex

The cortex, as can be seen in text-fig. 8, forms the anterior or cranial part of the gland, comprising about two-thirds of the entire organ. It envelops the medulla (chr.c.) except at the caudal pole of the gland, and it differs somewhat in its histological structure in the cranial and caudal portions. In the cranial region, the cortex consists of three zones (text-fig. 3, 2.7.1, z.f., 2.7.2),

the outer zone (z.7.1) and the deepest zone (z.7.2) appearing reticular in form and resembling the zona reticularis of the Eutherian organ. These two zones become continuous with each other in the caudal part of the gland (Plate III, fig. 7, j.), whilst between these two layers is found a more compact inner zone (text-fig. 3, z.f.), the zona fasciculata of the Eutheria.

The appearance presented by the cortical tissue here is suggestive of an invagination caused by the approximation of the medullary tissue to the cortical cells. The deepest layer of the cortex lying next to the medulla being originally part of the outer cortical layer, whilst the inner compact layer formed the centre of the originally spherical cortical mass (text-fig. 3, chr.c.).

The medulla is covered by a prolongation of the zona reticularis of the cortex (text-fig. 8, z.r.1) which spreads over the chromaffin tissue except at the caudal pole. Round the hilum there is present an area of cortical tissue which differs in histological detail from that forming the other cortical zones. This layer resembles the zona glomerulosa of the Eutherian suprarenal, and I propose to use this name for it. The cells of this layer (text-fig. 3, z.g., Plate III, fig. 8, z.g.) spread forwards cranially as a thin sheet between the capsule and the outer reticular zone, and gradually thin out so that they are absent from the central and cranial parts of the gland. Sometimes, however, isolated groups of cells belonging to this zone can be recognised under the capsule in these regions. In the region of the hilum this layer becomes infiltrated with connective tissue and round the hilum itself (text-fig. 5, h.) the connective tissue almost completely replaces the cortical cells.


The outer reticular zone of the cortex (text-fig. 3, 2.7.1) lies directly under the thick fibrous capsule and forms about two-thirds of the cortical tissue of the gland. It consists of an anastomosing network (Plate III, fig. 7, 2.7.1) formed by columns of polygonal-shaped cells which are indistinctly delimited from each other. Between the cell columns are spaces which are occupied by blood vessels (Plate III, fig. 9, c.) and connective tissue, derived from the septa which grow in from the capsule. The cells forming the cell columns have vacuolated cytoplasm, whilst their nuclei are small and spherical, and stain intensely with haematoxylin. This zone resembles the zona reticularis of the suprarenal of the Eutherian Mammal in its histological detail, although it differs from this in its position directly under the capsule.


At the cranial end of the gland is found a peculiar conical area (text-fig. 3, c.pt.) which projects into the connective tissue of the capsule (cap.). In this region the connective tissue of the capsule appears lamellar in structure, and in the meshes of this are found groups of cells which stain somewhat darker than those of the outer layer of the cortex, although in general detail they resemble these cells. They form small groups or lamellae of irregular size which are separated from each other by well-defined connective tissue septa. Blood vessels characteristic of the outer cortical zone are not present.


The inner zone of cells or zona fasciculata (Plate ITI, fig. 10, z.f.) consists of rather coarse strands of paler staining polyhedral cells which run more or less parallel to each other and which are separated by reticular fibres (Plate III, fig. 10, ret.f.) and small capillaries. These cells are larger than those of the outer layer and stain less intensely. Their nuclei (nuc.) are round, whilst the chromatin is clumped in small granules lying close to the nuclear membrane. The cytoplasm contains vacuoles, larger than those of the outer layer, so that it seems likely that this region is rich in lipoids. ,

