Microscopic Studies in Living Mammals with Transparent Chamber Methods*

Microscopic Studies in Living Mammals with Transparent Chamber Methods* ROY G . WILLIAMS Departmen.t of Anatomy, University of Pennsylwnia. Philadelphia. Pamsylmtiia I . Introduction ................................................ I1. Construction and Installation of Chamhers .......................... 111. Blood Vessels ..................................................... 1. Growth ........................................................ 2. Caliber Changes ................................................ 3 . Anastomoses ................................................... 4. Effects of Various Agents ...................................... I V Lymphatic Vessels and White Cells ................................ 1. Growth of Vessels and Development of Function ................ 2. White Cells ................................................... V Other Tissues of the Ear .......................................... 1 . Nerves ........................................................ 2 Connective Tissue .............................................. 3 . Fat ............................................................ 4. Epidermis ..................................................... 5. Cartilage and Bone ............................................. VI. Grafts ............................................................ 1 . Autogenous and the Technique of Making ........................ 2. Homologous .................................................... 3 Tumors ........................................................ 4. Parasites and Eggs ............................................. VII . Tuberculosis ....................................................... VIII. Conclusion ........................................................ I X . References ........................................................

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I. INTRODUCTION The possibility of creating in mammals a thin region suitable for prolonged study with transmitted light and high magnification was a concept of Dr . E . R . Clark. Naturally occurring thin regions such as the tails of frog larvae have been used for histologic study since the beginning of microscopy. For many years, Dr . Clark himself used the tadpole tail for his investigations of blood vascular and lymphatic growth . H e has stated (1931) that he conceived the transparent chamber method as a means for extending his observations to mammals. Such a method depends upon the capacity of blood vessels. connective tissue. and other cells to grow into and fill actual spaces in the body if the spaces are *The work of the author is being supported by a grant from the National Institutes of Health. United States Public Health Service.

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sufficiently small. Clark (1931) further stated, “In 1875, Ziegler made studies on the new vessels and tissues which had grown into a space between two coverslips inserted under the skin of mammals, and in 1!2002 Maximow gave a beautiful description of the new tissue present in celloidin chambers which were inserted and removed at different intervals and then fixed and stained. Although both of these investigators made studies on fixed material, their results showed conclusively that new tissue, including blood vessels, will invade thin artificial spaces.” Although Dr. Clark had speculated about the feasibility of the method as long ago as 1910, actual work did not begin until 1922 when the problem was suggested to one of his students, J. C. Sandison. Sandison (1924) made a preliminary report of his studies and in 1928b published a more detailed account of a chamber together with a general survey of the growth and behavior of living cells and tissues as they appeared in it. According to Clark (1931), Sandison then decided to carry out his original intention to complete his surgical training and did nothing further about perfecting the method. However, since then, Clark and others working in his laboratory and elsewhere have perfected and adapted the technique until it may now be applied to all the common laboratory mammals and, depending on the type of chamber used, histologic and histophysiologic studies can be made in the same animal for a period of years if necessary. The transparent chamber technique might in its broadest sense include all transparent devices for exteriorizing internal organs so that they may be studied microscopically for more than a few hours, e.g., intestine (Zintel, 1936) ; pancreas (Flory and Thal, 1947) ; ovary and tube (Estable, 1948), or for replacing part of the body surface with a window so that underlying parts may be visualized, e.g., the skull chamber (Wentsler, 1936) or the transparent calvarium of Shelden et al. (1944). But, in this review consideration will be given only to those methods concerned with establishing a thin area of living tissue suitable for microscopic work at the surface of mammals and the uses to which such preparations have been put.

11. CONSTRUCTION AND INSTALLATION OF CHAMBERS The design and construction of most chambers for use in rabbits’ ears stem from one or another of the four types described by Clark, KirbySmith, et d. (1930). Of these four types, two, the “preformed tissue” and “round table,” are still in general use without fundamental changes ; one, the “bay chamber,’’ is obsolete, and the other, the “combined chamber,” is useful only for the limited purpose of comparing the appearance and behavior of newly formed vessels with original ear vessels.

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The preformed tissue chamber is used chiefly for visualization of the original ear vessels with their nerve supply intact. The principle of this chamber is the retention of a layer of skin and subcutaneous tissue on one side and the substitution of a thin mica covering for the cartilage and skin of the other. The parts of the chamber and their relationship to the tissues of the ear are shown in Fig. 1B. The thinness of the retained tissue is maintained by pressure exerted from both sides. Details of construction are given by Clark, Kirby-Smith, et al. (1930). This chamber has been adapted to the mouse by Algire (1943a, 1946b, 1947). The round table chamber is a device for studying the growth and behavior of blood and lymphatic vessels, nerves, connective tissue, and other tissues and cells. It was originally described by Clark, Kirby-Smith, et al. (1930) and has been modified in various ways by Williams (1934a), Ebert, Florey, and Pullinger (1939), Ebert, Ahern, and Bloch (1948), Essex (1918), Ahern et d . (1949), and Robertson (1951, 1952). It has been adapted to a surgically made skin flap by Williams (1934b), to the dog’s ear by Moore (1936), and to a dorsal skin flap on the mouse by Algire (1943a) and Algire and Legallais (1949). J o s h (1952) has modified Algire’s procedure in the mouse. The parts of a round table chamber and their relationships to the tissue of an ear are shown diagrammatically and not to scale in Fig. 1C. In installing this chamber, the skin but not the subcutaneous tissue is removed from both sides of the ear over an area the size of the chamber. A hole is cut through the center of the denuded area to take the table of the chamber. Holes are punched through the ear for the three bolts that hold the base and cover together against projections from the table, the projections being the feature that insures an observation space of uniform thickness, 40 to 75 p , which growing tissues can invade. When the chamber is installed in an ear, access may be had in two ways to the tissue filling the observation space : (1) through a hole in the table, closed with a plug when not in use (Clark, Kirby-Smith, et al. 1930; Ebert, Florey, and Pullinger, 1939), or (2) incorporating in the chamber devices that permit removal and replacement of the cover (Williams, 1934a: Williams and Roberts, 1950). The useful life of preformed tissue and round table chambers as originally described or in any of their modifications is something less than one year although now and then a round table chamber will stay in place longer, but that cannot be counted on. Neither of the chambers described nor any of their modifications can be used satisfactorily without external protection. There is no firm contact union of chamber and tissue, and the mechanical interlocking of the

