Talk:Book - Contributions to Embryology Carnegie Institution No.42

By Charles R. Essick, Captain, Medical Corpx, U.S. Army, Army N ritro-surgical Laboratory, Baltimore.

With one pliite.

FORMATION OF MACROPHAGES BY THE CELLS LINING THE

SUBARACHNOID CAVITY IN RESPONSE TO THE STIMULUS

OF PARTICULATE MATTER.

By Charles R. Essick.

In the course of a study of the processes involved in the locaHzation of an infection within a focus in the nervous system, certain physiological reactions of the cells lining the subarachnoid space have been noted. When active or inert particles of matter are injected into the subarachnoid cavity of a hving animal, the cells Uning the space hypertrophy, lose their normal attachments, and engage in remov- ing the debris. The importance of such a formation of free-living cells from fixed elements in any process involving destruction and repair in the meninges (infection, hemorrhage, etc.) becomes apparent. The control of cell-reaction promises much in the ultimate therapy of such conditions.

Physiological activity of cells has always been an attractive study, two functions of which may be readily demonstrated in fixed preparations, — i. e., phagocytosis and amoeboid wandering. We are accustomed to think of cells as peculiarly fitted to the specialized work in which they are normally engaged ; for example, the peri- toneal and pleural surfaces are membranes of cells specifically adapted to the free movement of viscera; endothelium of blood-vessels forms a closed tube for con- ducting the various chemicals used in tissue economy; connective tissue furnishes a supporting framework, and so on. As a corollary to this idea we have to employ a special set of unattached cells to remove products formed during the normal wear and tear of the tissues, and to overcome and remove any noxious stimulants.

The kaleidoscopic changes which take place in inflammation have attracted many observers to the role played by the so-called fixed cells and have given rise to a large number of conflicting views. It seems unnecessary, for a clear under- standing of this paper, to go into these conceptions in detail. Accumulated evidence leaves little doubt that, under certain conditions, the normal specialized function becomes a secondary characteristic and the more primitive attributes of the uni- cellular organism become the predominant features. In other words, unless a cell has become too highly specialized the primitive functions of free amoeboid move- ment and phagocj-tosis may be elicited by the proper type of stimulation in cells which normally are regarded as sessile or fixed elements. The connective tissues have furnished Maximow with a host of cells (polyblasts) , normally sessile and in fact almost indistinguishable from their neighbors; such polyblasts under stimula- tion become amoeboid and phagocytic. At times even the fibroblasts may round up and behave toward irritants in the same way that the polyblasts do. Schott (1909), confirmed by Goldmann (1912), showed that the mesothehal Uning of the pleural and peritoneal cavity could furnish free-moving phagocytic cells. In exactly the same way, when the destruction of brain tissue occurs, neuroglia cells

379

380 MACROPHAGES FROM ARACHNOID CELLS.

pull in their protoplasmic processes and become globular and phagocytic (Alzheimer, 1910). The endothelium of blood vessels has been recognized for a long time as furnishing large phagocytic cells in areas of inflammation. Evans (1915), bj^ oflfering trypan-blue to the endothelium of the liver, lymph glands, and spleen, has observed the formation of a new circulating mononuclear blood element (macrophage) which buds off the lining of the vessel lumen, but only after prolonged irritation.

To INIetschnikoff (1S92) we owe the physiological term macrophage. This term was adopted becaus(» it did not imply a fixed ancestry. An excellent presentation of the biological activities of the phagocytic cells in inflammation of the brain is given by Mertzbacher (1909). His term Abrdumzell is much more suggestive in that it calls to mind the attempts at repair which go hand in hand with efforts to nullif.y the destruction of tissue continuity. This study is concerned with the efforts of the arachnoid membrane towards such removal of foreign material. To include the polyblast of Maximow, the pyrrol-zell of Goldmann, the clasmatocyte of Ranvier, the endothelioid cells, and Kornschenzellen in the class of macrophages is to simply express a histological similarity combined with a common physiological behavior. The common histological properties are the single nucleus eccentrically placed and a cytoplasm usually reticulated because of the intracellular inclusions. The cells may contain fat-globules, inert bodies, blood elements (either red or white), and albuminous granules, as well as vacuoles. Among their physiological functions are independent freedom of movement and ingestion of particulate matter. In the very first weeks of embryonic life one comes upon such cells fully developed, before any others, to their adult form, first in the placenta and later in the body of the embryo (Essick, 1915). Certainly we are dealing with a universal expression of the need, in the physiological economy of the organism, for cells whose function is to remove the products of tissue destruction. A very good resume of the hterature up to 1909 is given by Schott, who discusses the relation of fixed tissue and macrophages.