Separating this zone from the medullary tissue is a region composed of anastomosing strands of cells (Plate III, fig. 7, z.r.2) which resembles the zona reticularis (z.7.1) in its histological structure. This zone is considerably thinner than the outer reticular zone (z.r.1), although it becomes continuous with that in the caudal part of the gland. These two layers of the zona reticularis form a covering to the medullary tissue in the caudal part of the gland. Towards the hilum, however, they are replaced by a layer of darkly staining, coarsely vacuolated cells forming the zona glomerulosa (text-fig. 3, z.g.) which for a short distance spreads anteriorly over the cells of the zona reticularis and lies between this zone and the connective tissue capsule. These cells of the zona glomerulosa are arranged in irregular or oval groups (Plate III, fig. 11, 2.g.) which are separated from each other by anastomosing connective tissue septa (c.t.) derived from the capsule (cap.). These connective tissue septa become very numerous in the region of the hilum and often completely replace the cortical cells. The cells are columnar in shape (Plate III, fig. 11, 2g.) and distinctly separated from each other, whilst their nuclei are oval or spherical, and stain deeply with haematoxylin. This zone, as I have already described, spreads over the cortical cells towards the middle of the gland, whilst isolated groups of these cells can be often seen in the more cranial part of the gland.

The Medulla

The medulla consists of cords of cells (text-fig. 4, chr.c.) separated by fine strands of reticular connective tissue (ret.f.). The latter anastomose to form a network, in the meshes of which are found blood capillaries and blood sinuses.


The chromaffin tissue consists of large polyhedral cells which are nearly twice as large as the cortical cells, whilst they are not sharply delimited from each other. Their nuclei (nuc.) are large, spherical in shape, with a prominent nuclear membrane, whilst the chromatin is aggregated into one or two granules. The cytoplasm is finely granular and contains numerous large vacuoles. These, as far as I am aware, have not been described in any other animal.

The medulla comes to the surface at the hilum of the gland (text-fig. 5, h.) and in this region just as Elliott and Tuckett (14) described there is present a small nerve ganglion (g.n.). This ganglion is enclosed in a connective tissue capsule which separates it from the medullary cells. The nerve cells (n.c.) are multipolar and their processes ramify amongst the cells of the chromaffin tissue.


Text-fig. 3. Diagram to show the relation of the cortex and medulla in the suprarenal gland of Echidna. The cortex can be seen to form the cranial part of the gland and it forms a covering to the medulla (chr.c.) except over the caudal pole of the gland. The cortex consists of three zones(z.7.1,z.f.,2.g.). Of these the zona reticularis (z.r.1) and the zona fasciculata (z.f.) are found aggregated at the cranial end of the gland. The zona reticularis also forms a narrow zone (z.r.2) adjacent to the medulla which is continuous with the outer zone (z.r.1) in the caudal part of the gland. The zona glomerulosa (z.g.) forms a thickened mass at the caudal end of the gland which runs forwards for a short distance over the rest of the cortex towards the middle of the gland. The medulla (chr.c.) is oval in shape and projects on the surface at the caudal pole where a small sympathetic ganglion (g..) is found embedded in the medullary tissue. On the cranial end is found a small conical area (c.pt.) formed by cells arranged in groups oF lamella between the layers of the capsule.

The Supporting Framework

The suprarenal is invested by a thick fibrous capsule which is formed of two parts, an outer compact layer of fibrous connective tissue and an inner looser layer composed of reticular cells and fibres. The capsule becomes continuous with the connective tissue covering the blood vessels in the region of the hilum, and in the cranial region it forms a covering to the small conical projection which I have shown in text-fig. 3, c.pt. The septa which run into the cortex from the capsule are of two types, the larger ones extend through the greater part of the cortex and are formed of connective tissue and reticular fibres, whilst the smaller ones are formed chiefly of reticular fibres. These latter spread round the cell columns in the zona reticularis, whilst the longer septa reach the zona fasciculata, where they separate into thin strands or threads which ramify round the cellular strands. The reticular fibres are not so coarse here as in the zona reticularis, whilst the cellular strands are more closely packed together.


Text-fig. 4. High-power drawing of the suprarenal of Echnida, showing the medulla (chr.c.) and the zona reticularis (z.r.2) lying adjacent to it. Mag. x 130. The chromaffin cells (chr.c.) are arranged in irregular cords, separated by strands of reticular fibres (ret.f.). The nuclei (nuc.) of these cells are large and spherical in shape, whilst the cytoplasm contains large vacuoles. Blood sinuses (bl.s.) are also seen in the chromaffin tissue. The zona reticularis (z.r.2), formed of anastomosing cell-cords, lies separated from the medulla (chr.c.) by a layer of connective tissue (c.t.). These cells are smaller than those of the medulla and stain darker with hsematoxylin.