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A Outer skin and subcutaneous tissue Cartilage

Inner skin

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C. PI

Mica

Mica

Plastic

Tantalum

--_ ___Tantalum

gauze

Paper

Lucite

Other metals

FIG.I. Diagrams showing the parts of three commonly used chambers and theii relationships to each other and to the tissues of an ear. A, section through a portion of an ear. B, cross section of a “preformed tissue” chamber. C, cross section of a “round table” chamber. D, cross section through the long axis of a tantalum chamber. Diagrams are not to scale. The bolts are made of tantalum and the nuts of brass.

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two is inadequate to prevent some movement of the device in the ear or wherever it is installed. By using plastic splints and removable shields so placed that they nowhere come into contact with the chamber, a satisfactory degree of chamber stability can be obtained (Clark and Clark, 193Zb). Unanesthetized rabbits are immobilized for study by placing them on their backs on an animal board equipped with a head stock (Clark, Sandison, and Hou, 1931). Essex (1948) immobilizes them in their natural sitting position, and Algire (1943a) places mice in a brass cartridge containing a slitlike opening through which the flap containing the chamber protrudes. To expose tissues in chambers to fluids of known composition or to withdraw fluid for analysis, two means have been devised. A glass chamber (Abell and Clark, 1932b) may be made so that it contains a well or “moat” accessible to the outside through silver tubes and internally in communication with the space into which tissues grow, or a mica cell with inlet and outlet holes can be used to replace the cover on a removabletop chamber (Williams and Roberts, 1950). Injection ducts and “lacunae” may be built into a round table chamber (Ebert, Sanders, and Florey, 1940). Of the chambers so far devised, only one consistently permits studies for longer than one year and provides free access to the contents (Williams and Roberts, 1950). It is made of tantalum and mica and, when properly installed between perichondrium and cartilage, cannot be extruded. It may be used for at least four years and probably for the animal’s remaining life. When equipped with an internal reflecting surface, it may be installed in the skin of any laboratory mammal as large or larger than a rat without the necessity for making a skin flap. Light-reflecting chambers must be studied with vertical illumination, the Leitz Ultrapak being most suitable. The principles of constructing and installing tantalum and mica chambers are entirely different from those previously described. The parts and their relationships to each other and to the tissue of an ear are illustrated diagrammatically in Fig. 1D. The body of the chamber is composed of a flat tantalum ring enclosed in a double layer of tantalum gauze which projects well beyond the ring. Four bolts carry devices that maintain the observation space and permit removal of the mica cover. I n installing this chamber, immediate stability is achieved by the killing compression of skin and cartilage under the upper tantalum ring. This holds the chamber firmly until connective tissue and vessels have time to grow through the metal gauze from side to side and effectively anchor it in place. External protection is at no time necessary.

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Because this chamber is placed in a naturally occurring cleavage plane, its installation in an ear is easily and quickly done, Through a short incision in the skin and cartilage on the inner ear surface, the perichondrium and overlying tissue are elevated away from the cartilage by means of a semisharp instrument designed for the purpose, over an area slightly larger than the chamber. Incisions at right angles to each other are then made through inside skin and cartilage and the partly assembled chamber laid between perichondrium and cartilage. Inside skin and cartilage cover the chamber. The remaining rings are installed and forced firmly against the included tissue by tightening the nuts on four bolts. Tissue over the observation space is trimmed away and the cover and retaining devices applied. The chamber then requires no further attention. Vessels appear in the observation area somewhat later than they do in round table chamber because of the greater distance they have to grow, and nerves do not grow into them, but events transpiring as vascularization develops are otherwise the same. Epidermis which occasionally ruins round table chambers cannot grow into the tantalum variety. The tissue that grows into tantalum chambers, or probably any chamber, is, after some weeks, laminated, consisting of three discrete layers, the outer ones composed of avascular connective tissue, 5 to 10 p thick, and between them the vascular layer, the total thickness being uniform in any single chamber. It is the inner avascular connective tissue layer that permits opening the chamber with no extravasation of blood or other obvious damage to the contents. Minimal thickness of tissue that can be achieved in the observation space is 18 p . Vessels will not grow into a thinner space. Optimal thickness for most purposes is from 40 to 75 p.

111. BLOODVESSELS 1. Growth The presence of a fluid-filled space in the body is not of itself an adequate stimulus to vascular growth (Clark, 1936aa). There must be present in the environment growth-promoting conditions which involve both physical and chemical properties. Some of the physical factors that stimulate or influence vascular growth are : space relationships in the region being vascularized, temperature conditions, consistency of the surrounding medium, rate of blood flow, and amount of interchange through the capillary wall. It is unlikely that there is in the body a chemical which under any circumstances is specifically responsible for vascular growth. Embryonic tissue extracts and inflammatory exudates contain substances that favor growth of lymphatics, nerves, and connective tissue as well as blood vessels and other tissues (Clark and Clark, 1936a, 1939). It is therefore reason-