For a better understanding of the results of experimental introduction of minute particles into the subarachnoid space with the subsequent formation of macrophages, it seems necessary to give a brief description of the anatomy of the leptomeninges and the mode of preparing specimens for study.

The pia mater and arachnoid may be ajitly compared to a living sponge accurately filling the irregularities between the brain and the dura, the exterior surface of which is formed by a closed semi-permeable membrane, while the surface approximating the nervous tissue is perforated by the entrance of the perivascular spaces. Vessels, nerves, and ligaments pass through tliis layer of tissue without lying free in the cavity. Just as the spaces in a sponge are in free intercommu- nication, so the cerebro-spinal fluid is normally continuous everywhere. The connective-tissue framework is largely made up of white fibrous tissue, covered by a continuous layer of flattened cells externally, where they form a comparatively simple uninterrupted surface looking toward the dura. The underlying space, known as the subarachnoid cavity, is broken up by anastomosing strands, but the lining presents a continuous unbroken surface of cells cxcei)t where the perivascular

MACROPHAGES FROM ARACHNOID CELLS. 381

channels open into it. The spinal cord has been chosen as a place for studying the arachnoid tissue, liccause of the ease in approaching it experimentally and pre- paring specimens for microscopic study.

In these observations cats of various ages were used, but the results were uniform. After sacrificing the animals with ether the thorax was opened and 10 per cent formaUn was injected immediately into the aorta. A few hours later the bony covering of the central nervous system was removed en bloc and the dorsal region of the ]:)rain and cord was exposed, care being taken not to rupture the dura. Those specimens, partially exposed, were immersed for 5 days in 10 per cent for- malin to insure good fixation, after which all of the remaining bone was removed. The dural covering of a section of the cord was carefully removed and the arachnoid separated from the pia as an intact membrane. This was best accomplished by the aid of a binocular microscope, as great care had to be exercised to avoid undue tension on the delicate strands uniting the pia and arachnoid. Eye-scissors were used to cut the trabeculse. The membranes thus obtained were best studied by immersion in a weak aqueous solution of toluidin blue. Permanent preparations were made by the technical methods usually applied to celloidin sections. The conventional microscopic section of the pia arachnoid, in adcUtion to its shrunken and distorted picture, gives one a very limited field to study, as only a small frag- ment of the rich tl-abecular system appears in any one specimen. To this hmitation is added the fact that the strands are usually cut in such an oblique direction as to render them almost unintelligible. When examined in a dissected specimen the cells clothing the smaller trabeculae are almost completely isolated from their neigh- bors and furnish a brilliant opportunity^ for studjdng them in profile or for noting their cellular contents without confusion. This clearness of i)icture makes the study of cell hypertrophy and proliferation more convincing because of its comparative isolation, and it approaches more nearly the conditions seen in ti.ssue cultures where cells become amoeboid and separate themselves from their normal environment.

The cells covering the membranous expansion of the arachnoid have large, pale, oval nuclei with very indistinct chromatin network (fig. 11). With the ordinary cytological stains the cell-boundaries can not be made out, j-et their irreg- ular arrangement may be demonstrated by silver precipitate. Distributed through- out the brain and cord are clusters of closely placed nuclei within the arachnoid membrane. These are well shown in the upper left corner of figure 11. Such areas are irregular in shape, size, and distribution. Histologically they correspond to the arachnoid cell clusters found by Weed (1914, p. 64) in the dura. They represent normal structures and, like the arachnoid trabeculse, become the seat of calcium deposits with the advancing age of the animal. In no sense should they be mis- taken for a cellular proliferation in response to a degenerative process. One does not choose by preference the membranous expansion of the arachnoid in studjdng the formation of macrophages. One meets here the same difficulties that are encountered in the flat serous surfaces, such as the peritoneum. It is hard to elimi- nate doubt concerning the exact relations of a single cell to the membrane spread out as a flat preparation. Analogous processes can be made out, but not with the

382 MACROPHAGES FROM ARACHNOID CELLS.

same convincing clearness, as one finds on the thin connective-tissue trabeculae uniting the arachnoid membrane to the pia mater.