In the zona glomerulosa round the caudal part of the gland, the connective tissue septa are much coarser than in the other cortical zones, and they ramify between the cortical cells, separating them into small, round or oval masses (Plate III, fig. 11, z.g.). Towards the hilum, the invasion of the cortical zone by the connective tissue is so great that the cortical cells form isolated groups among the connective tissue strands and they are often almost completely replaced by dense fibrous connective tissue.

In the deepest cortical zone (i.e. the zone continuous with the outer reticular zone which lies adjacent to the medullary tissue), the septa are thicker and spread out between the cells. They run into the connective tissue layer between the cortex and medulla.



Text-fig. 5. High-power drawing of caudal end of the suprarenal. Echidna. Mag. x115. The medulla (chr.c.) comes to the surface of the gland in this region (4.) and close to it can be seen a small sympathetic ganglion (g..). Some of the nerve-cells (n.c.) of this ganglion can be seen embedded in the chromaffin tissue (chr.c.). A bundle of nerve fibres (n.f.) can be seen in the left lower corner of the figure.


The supporting framework of the medulla is derived partly from the layer of connective tissue (Plate III, fig. 7, c.t.) which separates the cortex and the medulla, partly from the connective tissue of the hilum and partly from the connective tissue surrounding the prolongations of cortical tissue which run into the medulla. The connective tissue and reticular fibres (text-fig. 5, ret.f.) spread out between the cell strands, forming a well-defined network round them. This network is much coarser and better developed than in the cortical regions.


The Blood Supply

(Text-Fig 6)

The suprarenal receives its blood from an artery (a.) which runs into the organ at the hilum. This vessel divides into numerous branches, some of which {a.cap.) ramify in the capsule, whilst the majority run directly to the cortex (a.cor.) and the medulla (a.chr.c.).


Fig. 6 (see legend opposite).

(1) Arteries of the capsule (a.cap.)

The capsule is supplied by a number of small branches from the main artery (a.) which form an ill-defined capillary plexus (cap.pl.) in the inner connective tissue layer of the capsule. This capillary plexus is very irregular and is best developed in the conical projection at the cranial end of the gland. The blood from the capillaries is collected into large sinuses (bl.s.) from which it runs into the surrounding connective tissue. Some of the blood from the capillary plexus also drains from the cortex into the cortico-medullary blood sinuses (bl.s.) and the central venous channel (c.v.ch.).

(2) Arterioles of the cortex (a.cor.)

The cortex is supplied by numerous small arterioles (a.z.7r.1, a.2.f., .2.g.) which run into the cortical substance direct from the hilum. In the cortex the capillaries derived from these arterioles run into the connective tissue septa derived from the capsule, and ramify between the anastomosing cellular strands forming a complicated sinus-like plexus (z.7.1.pl.).

In the outer zone the capillaries form wide anastomosing channels, the size of the vessels ‘being dependent on the mesh-like character of the spaces between the cellular strands. In the inner zone where the cell strands are thicker and more uniformly arranged, the capillaries (z.f-pl.) run parallel to one another, whilst they are joined by numerous transverse anastomoses (t.ans.). On reaching the deeper zone adjacent to the medullary tissue the capillaries (z.7.2.pl.) become larger and form an irregular meshed plexus.

- Some of the larger arterioles (a.z.f.) which run through the outer reticular

zone to the zona fasciculata are accompanied by an invagination of cells of the zona reticularis which form a thin covering to the vessels. This covering has been observed by Elliott and Tuckett (14) in the suprarenal of Ornithorhynchus.


Text-fig. 6. Diagram to show the blood-supply of the suprarenal. Echidna. Note the suprarenal is supplied by an artery (a.) which enters the gland at the hilum and divides into numerous branches (a.cap., a.cor., a.chr.c.). Some of the branches (a.cap.) enter the capsule (cap.) and form a capillary plexus (cap.pl.) in the deepest layer. The blood from these capillaries may drain into the subcapsular blood-sinuses (b/.s.) and so into the surrounding connective tissue and away from the gland or into the capillaries (z.g.pl., z.r.1.pl., z.f.pl., z.r.2.pl.) of the cortex. Other branches (a.cor.) pass directly into the cortex and run in a longitudinal direction through this giving off numerous small branches (a.z.g., a.z.r.1, a.z.f.) which ramify in the connective tissue septa between the cells and form a capillary plexus (z.r.1.pl., z.7.2.pl., z.f.pl., z.g.pl.). In the zona reticularis (z.r.1, z.r.2), the capillaries (z.r.1.pl., z.r.2.pl.) are large and irregular in shape, whilst in the zona fasciculata, they (z.f.pl.) are narrow and lie parallel to one another, joined by numerous transverse anastomoses (t.ans.). The blood from the superficial cortex may drain into the subcapsular blood-sinuses (6J.8.) of the cortico-medullary zone or into the central venous channel (c.v.ch.).