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ably certain that there are chemical substances which can stimulate local growth in general and that these substances are potent components of embryonic extracts and inflammatory exudates, but just what they are is by no means clear at present. Capillaries form from endothelial sprouts arising from pre-existing capillaries. Solid, pointed endothelial projections extend out from the side of a circulating capillary or venule. In the base of these projections, a liimen develops which is continuous with that of the parent vessel. Endothelial nuclei migrate into the process by ameboid activity of their surrounding endoplasm and also increase in number by mitosis. No outside cells contribute to these endothelial extensions. The growing sprout eventually comes in contact with another sprout or capillary whereupon it forms a connection through which the lumen gradually extends. Capillaries appear in chambers after about seven days and advance at rates varying from 0.2 to 0.6 mm. per day (Clark, 1936a; Clark, Clark, and Abell, 1933). Newly formed capillaries are generally larger with walls softer and more fragile than older ones. These differences last for only a day or so, although they may return in older capillaries following chemical or mechanical stimuli not necessarily involving inflammation. In early stages of growth, the endothelial cells are arranged as a syncytium within which nuclei move around, sometimes passing one another in the wall or migrating across the lumen. There is evidence that the endothelium consists of an exoplasm and an endoplasm, the former as a continuous homogeneous layer and the latter containing the nuclei and surrounding cytoplasm (Clark and Clark, 1939). Capillaries beget capillaries in the initial vascularization of a region and are produced in great excess of those found in the more stable vasculature that develops as the stimulus to growth subsides. Within the indiscriminate capillary plexus resulting from the growth stimulus, arterioles and venules develop. Arteries conveying blood to the plexus influence the development of new arteries within it. Whether a capillary becomes an arteriole or not depends on the blood pressure and volume of flow and the times over which those factors are applied. Pressure regulates the thickness of wall, and volume of flow determines the size of lumen-these are parts of the histomechanical principles of Thoma (Clark, 1936b). The various features of capillary growth and circulatory changes previously mentioned are not limited to conditions in which a whole area is being newly vascularized. The normal vasculature of the adult mammal is, except for larger vessels, in a labile state and subject to change in form and endothelial consistency in response to minute chemical, mechanical,

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and thermal stimuli (Clark and Clark, 1932a; Clark, Hitschler, et al., 1931). Gradually, over long periods, vessels functioning as venules may become arterioles, or the reverse may occur. Both arterioles and venules may revert to capillaries, and, in response to mild inflammations, these vessels may produce a new plexus in which new arterioles and venules differentiate (Clark and Clark, 194Oa). Loss of capillaries occurs at the same time new ones are forming. In some instances the process is the reverse of sprouting, and in others it appears to result from dissohtion of endothelium. If a capillary does not have flow in it for a sufficiently long period, occasionally no longer than 24 hours, it will disappear. Loss of capillaries is greater in the neighborhood of arterioles than near venules (Clark and Clark, 1935, 1939). Capillary sprouting is, perhaps, not the only mechanism concerned in the vascularization of a region. Chalkley, Algire, and Morris (1946), in studying wound repair in mice, observed that as much as 49% of the vascular bed was restored before vascular sprouts appeared. This suggested three things to the authors, one of which was that there could have been an increase in length of formed vessels and an extension of them into the wound area without the vascular sprouting process playing a part. This was an interesting and astute explanation of a finding that could have been made in a living animal only by the use of an accurate method for determining the level of vascularity (such a method was devised by Chalkley, 1943). How the increase in length could have been produced was explained hypothetically. Although the authors did not mention it, the process may well have been similar to that observed in tissue culture by Lewis (1931), who with time-lapse motion pictures demonstrated conclusively that, in Vitro, a formed capillary plexus can become exteEded in the absence of hemodynamic factors and without endothelial sprouting. The mechanism by which this was achieved was not explainable from the motion picture film. In typical first vascularization of parts, embryologically, the first veins are at a distance from the arteries or, as in the brain, at the opposite side of the organ or alternating, as in the lung. In the vascularization of chambers, arteries and veins tend to alternate and venae comites develop only as a secondary formation. Some of the conditions leading to the formation of companion veins are thought to be: the development of a growth-promoting medium in which a secondary growth of capiIlaries takes place in the interstices of the original set; the existence next to the artery of an unimpeded growth space; the thick layer of muscle cells on the artery making it impervious to lateral growth of its own endothelium or invasion from outside endothelium; the splinting effect of the artery which lessens outside pressure on adjacent veins (Clark and Clark, 1943b).

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2. Caliber ChaPiges Adventitial (Rouget) cells on regenerating capillaries form from connective tissue cells. Sparsely distributed inert cells resembling fibroblasts assume a flattened, longitudinal position with their processes parallel with the vessel wall. In capillaries that regress, these cells remain behind. On vessels becoming venules, the number may be slightly increased. Vessels differentiating into arterioles tend to straighten, lose side branches, reduce their caliber, and increase the strength and thickness of endothelium. During these changes, the number of adventitial cells rapidly increases. At the same time their axes change from longitudinal to transverse, and they become converted to smooth muscle cells. None of the vessels having only longitudinally arranged adventitial cells show active contractility. Vessels having transversely arranged cells develop active contractility if they are reached by a regenerating vasomotor nerve (Clark and Clark, 1937a, 194Oa; Clark, Clark, and Williams, 1934; Sandison, 192&, d, 1929, 1931, 1932). Beecher (1936a) stated that capillary contractility is present and common in the rabbit and that it is produced by the Rouget cells and by swelling of endothelial nuclei, both of those elements being controlled by the sympathetic nervous system (Beecher, 1936b). Clark and Clark (1940a), who, after many years of study, could find no evidence for active contractility of capillaries in the mammal, state that “the real factors responsible for control of peripheral circulation in the mammal have been shown to be the smooth muscle cells on arteries, arterioles, arterio-venous anastomoses and a few of the larger veins mediated through the sympathetic nervous system.” Sanders et al. (1940) concluded that capillary contractility does occur in rabbits’ ear chambers and that it is under control of sympathetic nerves. They believe it is produced by endothelial swelling but could find no evidence that the Rouget cells assist in the contraction. While Clark and Clark (1933b, 194Oa, 1943a) nowhere deny that caliber changes occur in capillaries of mammals, they feel that such changes are so “slight, infrequent and passive” as to be completely outweighed in importance by the “positive, violent, spontaneous and rhythmic” contractility of vessels with muscle cells on them.