The cells clotliing the trabecular are very sensitive to changes in the cerebro- spinal fluid which bathes them. Weed (1917, p. 467) calls attention to tliis fact by remarking that the "general morphology * * * depends apparently * * * on their physiological state." Particulate matter, either resulting from cell destruction or introduced directly into the cerebro-spinal fluid, calls forth a most remarkable reac- tion. Inert particles, such as carbon or cinnabar, as well as active matter, such as fragmented red blood-corpuscles or dead leucocytes, initiate morphological changes in the arachnoid cells. The reaction of the cellular membrane to such particulate matter is a slow one and appears to be well under way only after the first 24 hours; dead bacteria may be taken up and removed by the leucocytes before the arachnoid cells show any signs of activity. The most striking results have occurred after stimulation with laked blood, due probably to the fact that it has no toxic effect on the ceUs and may be utiUzed by them as food. Partial laking with cUstilled water was resorted to because the red blood-cells seem to live for some time if in- jected immediately into the subarachnoid space, whereas laked corpuscles cause a very much more rapid response on the part of the arachnoid cells. This fact suggests a degree of protection against phagocytosis by the living erythrocyte.

The blood was prepared for injection with due precautions to keep it sterile. Twenty cubic centimeters of blood, either homologous or autogenous, were defibrin- ated by shaking up with glass beads. If massive doses of erythrocytes were desired the defibrinated blood was centrifugaUzed, and subsequently the isotonicity of the mixture was restored by adding 10 X normal concentration of sodium chloride, potassium chloride, and calcium chloride. One or two cubic centimeters could be slowly injected into the lumbar subarachnoid space, or (if a heavy dose were desired) a replacement of the cerebro-spinal fluid over the cord was done in the following manner: A needle was introduced through the occipito-atlantoid ligament and one into the lumbar subarachnoid space. Laked blood was allowed to flow by gravity into the lumbar needle while the displaced fluid made its escape from the occipital region until laked blood appeared. In this way one gets possibly 5 or 6 c.c. of corpuscles around the spinal cord, and if the irrigation pressure is maintained below 300 mm. of water the animal never shows any symptoms referable to the experiment when once it recovers from the anesthesia. The various steps were controlled bac- teriologically to rule out a possible confusion with a septic meningitis.

Within 6 hours after the hemolyzed erythrocytes are introduced into the subarachnoid space a full-blown sterile meningitis is in progress; 6,000 to 10,000 leucocytes, composed almost entirely of the polymorphonuclear and transitional variety, are present in a cubic milUmeter of the blood-tinged cerebro-sphial fluid. Examined on a warm stage these leucocytes exhiljit a most surprising amoeboid activity and their cytoplasm is literally stuffed with the small particles of frag- mented red blood-cells. At the end of 24 hours the leucocyte count in the spinal fluid has dropped to 2500-1500 per cubic milUmeter and in 48 hours has reached the neigliborhood c)f 100. With the decrease of smaller cells from the fluid a new

MACROPHAGES FROM ARACHNOID CELLS. 383

mononuclear element is found in the cerebro-spinal fluid in increasing numbers. It, too, is actively phagocytic and when studied on the warm stage presents the well-known morphological characteristics of the amoeboid macrophage. The nucleus is large, measuring 7 to 9 microns, and usually has a well-developed nucle- olus. The cnoplasm shows a large number of inclusions and vacuoles which take up the main body of the cell and flatten out the nucleus against the limiting mem- brane. Such inclusions consist mainly of fragmented erj-^throcytes or pigment granules of reduced hemoglobin. The number of these large cells may reach the neighborhood of 50 per cubic millimeter of cerebro-spinal fluid, withdrawn at the end of 48 hours.

IVIacroscopic examination of the spinal cord at the end of 48 hours reveals Uttle evidence of the injection of laked blood. A diffuse, pale salmon-pink may be seen through the dura, but unless one were looking for blood the cord might be easily regarded as perfectly normal in color. After five injections of blood, made at 48-hour intervals, a brownish tinge was evident macroscopically, but here again it was not pronounced, except in the immediate site of the injection.