The medulla is supplied by blood from two sources, by branches (a.chr.c.) from the main artery (a.) or indirectly from capillaries (a.cor.t.) derived from the cortical branches (a.cor.) of the main vessel (a.) which runs along cortical trabeculae. On reaching the medulla they form a capillary plexus (chr.c.pl.) which is ultimately drained into the blood sinuses (6l.8.) of the cortico-medullary zone or into the central venous channel (c.v.ch.). At hilum (4.) the central venous channel (c.v.ch.) gives rise to two or more veins (v.).

The blood from the capillaries of the zona reticularis (z.7r.1.pl.) drains either into subcapsular sinuses or through the capillary plexus of the inner zones into the central venous channel (c.v.ch.) which runs from the centre of the cortex through the medulla to the hilum. In the deeper zones the blood drains either into the small irregular blood sinuses (bi.s.) of the cortico-medullary boundary zone or into the central venous channel (c.v.ch.).

(8) Arterioles of the medulla (a.chr.c.)

The medulla is supplied by small arterioles (a.chr.c.) entering it directly from the hilum or by capillaries (a.cor.t.) which run into it along the cortical trabeculae. On reaching the medulla these vessels form an irregular plexus (chr.c.pl.) in the connective tissue reticulum round the cell columns, The vessels of this plexus are very much larger than those in the cortex and they drain either into the central venous channel (c.v.ch.) or into the large sinuslike branches (bl.s.) of this channel. From this channel the blood is collected into veins (v.) in the region of the hilum. The venous blood therefore from the cortex and medulla are intermixed in this central venous channel (c.v.ch.), for although a number of venules run into the capsule from the superficial zone of the cortex, most of the blood is collected in this central venous channel (c.v.ch.).

The walls of the capillaries, blood sinuses (bl.s.) and the central venous channel (c.v.ch) are formed by a single layer of endothelium resting on the reticular fibres. In the region of the hilum (h.) the veins (v.) which arise from the blood sinuses are enclosed in a single layer of longitudinally running smooth muscle fibres. A circular muscle coat was always absent from these vessels, This peculiar arrangement of the muscle coat on these veins has also been described by Flint (5) in the suprarenal of the {{dog++.

Discussion

The suprarenal of the Monotreme forms an intermediate stage between that found in the lower Vertebrates and that of the higher Mammalia. In the Bird and in some Reptiles the cortical and medullary cells are found intimately intermixed with each other. This is perhaps best seen in the Bird’s suprarenal, where the entire organ is formed by an intermixing of cortical and medullary tissue; in the Reptilia the intermingling of the two layers is less than in the Bird; and in the lizard and snake, according to Poll (16), there is a separation of the cortical tissue to the dorsal surface of the gland. In the cortical region, the medullary tissue is still intermixed with strands of cortical cells.

In the Monotreme suprarenal, this separation is almost complete, for the medullary tissue is aggregated at the caudal end of the gland, whilst the cortical cells form a covering to the medullary cells except over the caudal pole. In this region, cellular trabeculae run into the medulla from the cortex, but the intermingling of the cortical and medullary tissue is of very slight extent compared with that in the Reptilia.

The appearance presented by the Echidna suprarenal is suggestive of the invagination of the cortical tissue by the medullary cells, for, as can be seen in text-fig. 3, the outer zona reticularis is continuous with the inner reticular zone of the cortex which lies adjacent to the cortico-medullary boundary. At the caudal end of the gland the zona reticularis has secondarily, so it appears, grown round the medullary tissue to enclose it except over the caudal pole. The zona glomerulosa appears only at the caudal end of the gland and grows forward cranially for a short distance over the zona reticularis.