3. Anastomoses The governing factor for the formation and maintenance of arteries and arterial anastomoses at the periphery appears to be Thoma’s histomechanical principle that size of lumen and thickness of wall are determined by amount of blood flow and pressure respectively. Tn absence of flow for

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sufficiently long, an artery becomes obliterated, For maintenance of small arterial anastomoses, frequent reversal of flow is essential. This is produced by unsynchronized contractions of vessels or parts of vessels and by varying external pressures. Between vessels protected from external pressure or in which unsynchronized contractions do not occur, there are few anastomoses (Clark and Clark, 1946, 1947). Arteriovenous anastomoses occur normally in many regions of which the rabbit’s ear is one. These operate as mechanisms for partially shunting blood around the capillary bed. They increase rapidly in number in response to sudden and sustained increase in blood flow. They may be retained, or they may disappear completely when the flow returns to normal. These connections have well-developed cellular walls that contract vigorously, being able to close the lumen completely. The diameter of lumen in any single contracting arteriovenous anastomosis may vary from 0 to 60p Anastomoses may have their own nerve supply and contract independently of the arteries or in conjunction with them, or they may have no nerves and contract only on local stimulus (Clark, 1938; Clark and Clark, 1934a, b). 4. Effects of Varioiis Agents The transparent chamber method affords an excellent means for studying changes in the peripheral circulation and the effect of drugs and other agents on blood vessels. Hou (1932) studied the action of ephedrine and related substances, as did Levinson and Essex (1943a) and Vigran and Essex (1950). Wilson (1936) used Adrenalin, ephedrine, ergotoxine, histamine, nitroglycerin, and tyramine, and Solis and Essex (1951) studied the action of protarnine sulfate. Abell and Page (1941) investigated the effect of angiotonin in hypertensive rabbits and in 1942a, b reported that angiotonin and rennin elevated arterial pressure with little reduction in blood flow by constricting the arteries and augmenting the force of the heart beat. Tyramine and methylguanidine sulfate acted similarly. Angiotonin, unlike epinephrine or pitressin, acted on ear vessels in a manner suggesting that it may be capable of producing chronic hypertension. Seldon et al. (1942) studied the effect of Pentothal sodium, cyclopropane, nitrous oxide and oxygen, ethylene, and ether on minute peripheral vessels, All these anesthetics produced a sustained increase in systolic blood pressure except ethylene, with which the increase was slight. Pentothal sodium produced dilatation of both arteries and capillaries, and all others caused arteriole and capillary constriction except cyclopropane, with which the capillary bed only was dilated. They concluded that Pentothal sodium because of its dilating effect on both arteries and capillaries may be

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responsible for the increased oozing of blood observed in operations with that anesthetic. Ergonovine maleate, a drug used to prevent postpartum hemorrhage and in the relief of migraine, caused no obvious injury to blood vessels with forty times the usual dose (Abell, 1946a). Massive doses of the drug, unlike ergot, ergotoxine, and ergotamine, did not cause gangrene, although small thrombi and endothelial stickiness occurred (Abell, 1946b). Cortisone (Ebert and Barclay, 1952) tended to protect the vasculature against the effects of inflammation, improving arteriolar tone, diminishing damage to endothelium, and decreasing diapedesis and exudation. I t was concluded that if the results of inflammation are useful, as they would be in combating a bacterial invader, then the use of cortisone is harmful. But in a useless inflammation, such as rheumatoid arthritis, cortisone would be beneficial. Abell and Clark (1932a, b, 1933, 1937a, b) and Abell (1934, 1935, 1937a, b, 1939, 1940) used a glass chamber so constructed that blood vessels and related tissues could be exposed to the action of various fluids and the fluid withdrawn for analysis. When zinc-free methylene blue was placed in the well of the chamber, it was rapidly toxic to vessels if the concentration was 0.5% or greater. The dye diffused into the tissue for only about 0.2 mm. By variations in staining intensity, it was determined that a gradient of oxygen tension was present at the periphery of the vascular tissue. Abell (1934) placed buffered phosphate solutions at p H 7.4 and 6.2 in the moats of chambers and found that the solutions injured the vessels and were precipitated at the injury sites. These solutions entered the capillaries in concentrations diminishing as the distance from the source increased. Precipitate from pH 6.2 solution was gradually absorbed, but that from pH 7.4 solution was much less soluble. Urea (Abell, 1937a, b) passed by diffusion from the chamber moat into blood vessels. Amount of diffusion was proportional to the concentration. The amount of decrease of urea concentration was directly proportional to the concentration and to the capillary area but inversely proportional to the volume of solution. Phenol red in 0.4% isotonic solution diffused into tissue a visible distance of 1 mm. I t was not concentrated and not toxic. With the circulation free, the indicator pointed to a pH of 7.2 for the intercellular substance. With the circulation stopped, the p H dropped to 6.8 within 10 to 15 minutes, indicating the accumulation of acid metabolites. Following restoration of the circulation, the pH returned to 7.2 within 1 or 2 minutes (Abell and Clark, 1937b).