Our chief interest lies in the microscopical appearance of the cells lining the arachnoid space when filled with laked red blood-corpuscles. The normal arachnoid membrane has been beautifully pictured by Key and Retzius, whose technique of preparing specimens for microscopic study was in most respects similar to that employed in these experiments. All of their illustrations show a finely granular protoplasm, becoming coarser around the poles of the oval nuclei. Their figure 1, plate X, corresponds very nearly to the resting normal trabecula which is pictured in figure 3. Their interpretation of this appearance is expressed in the following quotation (p. 127) :

"Um die Kerne, besonders aber an ihren beiden Polen liegt ein Haufen von Kornchen, welche theils feiner, mehr protoplasmatisch sind, theils aber grossere glanzendere Kugeln ausmachen. Diese Kornchen kommen fast an jedem Kerne vor, sind aber zuweilen nur sehr sparsam vorhanden, zuweilen aber auch sehr zahlreich, die Enden der Kerne fast verdeckend. Diese Kornchenzone, welche bei jungeren Indi\'iduen im Allgemeinen reich- licher erscheint und als mehr oder weniger veranderter Ueberrest des ursprunglichen Pro- toplasma zu betrachten ist, streckt sich in verschiedener Entfernung vom Kern auf die Oberfliiche der Scheide, sich allmahlig verdunnend und verschmalernd, hinaus, bald hat sie eine bestimmtere Begrenzung, bald erstreckt sie sich in verschiedener, zuweilen phantastischer Form, als Seesternanne u. s. w. nach verschiedenen Richtungen, am gewohnlichsten aber bipolar vom Kern hinaus."

These granules could be stained with rosanihn. Other granulation, similar to fat but not staining so deeply with osmic acid, were observed by these authors in the normal arachnoidal cells. Such appearances are shown in their Taf. x, fig. 3, and Taf. XI, fig. 1.

Specimens dissected from the arachnoid can be best studied in aqueous solu- tions. A faint stain with toluidin blue will help to differentiate the cell-structures, but the natural differences of refraction and normal color of the fragmented erj-thro- cytes give the most impressive preparations. The first effect of a change in cerebro- spinal fluid is reflected in the protoplasm of the cell. Normally very thm (fig. 1)

384 MACROPHA(iES FROM AKACHNOID CELLS.

SO as to be hardly demonstrable, it bef^ins to increase in thickness and the nucleus no longer stands out sharplj^ as viewed in profile (fig. 2a and fig. 3). Other cells conttiining small inclusions of c(!llular fragments (fig. 4a) are found. Their cyto- plasm now forms a resjjectable accumulation around the nucleus and the whole cell jirojects sharply from the trabecula. The nucleus is more (hstinctly circular in outhne and is seen to occupy iin eccentric position in the cell (figs. 2c and 4b). Still other cells are literally gorged with fragments of erythrocytes, some of which may be almost whole. In this condition (fig. 46) they are about ready to leave the sessile position always occupied and become amoeboid.

Histologically, these fixed cells do not differ from the free, round phagocytes wliich api)eared in the fiuid tai^pings and were identified as macrophages. They arc phj'siologically still a portion of the membranous lining of the fluid cavity, although their attachment becomes more and more restricted. After budding off they tend to become still further distended with erythrocytes and pigment, often reaching a diameter of 16 microns (figs. 6 and 7). Other cells (fig. 8) occur with relatively Uttle vacuolization of their protoi)lasm and few fragments of erythrocytes. They are smaller (9 to 1 1 microns) and represent cells losing their attachment while still in the stage represented in figure 3. Their protoplasm is finely granular and dense, showing a paler zone around the eccentric nucleus. The free-moving macro- phages gather in clumps (fig. 12) and are most numerous where the debris is greatest. Unless the quantity of matter is very large they quickly store it in their bodies. Occasionally a polymorphonuclear leucocyte, probably representing a dead cell, suffers the same fate as the red blood-cell (fig. 6). The cycle of development may be followed more easily on the trabecular, but the cells covering the membranous portion of the arachnoid, as well as those normally identified with the outer surface of the pia mater, undergo the same physiological reactions to the stimulus of the blood (lower portion of fig. 11). Intracellular inclusions are seen clearly enough when looking down upon a cell, but better evidences of the membrane's participa- tion are obtained in cross-section.