It is interesting to note that in the bird (pigeon), the cortical cells resemble in appearance the cells of the zona glomerulosa of the Eutherian Mammals. Cells comparable to those of the zona fasciculata and zona reticularis are not found in the lower forms. These zones can be distinguished in the Monotreme suprarenal, although their relative positions differ. As I have shown, the zone comparable to the zona glomerulosa is found at the caudal pole of the gland, that comparable to the zona reticularis forms the outermost cortical layer, whilst the zona fasciculata, generally the best-developed zone of the cortex in the Eutheria, is poorly developed and forms the innermost layer of the cortex.

The chromaffin tissue appears similar to that found in the Eutheria except that in the cytoplasm of these cells there are found vacuoles from which it is probable that lipoids have been dissolved during the fixing process. Such lipoids are not found in the medulla of the Eutheria.

One of the most interesting features of the suprarenal of the Monotreme is, however, the incomplete surrounding of the medulla by the cortex and the presence of a small ganglion mass lying close to or embedded in the medullary tissue. According to Vincent(17), in the suprarenals of young animals, the medulla is not completely surrounded by cortex, but comes to the surface at some point. Further, he says that in the suprarenal of the young rabbit “near that part of the circumference where the medulla reached the surface was a sympathetic ganglion outside the capsule of the organ.” This similarity between the developing suprarenal of the young rabbit and that of the adult Monotreme is suggestive in that perhaps here we have a definite step in the evolution of the Eutherian suprarenal.

Summary

  1. The suprarenal of Echidna is ovalish in form, and consists of a welldifferentiated cortex and medulla. The cortex lies at the cranial pole of the gland and forms an incomplete covering to the medullary tissue. This latter tissue comes to the surface at the caudal end of the gland and constitutes the hilum where the blood vessels enter and leave the gland,
  2. The cortex, situated at the cranial end of the gland, consists of two zones, an outer reticular zone and an inner or zona fasciculata. Between the zona fasciculata and the medulla is found a narrow reticular zone which becomes continuous with the outer zona reticularis in the caudal region. This reticular zone spreads backwards over the medulla for some distance, but caudally it is replaced by a mass of cells, the zona glomerulosa. The cells of this zone spread cranially over the zona reticularis for a short distance, separating it from the capsule.
  3. The zona reticularis consists of a network of anastomosing cellular strands, in the meshes of which there are wide blood capillaries, whilst the zona fasciculata consists of more regular coarser strands between which capillaries run parallel to each other. The zona glomerulosa consists of groups of columnar cells, separated from each other by connective tissue septa. These septa in the region of the hilum often completely replace the glandular tissue.
  4. The cortex receives its blood supply from the capillaries of the capsule and also from arterioles which run direct from the hilum to the cortex and there break up into capillaries in the connective tissue septa. From these the blood passes either into the cortico-medullary sinuses or into the large blood channel which runs from the cortex through the medulla to the hilum, and there gives rise to the veins.
  5. The medulla forms the caudal part of the gland and is separated from the cortical tissue by a layer of connective tissue and irregular blood sinuses. It consists of groups of large granular cells with numerous large vacuoles in their cytoplasm. Intermixed with these cells are found prolongations of cortical tissue.
  6. The medulla is supplied by blood vessels from the hilum which ramify among the cells and are collected either into the cortico-medullary sinuses or into the main venous channel.
  7. A small ganglion composed of multipolar cells is found in the region of the hilum, often embedded in the medullary tissue. Processes from these cells ramify among the medullary cells.


This work was carried out in the Department of Histology and Embryology, under the direction of Prof. J. P. Hill, F.R.S., to whom I wish to express my thanks for permission to use the material from his collection and for his help and guidance throughout the investigation. I am indebted to Dr D. Krauz for the original drawings of text-figs. 4, 5 and 6; to Mr A. K. Maxwell for retouching the microphotographs from which Plate ITI, figs. 7 and 11 were made, and to Mr F. J. Pittock for making these microphotographs.

References

(1) Bannwarth (1891). “Untersuchungen iiber die Milz.” Arch. f. mikros. Anat. Bd. XXXVI.