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Carbon dioxide perfused through a chamber in the presence of phenol red caused the pH of the intercellular substance to drop from 7.2 to 6.6, without damage to the endothelium. If the endothelium was sticky to leukocytes before perfusion, the gas eliminated the stickiness. It had no effect on the caliber of denervated vessels. When the circulation was poor, the acid change was more rapid and produced thickening and vacuolation of endothelium in arteries. If the COz perfusion was then stopped, the vacuoles disappeared rapidly, but if continued, plasma hemorrhage occurred in all vessels with stoppage of flow in some. The changes were reversible if COZ perfusion was not too prolonged (Abell and Clark, 1937a). Abell (1939, 1940, 1 9 4 6 ~ )found all capillaries permeable to protein, mature ones being much less so than those newly formed. Metaphen, a mercurial antiseptic, in concentrations of 1 :2500 produced no serious damage to blood vessels after 12 hours of contact. Four days of continuous contact destroyed the vessels but only for a distance of 1.5 mm. from the source. In contrast, 70% alcohol produced extensive hemorrhage and other damage within 15 minutes (Abell, 1941). Burns (Abell and Page, 1943) produced vasoconstriction of all arteries and larger veins. This resulted in reduced blood flow to the tissues and inadequate return of blood to the heart. It was thought likely that the burned tissue produced a substance that entered the blood plasma and produced the vasoconstriction. When rabbits were sensitized to horse serum, injection of that antigen or its introduction into the moat of an ear chamber resulted in arteriolar contraction, injury to endothelium, emigration of leukocytes, and emboli formation in capillaries and venules. Repeated introduction of serum into the moat exaggerated the changes and resulted in areas of complete endothelial destruction. However, reparative processes began immediately, eveti in the continued presence of serum (Abeil and Schenck, 1938). Ebert and Wissler (1950, 1951a, b) observed similar changes. They determined that cortisone tended to protect against the damage. Levinson and Essex (1943b) reported that shock resulting from intestinal manipulation produced vasoconstriction which was altered in denervated parts. Algire ( 1946a, c ) produced tourniquet shock in mice and compared the result with that following injection of bacteria polysaccharide. The results were similar in that both produced decrease in capillary circulation hut were dissimilar to the extent that edema was produced by the bacterial derivative but not by tourniquet shock. These experiments did not support the concept that in tourniquet shock there is increase in systemic capillary permeability. Algire, Legallais, and Park (1947) also found that a bacterial polysaccharide caused progressive decrease in rate of blood flow

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and decrease in percentage of functional capillaries in striated muscle. Occlusion of vessels reached a maximum within 3 to 4 hours and recovery occurred within 18 hours. No hemorrhage or necrosis was observed in muscle, but as circulation decreased, contraction was retarded and finally ceased. Algire and Chalkley (1945) and Chalkley et al. (1946) studied vascular reactions to traumatic wounds and the effect of dietary protein on vascuiar repair of wounds in mice. The growth sequence in wound repair was similar in most ways to that already described as chambers became vascularized. Both low and high protein diets retarded vascular growth, but the latter did so only if fed for long periods before wounding. A high protein diet for only fifteen days before injury did not delay vascularization and might have been beneficial. When thioflavine S in 4% aqueous solution is injected into the blood stream during ultraviolet irradiation, the circulating blood becomes yellow fluorescent, large emboli develop, and arteries are constricted. The emboli are secondary to dye aggregation, but the arterial constriction is a photodynamic reaction associated with radiation between 3200 and 38QO A., the spectral range strongly absorbed by the dye. The reaction is blocked by reduction of tissue oxygen tension or by filtering out the spectral band (Algire and Schlegel, 1950). Silica granules implanted in chambers are taken up by macrophages where they remain for months. The silica-laden macrophages produce no visible effect on connective tissue, lymphatics, or blood capillaries, and there is no tendency toward the formation of silicotic nodules (Gark and Haagensen, 1939, 1940).

VESSELS AND WHITECELLS IV. LYMPHATIC Grvwth of Lymphatic P’essels and Development of Function 1. All new lymphatic endothelium comes from pre-existing lymphatic endothelium. The growth process is similar to that in blood capillaries. Endothelial sprouts are sent out into the bases of which a lumen gradually extends. The lymphatic system is normally closed during growth and subsequently (Clark, 1 9 3 6 ; Clark and Clark, 1931, 1932b, 1933a, C, 1937b, c ) . Ingrowth of new lymphatics is always later and more sporadic and the number of vessels less than is the case for blood vessels. Rate of growth may equal that of blood vessels, but anastomoses are less frequent and the vessels are less labile. “The adult mammalian lymphatic system, like the blood-vascular system, retains the same growth properties present in its

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embryonic development, after the stage of primary differentiation" (Clark and Clark, 1932b). The growth of lymphatic vessels is influenced by the consistency of the medium into which they grow. When growth of blood vessels precedes that of lymphatics, the latter tend to occupy the clear space surrounding veins. Since there is little pressure in lymphatics, they are easily compressed and portions of them may be cut off and isolated. Isolated lymphatics persist for months and retain their growth properties. They may eventually reunite with others or gradually disintegrate (Clark and Clark, 1933a, c, 1937b, c). Because of the close proximity of peripheral lymphatics to blood vessels and the fragility of the endothelial walls, it is common to have extravasation of blood into lymphatics. Holes in the walls of vessels heal quickly. Blood cells in lymphatic vessels may pass along and disappear within 24 hours, but if trapped in the system they can exist unchanged for weeks. Fluid passes into newly formed lymphatics but only slightly, and the flow of lymph is extremely slow or absent for long periods. The lymphatics play no significant part in the removal of extravasated erythrocytes or of other extraendothelial debris. During edema, fluid in lymphatics increases, but flow may be diminished. Regions without lymphatics show no disturbed physiology. In the absence of external pressures, lymph flow is extremely sluggish (Clark and Clark, 1937b, c). Henry (1933) found that the total area of lymphatics of the ear about equals that of the blood vessels. Lymph flow was variable but extremely small even with massage. Maximum flow was 0.0845 cu. mm. per square millimeter of lymphatic surface per hour. Following injuries, lymphatic vessels may develop holes in the endothelium that may persist for as long as thirteen days, if there is fluid outside, and if pressure and suction alternate (Clark and Clark, 1933a, c).