The cells of the arachnoid facing the dura mater show similar changes, but in only one instance was a subdural extravasation of blood produced in the region of the spinal cord without the hemorrhage involving the subarachnoid space. Over the cerebral cortex it is rather common to find a hemorrhage separating the dura and the arachnoid membrane. It is then possible to obtain fixed prei)arations of macrophages in a confined space, with their ixseudopodia thrust out for a consider- able distance. Such a cell is illustrated in figure 10, showing the characteristic vacuolated appearance of the protojjlasm.

This brings up the question of the specificity of certain cells to produce macro- phages, and their response to a stimulus applied at a distance. No portion of the arachnoid membrane shows any differences in its behavior towards particulate matter; to produce; a resjjonse, actual physical contact alone seems necessary. Thus, wh(Te the collections of d(bris are tliick almost every cell shows signs of swelling up, while adjoining regions look relatively (luiet. This is illustrated more clearly by the different reactions of the cells situateil on the two sides of the arachnoid

MACROPHAGES FROM ARACHNOID CELLS. 385

membrane. If blood is absolutely confined to the subdural space, the layer of arachnoidal cells facing the dura exhibits the characteristics welUng-up with macro- phage formation, while the layer of cells hning the subarachnoid space, although separated from those of the subdural space by only a very thin layer of white fibrous tissue, remains entirely unaffected. Vice versa, the arachnoid cells compos- ing the membrane looking toward the dura never participate in the reaction towards a stimulus appUed to the subarachnoid space. The collection of cells forming the plaques noted above never has been seen to furnish macrophages or even to phago- cji:ize particles of matter.

Permanent specimens stained with the ordinary methods furnish Uttle addi- tional information. Quite often we have all of the inclusions of the cells dissolved out or not counterstained. This gives us a chance to study the protoiolasm of the cells as they rest on the trabeculae. The typical foamy structure occurring in the macrophages is shown in figures 26 and 2c. As ordinarily seen (figs. 9 and 10) this network is the result of the vacuoles and ingested material within the cell. The loose meshwork has been frequently misinterpreted as a sign of degeneration, but it certainly suggests, in these experiments, hungry active cells.

A very natural question arises concerning the fate of the denuded trabeculae and those regions of the membrane which have lost likewise their covering of cells. It is quite easy to convince one's self that the number of cells covering the connective-tissue strands is reduced or, in specific instances, that the entire cellular covering of the trabeculae is gone. Carelessness in preparing the specimen may result in a flaking off of the cells clotning the trabeculse, and undue tension brings about a loosening of the cells or even produces a naked bundle of fibrous tissue. On the other hand, the number of cells per unit of area is by no means constant in the normal arachnoid, and the personal element of interpretation can be exercised to any extent. The efforts to replace cells which have budded off are widely dis- tributed at the time when the production of the free-mo\'ing macrophages is at its height. ^Mitotic figures occiu- with marked frequency, both among the cells covering the trabeculae (fig. 5) and the membranous expansion of the arachnoid (fig. 11). The locaUzed occurrence of dividing cells corresponds to the areas of particulate stimulation, and in all probabihty there never exists a true denuding of the surface. During the process of division the protoplasm takes on a denser stain, partaking more strongly of the basic dyes. The protoplasmic bodies of the ceUs which have not detached themselves close over the gap and very shortlj'^ a new division takes place. An actual proUferation of arachnoid cells, resulting in the formation of a regular morula mass, has not been observed in comiection with blood stimulation alone, but the combination of infection and blood gave a remarkable picture of this phenomenon.

Experiments were carried out with dilute suspensions of carbon and cinnabar granules. Phagocytosis of inert matter by the cells comprising the membrane could not be expected to be as vigorous as is the case with erjlhroc^-tes, inasmuch as the stimulant may be slightly toxic and in no wise can be used for food. Removal of the last traces of such particles involves months. The actual production of

386 MACROPHAGES FROM ARACHNOID CELLS.

macrophages is not so great as in the above experiments, but one comes upon the same swelling up of the jirotoplasni and phagocytosis of granules by the cells found on the trabecular. The arrangement of the ingested particles tends to be close to the nucleus and consists of the .smaller granules. Weed (1917, p. 470) noted, a few hours after injection, "i)articles of carbon in the cuboidal cells of the arachnoid and similar pictures after the injection of cinnabar." Inert particulate matter which could be easily identified in the tissue has been employed by many observers in the study of the drainage of cerebro-spinal fluid, by injection into the sub- arachnoid space. Quincke (1892, p. 159) remarks:

"Ausserdem fand sich Zinnober in rundlichen oder unrcgelmassig gestalteten Zellen, die, etwas grosser als Lymphkorperchen regellos verstreut Ltn Subarachnoidalgewebe vorkommen, bald einzelen bald gruppenweise : und die wohl als Bindegewebszellen von veriinderlicher Form anszusehen sind."