(2) Foor, N. C. and Minarp, M. C. (1927). “Laboratory method and technical notes.” Arch. of Pathol. vol. rv. (3) (4) (5) (6) (7)

(8) (9)

(10)

(11) (12)

(13) (14) (15) (16)

(17)

Hetty, K. (1902). “Die Blutbahnen der Milz und deren funktionelle Bedeutung.” Arch. f. mikros. Anat. Bd. Lx1.

Kutrscuitsxy, N. (1895). “Zur Frage iiber den Bau der Milz.” Arch. f. mikros. Anat. Bd. xvi.

Macxenziz, W. C. (1916-17). “The shape and peritoneal relationship of the spleen in monotremes and marsupials.” J. Anat. vol. LI.

Mat, F. P. (1902-3). “On the circulation through the pulp of the dog’s spleen.” Amer. J. Anat. vol. 1.

Mottter, V. S. (1911). ‘Ueber den Bau der capillaren Milzvenen.” Arch. f. mikros. Anat. Bd. Lxxv1.

McNeEz, J. M. (1929). “Splenomegaly in Great Britian.” Glasgow Med. J. vol. m1.

Nevsert, K. (1922). “Der Ubergang der arteriellen in die venose Blutbahn bei der Milz.”’ Zeit. f. d. ges. Anat. Abt. 1, Bd. Lxv1.

Sozorta, J. (1914). “Anatomie der Milz.” In Bardeleben’s Handbuch der Anatomie, Bd. 111, iv, Anhang.

Tuoma, R. (1899). “Ueber die Blutgefisse der Milz.” Arch. f. Anat. u. Physiol. Anat. Abt.

WEIDENREICH, F. (1901). “Die Gefasssystem der menschlichen Milz.” Arch. f. mikros. Anat. Bd. Lyi.

Wauirtine, A. J. (1897). “Comparative histology and physiology of the spleen.” Trans. Roy. Soc. Edin. vol. xxXxv1.

Exuiott, T. R. and Tucxert, I. (1906). “Cortex and medulla in the suprarenal gland.” J. Physiol. vol. XxxIv.

Fut, J. M. (1900). “The blood vessels, angio-genesis, organo-genesis, reticulum and histology of the adrenal.” Johns Hopkins Hosp. Rep. vol. 1x.

Pott, H. (1906). “Die vergleichende Entwicklungsgeschichte der Nebennierensysteme der Wirbeltiere.” Hertwig’s Handbuch der vergl. und exper. Entwick. der Wirbeltiere, Bd. 11.

Vincent, S. (1898). “The comparative histology of the suprarenal capsules.” Internat. J. Anat. and Physiol, vol. xv.

Explanation of Plates

List of Abbreviations

A, Artery.

a Arteriole.

a.cap. Arteriole to the capsule.

a.chr.c. Anteriole to the medulla.

a.cor, Arteriole to the cortex.

a.cor.t, Arteriole derived from the cortical branch which runs along cortical trabecula.

a.z.f. Arteriole to zona fascicularis.

0.2.9. Arteriole to zona glomerulosa.

a.z7.1 Arteriole to zona reticulata.

6. Body of the spleen. ol, Blood.

bls. Blood sinus.

C Capillary.

Ca. Central artery.

Plate I

Fig. 1. Low-power drawing of Malpighian corpuscle (M.c.). Mag. x93. Mallory’s connectivetissue stain. Note the Malpighian corpuscle is surrounded by a capsule (cap.M.) formed of reticular fibres (ret.f.) inside which is seen the peripheral blood-space (l.s.), the blood from which is drained into the splenic pulp by means of fine capillaries (c.). Surrounding the Malpighian corpuscle (M.c.) is seen the splenic pulp (sp.p.) containing a small venous sinus (v.8.).

Fig. 2. Low-power drawing of Malpighian corpuscle (M.c.) to show central artery (c.a.). Mag. x 135. Mallory’s connective tissue stain. Note the central artery (c.a.), surrounded by connective tissue (c.t.) and the blood space (6l.s.) at the periphery of the corpuscle, just inside the capsule (cap.M.). The central artery (c.a.) can also be seen, lying in the splenic pulp (sp.p.) before it enters the corpuscle (M.c.).