2. White Cells Monocytes migrate from the circulating blood into surrounding tissue and become macrophages or tissue histiocytes. They increase in size and are phagocytic. Their ability to change their position is limited; hence they tend to stay in the neighborhood where they first appear. Many of them become oriented along blood vessels with the long axes parallel to the vessels (Ebert and Florey, 1939). Clark and Clark (1948) made similar observations and also saw macrophages divide, although the circulating blood seemed to be the main source of macrophages for combating infection. A single mocrophage may ingest as many as twenty

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erythrocytes. After an active period of phagocytosis, the cells diminish in size and remain as tissue components for many months. In certain locations, macrophages may fuse and form multinucleate giant cells (Sandison, 193i ) Macrophages shed portions of their peripheral cytoplasm 4.5 hours after taking up antigenic azoprotein ( Evans blue linked to horse serum), and the process may continue for many days (Robertson, 1952). Lymphoid cells that ingest the antigen are not seen after the first day. The shed cytoplasm is ingested by neighboring macrophages, and it is possible that this may be a process by which the antigen is shared by macrophages in a region. The dye-protein complex is not taken up by endothelial cells. Small lymphocytes are very actively motile cells. Ebert, Sanders, and Florey (1940) obtained no evidence that these cells may be converted to cells of some other type. Clark and Clark (1936b) and Clark, Clark, and Rex ( 1936), under circumstances that were particularly favorable for studying the fate of polymorphonuclear leukocytes, determined that such cells may change to small clear cells with round nuclei that might easily be mistaken for lymphocytes. However, they stated that the cells were probably undergoing degeneration and not transformation to a different type. They suggested that the small round cell infiltration frequently seen in pathologic conditions may be made up of these altered polymorphonuclear leukocytes. The injection of various substances into the blood stream, e.g., hydatid cyst fluid, acacia, glycogen, and dextran (Essex and Grana, 1949), causes leukocytes to cohere and form large clumps that adhere to the enduthelium. This condition is temporary, rarely exceeding 90 minutes in duration. Since leukopenia frequently precedes leukocytosis after the injection of such substances as glycogen, this observation on the behavior of leukocytes in the peripheral circulation may provide a clue to the reason for the letikopenia. I

OF T HE EAR V. OTHERTISSUES 1. Nerves

There is a marked variation in the extent of new formation of nerves in chanibers, depending on mechanical conditions, position of cut nerves with respect to the observation space, and whether or not growth-promoting conditions exist (Clark and Clark, 1938). I t appears that nerves regenerate where the growing ends are in a favorable growth-promoting environment and not because there is a need for them. The growing ends of regenerating ear nerves cannot he seen in chambers because of tissue density. However, the growth cones of fibers growing from autografts of sympathetic ganglia

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can be seen and the process of fiber formation observed. Once a medullated fiber has formed, it can be seen as a pair of highly refractile lines. Medullation begins at a Schwann cell and progresses in both directions. Isolated stretches of medullation may start at several Schwann cells simultaneously. Nodes of Ranvier are usually absent at first but may develop later. Myelin degeneration occurs rapidly over the entire length of nerve beyond a point of injurv. The Schwann cells may persist after injury and remedullation occur, at times centripetally (Clark and Clark, 1938). When nonmyelinated nerves are present but not visible in a chamber, they may be visualized by vital staining with methylene blue (Clark, Clark and Williams, 1934). By staining at various time intervals in the same animal, it was demonstrated that development of contractility in an artery progressed along the vessel at the same rate that accompanying nerves grew in length, whereas other vessels without accompanying nerves did not develop active controlled Contractility. As a preliminary to nerve repair after injury, Essex and deliezende ( 1943) emphasized the importance of vascularization. Repair does not rest solely with the nerve cell body and proximal part of the axone and distal sheath but also in the vascularity of the regenerating area. Thus, in manipulating nerves, special effort must be expended in maintaining their blood supply. 2. Connective Tissue Fibroblasts invade the observation space in round table chambers about six days after operation. Their time of appearance in the field parallels that of the blood vessels. Fibroblasts are essential for the development of connective tissue fibers (Stearns, 194Oa, b), and the cells are intimately associated with the actual formation of fibers. Orientation of fibroblasts appears to influence the orientation of fibers. There is no evidence in chambers indicating that connective tissue fibers are continuations of preexisting fibers or formed by transformation of a fibrin net or from any type of cell other than the fibroblast. Connective tissue fibers develop extracellularly as a result of fibroblast activity. The rate, amount, and direction of fiber formation is influenced by tensions. The presence of epidermis alters the tempo and pattern of fiber formation (Stearns, 1939). Intercellular substance is of a gelatinous rather than a free-fluid nature (Clark and Clark, 1933c) and, when blood circulation is free, has a pH of 7.2 (Abell and Clark, 1939).

3. Fa#

Fat is normally present in rabbits’ ears and may form in newly grown tissue. Its presence is not related to thickness of tissue or season of the

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year or to the age, sex, or state oi nutrition oi the animal. It frequently, but not invariably, appears first in cells close to blood vessels. There appears to be no relationship between fat and the presence of lymphatic vessels. Fat cells develop from cells indistinguishable from connective tissue cells. Minute refractile droplets develop in the cytoplasm, cell processes retract, the droplets increase in size, and the cell becomes rounded and larger. Loss of fat is the reverse-globules become smaller and break up into smaller and smaller droplets that finally disappear, leaving a granulated cytoplasm. Fat appears to enter cells in a soluble form and not by phagocytosis of visible fat droplets (Clark and Clark, 1940b).

4. Epidermis Epidermis sometimes grows into round table chambers from the surrounding normal skin. Clark and Clark (1944) have summarized the main points of its behavior and properties as follows : Migrating epidermal cells resemble fibroblasts in shape. The extension of a line of these cells is slightly more rapid than that of a growing line of fibroblasts and capillaries. During growth, small islands of epidermal cells may become completely surrounded by vessels. The cells become vacuolated and die, blood vessels then invade the islands, and the remains are disposed ot by macrophages. Fibrin dissolves in the neighborhood of growing epidermis, suggesting that the cells produce a fibrinolytic enzyme. Blood vessels do not invade living epidermis, and those near it are always wide, sinusoidal channels. Connective tissue that forms next to epidermis is coarse and arrayed in regular parallel rows in contrast with the finer irregularly arranged connective tissue fibers present elsewhere.