Unable to convince himself that the sessile cells took up any of the granules, he concludes (p. 176) :

"In den eigentlichen Epithelien der Dura oder Arachnoidea konnte der Farbstoff nie sicher nachgewiesen warden, wenn auch oft genug zinnoberhaltige Zellen der Epithel- schicht aufsassen. Ebensowenig fand sich Zinnober in den grossen spindelformigen Zellen, welche bei jiingeren Thieren die Bindegewebsbalken des Subarachnoidalgewebes bilden, noch in jenen blassen, epithelartig angeordneten Zellen, welche die bindegewebigen Mashen- raume dieser Membran auskleiden."

The failure of Quincke was probably the result of wiiiting too long to study the material — i. e., in periods of a week or more. It appears that the reaction of the membrane is less vigorous after a certain number of cells have become free in the locality; this phenomenon is strikingly illustrated where inert particles are used. Introduction of vital stains into the subarachnoid cavity has not shed any further hght on the physiological activity of the lining membrane. Goldmann (1913) makes no mention of vital staining of the meninges. The toxicity of the stain for the nervous S3^stem may account for this, as the animals die \Qvy quickly after the injection.

The experiments furnishing the material for this paper must be regarded as too acute to shed much hght on the fate of these cells which have separated themselves from their normal environment. The use of insoluble inert matter furnished a means of determining this and such a key is found in the work of Quincke. After months these wandering cells may be found, with their cinnabar inclusions, along the carotid sheath to cavernous sinus, along intercostal nerves several milhmeters beyond the junction of the sympathetic chain, i)lexus lumbalis, upper cervical lymphatic and submaxillary lymphatic glands. This shows that the process of migration is slow and de{)endent on the amoeboid activity of the cells themselves. The ultimate disposition of such insoluble matter must be a process similar to the storage of dust inhaled into the lungs. The soluble matter (in the laked corpuscles u.sed) is promi^tly digestcnl and oidy the iron pigment remains. No evidence was obtained in support of a view that the free cells after leaving the trabecuhc would again assume their former position.

MACROPHAGES FROM ARACHNOID CELLS. 387

The excitation of abnormal activities in cells has been variously interpreted and these experiments are subject to the same limitations. The only difference between the same stimulus applied to connective tissue, endothelial walls, or surface of a serous cavity, lies in the peculiarity of anatomical position. On the trabecule of the slender arachnoid strands the cells are often given but 4 microns to rest upon. There is Uttle surprise, then, that an increase of protoplasm brings about a pendu- lous appearance. Furthermore, the comparative isolation of these enlarging cells lessens the confusion with neighboring structures as well as permitting a clear view of the cellular inclusions. Material from these experiments, when subjected to the ordinary embedding and section technique, affords very disappointing specimens.

Although the stimulus of injected particulate matter is an excessive one, it points nevertheless to the physiological activities of this cell-membrane forming the walls of the subarachnoid space. Normally, a certain small quantity of debris finds its way into the subarachnoid cavity; this offers an explanation for the finding of a few macrophages as normal inhabitants of the cerebro-spinal fluid. These appear as the large mononuclear cells withdrawn at lumbar puncture. The capacity for developing macrophages occurs in the earliest months of embryonic hfe and is never lost by the adult pia arachnoid. These findings are comparable with the reactions in other cells of the body, provided the proper stimulus is given. Differen- tiated mesothelium, such as the arachnoid, peritoneum, and pleura, undifferentiated mesotheUum forming the supporting connective tissues, vascular endothelium, and finally neuroglia, may transform into amoeboid wandering cells capable of ingesting particulate matter. As a physiological class they may be embraced by the term macrophage, and as such are concerned with the reparative processes in the body.

SUMMARY.

(1) Particulate matter within the subarachnoid cavity causes the lining mesotheUal cells to round up antl bud off from their attachments.