Plate II

Fig. 3. High-power drawing of trabecula of Echidna spleen. Mag. x 297. Ehr. haem. and Pasini. The trabecula (7’.) consists chiefly of smooth muscle (s.m.) and a small quantity of connective tissue (c.t.). On either side of the trabecula is seen the syncytial network of reticulo-endothelial cells (ret.c.). Two large reticular cells (ret.c.) are seen intermixed with red blood corpuscles (7.6/.) at the right lower corner of the figure.

Fig. 4. High-power drawing of ellipsoid (el.). Mag. x 283. Ehr. haem. and Pasini. The terminal artery (t.a.) has divided into two penicillate arterioles (p.a.) which can be seen surrounded by a layer of reticular cells (ret.c.) and fibres (ret.f.) which constitute the ellipsoid (e/.). The penicillate arterioles (p.a.) are lined by a layer of endothelial cells (end.c.) whose nuclei project into the lumen (J.), which at the funnel-shaped opening (f.) become flattened and not clearly distinguishable from the reticular cells (ret.c.).


Fig. 5. Low-power drawing of a venous sinus. Mag. x 73. Mann’s methyl-blue-eosin stain. Note the large venous sinus (v.s.), containing blood (6/.) into which a number of small venous capillaries (v.c.) open from all sides, draining the blood from the surrounding splenic pulp (sp.p.). A part of the trabecula (7'.) is seen on the left of the venous sinus (v.s.). Below the trabecula (7'.) there is another small venous sinus (v.s.).

Fig. 6. High-power drawing of the granular cells (gr.c.) found in the pear-shaped enlargement of the dorsal limb of the spleen. Mag. x 439. Ehr. haem. and Dominici. Note the granular cells (gr.c.) lying intermixed with the reticular cells (ret.c.), lymphocytes (lym.), red blood cells (r.6l.) and reticular fibres (ret.f.) of the pulp. Compare the pycnotic nuclei (p.nuc.) of these cells with those of the cells of the surrounding tissue. ,


Plate II

Fig. 7. Medium-power microphotograph of the cortico-medullary portion of the suprarenal. Echidna. Mag. x56. Note the medulla (chr.c.) is separated from the cortex by irregular blood sinuses (b/.s.) and layers of connective tissue (c.t.). The layer of cortical cells (z.7r.2) surrounding the medulla is darkly stained and contains wide capillaries. At (j.) it becomes continuous with the zona reticularis (z.r.1). The inner zone or zona fasciculata (z.f.) stains paler and the cells are arranged in coarse parallel strands.

Fig. 8. High-power microphotograph of the cortex, about the middle of the suprarenal. Echidna. Mag. x90. Note the outer zona glomerulosa (z.g.) composed of oval groups of columnar cells separated by irregular anastomosing connective tissue septa, and the second layer or zona reticularis (z.r.1) which forms the outer layer of the cranial part of the cortex. This zone consists of an anastomosing network of cellular strands in the meshes of which are wide capillaries (c.). The innermost layer, zona fasciculata (z.f.) consists of paler staining cells arranged in coarse regular strands separated by fine reticulum.

Fig. 9. High-power microphotograph of the zona reticularis of the cortex. Echidna. Mag. x 180. Note the anastomosing strands of cells which form a wide meshed network, the spaces of which are occupied by large blood capillaries (c.). The cells are not sharply outlined from each other and their nuclei (nuc.) are small and spherical whilst their cytoplasm is finely vacuolated.

Fig. 10. High-power microphotograph of the zona fasciculata (z.f.) of the suprarenal. Echidna. Mag. x 180. Note the coarse regular strands of cells which are separated from each other by reticular fibres (ret.f.) and small capillaries. Portions of zona reticularis (z.r.1, z.r.2) are seen at the top and bottom of the figure respectively.

Fig. 11. High-power microphotograph of the zona glomerulosa (z.g.) of the suprarenal. Echidna. Mag. x 180. Note the arrangement of the cells in irregular rounded groups. These are separated from each other by anastomosing connective tissue septa (c.t.) derived from the capsule (cap.). The cells are columnar in form and their nuclei are oval or spherical. The cytoplasm contains numerous coarse vacuoles.



Cite this page: Hill, M.A. (2020, October 29) Embryology Paper - The histology of the spleen and suprarenals of echidna (1932). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_The_histology_of_the_spleen_and_suprarenals_of_echidna_(1932)

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