5. Cartilage and Bone Both cartilage and bone arise in chambers from time to time and for no certain reason. Cartilage arises from elongated motile cells resembling fibroblasts and containing a characteristically granulated cytoplasm. The cells finally lose their motility, enlarge, and become rounded. They lie in lacunae and contain commonly, in a mature cell, only one large fat droplet which more or less indents the nucleus. The cytoplasmic granules are uniform in size and in constant motion as is the cell itself, although it does not change position. The cell outline varies from minute to minute as short, blunt processes are sent out and withdrawn. Once the cells are fixed in position, a homogeneous ground substance appears between them (Clark and Clark, 1942). When the ground substance is well developed, the tissue then undergoes no change for many months unless bone develops in it. No mitoses were seen in chondroidal cells.

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Cartilage formation occurs in regions in which cessation of growth and retraction of capillaries is a conspicuous feature, although laying down oi the cartilagenous ground substance appears to precede the vascular change. Clark and Clark (1942) state, “it seems evident that some special localized chemical condition of the tissues must serve as stimulus for the metaplasia of connective tissue cells, or the differentiation of specific precartilage cells, into cartilage in the chamber areas, in view of the sporadic and restricted formation of cartilage.” Blood vessels appear to have only a secondary importance in the formation of new cartilage and bone. Within the chondral areas, dark, amorphous masses occasionally develop. These are composed of many minute granules thickly distributed in the hyaline intercellular substance. High magnification demonstrates the presence of typical bone lacunae and canaliculi in the granular areas. Cartilage itself may never be invaded by vessels, but if bone is laid down in it, capillaries then grow and invade the bone. However, vascularization of newly formed bone is not a very vigorous process. Bone formation and resorption occur simultaneously, but the process of resorption is obscure (Sandison, 1928a ; Kirby-Smith, 1933).

VI. GRAFTS 1. Autogemus and the Technique of Making There are three ways in which chamber methods have been used for the study of grafted tissue. (1) The grafts may be included in the chamber when it is installed. This is the method that was first used. It is now obsolete since better methods are available. ( 2 ) Tissue in the observation space of round table chambers may be approached from below through a small hole in the table, kept closed by a plug when not in use. This was first described by Clark, Kirby-Smith, et al. (1930) and improved by Ebert, Florey, and Pullinger (1939) and by Robertson (1951, 1952). ( 3 ) The chamber may be so built that the mica cover can be removed and replaced under fluid (Williams, 1934a; Williams and Roberts, 1950). This method provides easy access from above, requires no special apparatus, does not injure the exposed tissue, and is the method that has been most extensively used in the study of transplants. Methods 1 and 3 have been adapted to mice by Algire and Legallais (1949) and their adaptation has been used with changes by Conway, Joslin, and Stark (1951a) and by Joslin (1952). The method as used in mice permits studies of about two months’ duration. With tantalum chambers in rabbits, observations may be made in the same chamber for a period of years. In the following consideration of grafts, any statements or other data

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for which no literature references are given will be unpublished data from the reviewer's records. The technique of grafting is of primary importance in the study of transplants, for therein lies the greatest opportunity for introducing artifacts in the results. The literature is replete with contradictory statements concerning the survival, vascularization, and behavior of autografts. Any graft in any region not connected to the host by sewed anastomoses of fairly large blood vessels must be sufficiently small so that growing vessels may reach the center before cells die from lack of oxygen or, if blood vessels are slow in growing, so that the cells may be supplied by diffusion from host vessels. This requirement limits the size of grafts to fractions of a millimeter if they are not to have necrotic centers. For chamber work, there is an additional reason for small grafts, namely, the semirigid limited space in which they must lie. In rabbits, using the third method described above, optimal diameter of grafts after compression with the cover has been found to be from 50 to 300 p on host regions from 50 to 75 p thick. Thickness of grafts after compression with the cover is from 20 to 30 p. Grafts with those dimensions do not appreciably increase the thickness of tissue in chambers, since they rapidly become incorporated in it instead of existing as excrescences. Maximum diameter of grafts, except for thin tissue such as choroid plexus or omentum, is about 700 p. Compression by the cover of such small grafts does not occlude the underlying vessels because when the donor piece is removed from its circulating blood supply the cells undergo immediate softening. Microdissection of cells in vivo with the blood circulating and the nerves intact show the cells of thyroid, for example, to be much more viscous and turgid than they are when blood flow is stopped or than they are in excised cells surviving in Tyrode solution (Williams, 1944). For reproducible results, it is necessary to follow a standardized procedure in making giafts in chambers. A satisfactory sequence has been found to be as follows: (1) Exposure of the donor part, which is then covered to prevent drying. (2) Sterilization of the chamber cover and its removal under Tyrode solution. ( 3 ) Excision of 1 cu.mm. of tissue from the donor part and immediate immersion of the piece in Tyrode solution at 4"C. (4) Shaping of the grafts under a dissecting microscope, using instruments sharpened and pointed as for operating on embryos. ( 5 ) Pressure injury of the site at which the graft is to lie, using the point of a forceps. (6) Floating the graft on the point of a forceps and placing it, under a second microscope, on the injured site. (7) Replacement of the cover and retaining devices. (8) Closure of the wound through

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which the donor piece was obtained. Three or four grafts may be placed and covered by this means in less than 20 minutes from the time of excision of the donor piece. Injury of the host region at the graft site is not an important factor in vascularization of autotransplants. Stimulus to vascular growth comes from the graft itself and affects both the host vessels and endothelium carried over in the graft. The stimulus does not extend beyond the lateral graft margins and is uniform throughout the area of contact between host and graft. Table I shows a list of tissues that have been studied in grafts and TABLE 1 TISSUES STUDIED AS AUTOCRAFTS AND AS HOMCGRAITS Those marked S survived, those labeled N S did not survive, and those followed by P S survived but were altered in some way. Autografts Thyroid Adrenal Glomerulosa Fasciculata Medulla Ovary Follicles Interstitial cells Lymph node Bone Sympathetic ganglion Brain Brown fat Parathyroid Fat, abdominal Spleen Red bone marrow Epidermis Pancreas Omentum

-

~

N S

S

N S P S N S

N S N S

S S

N S N S

S

PS

N

S1

S S S

S N S

PS N S1 S'