(2) As free-moving amoeboid elements these cells fulfill in every way the criteria of the class of macrophages, and as such are concerned with the removal of debris.

(3) Normally the same type of stimulus, though in very greatly reduced force, results in the formation of the few large mononuclear cells occurring in the cerebro- spinal fluid.

BIBLIOGRAPHY.

Alzheimer, A., 1910. Beitriige zur Kcnntnis der patho- logischen Neuroglia und ihrer Bcziehungen zu den Abbauvorgiingen im Nervongewebe. IILstoI. u. Histopath. Arbeit Uber Grossliirnrindc (Nissl- Alzheimer) Jena, vol. 3, p. 401-562.

EssicK, C. R., 1915. Transitory cavities in the corpus striatum of the human embryo. Contributions to Embryology, Carnegie Inst. Wash. Pub. 222, p. 95-108.

Evans, H. M., 1915. The macrophages of mammals. Amer. Jour. Physiology, vol. 37, p. 243.

GoLDMANN, E. E., 1912. Neue Untersuchungen iiber die a«issere u. innere Sekretion des gesunden u. kranken Organismus. Tubingen.

GoLDMANN, E. E., 1913. Vitalforbung am Zentral- ncrvcnsj'stem. Beitrag zur Physiopathologie des Plexus Chorioideus und der Hirnhaute. Berlin.

Key and Retzius, 1875. Studicn in der Anatomic des Nervensystem und des Bindgewebes. Stockholm. Erste Hiilfte.

Maximow, a., 1909. Archiv. fur inikr. ,\nat. vol. 73, p. 444-561.

Metschnikoff, E., 1892. Le(jons sur la pathologic compar6e dc I'inflammation. Paris.

Mertzbacher, L., 1909. Uber die Mori)li(ilogi(> und Bio- logic der Abraumzellen im Zentmlnci'vcn-system. Histol. u. Histopathol. Arb. lib ( iro.ss-liirnrindc (Nissl-Alzheimer), Bd. 3', Jena, p. 1-142.

Qdinckb, H., 1872. Zur Physiologic der Cerebrospinal fliissigkeit. Arch. f. Anat. u. Phj-siol. (Du Bois Reymond), p. 153-177.

ScnoTT, E., 1909. Morphologische u. experimentelle Untersuchungen iiber Bedeutung u. Herkunft der Zellen der Serosen Hohlen u. der sogenannten Makrophagen. Arch. f. mikr. Anat., Bd. 74, p. 143-216.

Weed, L. H., 1914. Studies on cerebro-spinal fluid. Jour. Med. Research, vol. 31 (N. S. 26), p,21-117.

1917. An anatomical consideration of the cerebro- spinal fluid. Anat. Rec, vol. 12, p. 461-496.

DESCRIPTION OF FIGURES.

Fig. 1. Normal flattened appearance of cells covering the arachnoidal trabeculae. Hematoxylin and eosin. X 900.

Fig. 2. Three stages in transformation of arachnoidal cells into macrophages occurring after injection of erythrocji,es: (a) Primary incrcjise in protoplasm about the nucleus. (6) Beginning formation of a mtshwork. (c) Swelling up preparatory to budding off. Hematoxylin and eosin. X 900.

Fig. 3. Initial swelling of protoplasm as shown in prep- arations stained with aqueous toluidin blue. X 900.

Fig. 4. Phagocytosis of fragmented erythrocytes and blood pigment by cells covering arachnoidal trabec- uLc. Aqueous toluidin blue. X 900.

Fig. 5. Division of arachnoidal cell on trabccula. Tolui- din blue. X 900.

Figs. 6, 7, 8. Phagocytic macrophages from subarach. noid space. Stained with aqueous toluidin blue X 900.

Fig. 9. Macrophage from subarachnoid space. Stained with hematoxyUn and eosin. X 900.

Fig. 10. Amoeboid macrophage from subdural space. Stained with hematoxylin and eosin. X 900.

Fig. 11. Arachnoid membrane showing phiigocytosis of blood-pigment (lower portion of illustration). Reproduction of new elements is shown by mitosis. In the upper left corner appears a portion of an arachnoidal cell condensation. Staini^l with hema- toxylin and eosin. X 700.

Fig. 12. Photomicrograph of arachnoid membrane show- ing clusters of macrophages after subarachnoid injection of laked blood. X 280.