Leydig cells Tubules Pineal Choroid plexus Ciliary body V, carcinoma ~~

S

PS

Testis

~~

Homografts

N Sa N S N S N S N S

S

PS

N S1 S2 S1 S

~

~

and 2 studied in one or two animals respectively. All other tissues studied in not less than 3. Total number of animals used was 110. 1

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whether or not each survived. Survival was arbitrarily defined as occurring in any tissue which, at the end of four months, gave no signs of regression such as diminution in external dimensions, abrupt decrease in number of countable cells, decrease in blood vessels, and no leukocyte infiltration. The period of four months was selected because experience indicated that if a tissue was not going to survive it would disappear well within that time. Minimum time in which any surviving graft was studied was eight months and the maximum fifteen months. When autografts are made in chambers in the standard manner described above, there is great constancy in behavior and vascular response of each tissue. Tissues survive every time, or they do not survive at all, or they survive but are modified in some way, always the same way. Certain generalizations about surviving autografts can be made. Endotheliuni of the host will begin to invade them, and their own endothelium begin to grow within 24 hours. Endothelium of the graft survives and is an important factor in revascularization. Hemodynamic factors are not important in the first growth of vessels but are in determining the final pattern of vascularity. By 48 hours, a fully formed and blood-filled capillary plexus will be present within the graft, but it will contain no flowing blood. By three days, circulation will be free in all parts of the plexus. By four days, the circulation will reach its final form for each tissue with arterioles and venules well developed. A stable vascular condition will be reached by the eighth day. Further generalizations about surviving autografts can be made. Because of space limitations, the data on which they are based may not here be stated. The graft determines the nature of the vasculature in it, not the host vessels on which it is placed. The final vasculature resembles that of the whole part from which the graft was derived. The vascularization of surviving autografts tends to be a repetition of the same process as that by which the whole part was vascularized embryologically. Growth potentialities of endothelium characteristic of the embryo are retained in the adult and are similar in a wide range of species. The internal structure and organization of a surviving autograft tends to duplicate that of the whole part from which it was taken; e.g., grafts taken at random from red pulp of spleen reconstitute themselves to complete splenic lobules with central Malpighian bodies, penicillus arteries, ampullae of Thoma, sinusoids, etc.; thyroid grafts become oriented with small follicles a t the center and larger ones at the periphery; and adrenal glomerulosa grafts, under appropriate stimulation, reproduce the typical zonation of the gland. Agents that affect the donor tissue also affect the graft and in the same way. In other words, in tissues where nerves are

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ROY G. WILLIAMS

not functionally essential, if a graft survives without alteration of structure, it functions. Halstead‘s “law” which states that grafts of glands survive in proportion to the need for them does not apply for surviving autografts of glands given in Table I, since no deficiencies were created by the grafting operations. If a graft survives and is structurally unaltered with chamber methods, then it is available for histologic and histophysiologic experimentation. But if it does not survive, or if it is altered in some way, then it is up to the investigator to determine the extent to which the results were produced by technical difficulties or limitations of method. Among those autografts listed in Table I, the method was responsible for failure of survival of ovarian follicles and probably red bone marrow. I t was also responsible for the altered behavior of epidermis. But it was not responsible for results with adrenal medulla, brain, and pancreas, for those tissues did not survive, irrespective of the graft site. The changes in seniiniferous tubules resulted from excision of tissue for grafting and not from other limitations of the method. Routine magnifications used in the study of grafts are from 14 X to 600 x. For some cells in favorable locations, magnifications of lo00 x may be used, e.g., in zona glomerulosa of the adrenal cortex or testicular interstitial cells. But in other cells, e.g., parathyroid gland, where contrast between cells is poor in the living state, high magnification is generally unsatisfactory. In many cases, the same cells can be located day after day and the changes they undergo recorded by camera lucida tracings, photomicrographs, or cinephotomicrography. The tissues listed in Table I were not all studied in the same detail. Those not mentioned hereafter have been examined chiefly from the standpoint of survival only. Prolonged study of living grafts and microdissections in vivo have provided the information about thyroid that follows. Lateral limits of individual cells in follicles cannot be seen and nuclei are not often visible. The inner and outer cell boundaries are at most times very sharp in optical section, the inner more so than the outer. The colloid is clear, amorphous, and homogeneous and never presents the peripheral serrations so conspicuous in some fixed sections. I t has the same osmotic pressure as blood serum. Colloid pressure is about the same in all follicles irrespective of size and is less than capillary pressure. The viscosity of colloid varies in different follicles. Evans blue injected into small follicles disappears within 10 minutes, whereas in larger peripheral follicles it does not disappear in 12 hours, suggesting that the colloid in different follicles varies in other ways than in viscosity, probably in protein content. Methylene blue

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injected into colloid with no extravasation disappears within 20 minutes from all follicles, and extrafollicular cells become blue, indicating that diffusions from colloid can take place through the cell wall. The greatest visible activity of which a follicle is capable is a cyclic change that has been divided into four stages, the last and shortest stage being one of rapid collapse associated with great hut not complete loss of colloid (Williams, 1937). Just before collapse, some cells in the wall manifest pinocytosis. The coniplete four-stage cycle has been seen within 19 hours, but it may require many days, or follicles may never complete a cycle, activity being limited to oscillation between stages 2 and 3. Nothing leaves the follicles in a visible form. Normally, there are no holes or defects in the follicle wall. No evidence has been obtained from living follicles indicating that basal secretion occurs. When the gland is stimulated, the cells secrete toward the lumen in the apocrine manner. In unstimulated glands, secretion is very slow and is associated with the formation of droplets in the apical ends of cells and their extrusion into the colloid. When colloid volume changes occur, they are generally, but not always, accompanied by reverse volume changes in the cells. Colloid volume may increase without cordesponding decrease in cell volume. The evidence from living grafts is that the follicle is the functional unit of the gland, not individual cells. The cells secrete only into the lumen, where various changes in the secreted material can occur. The colloid cannot be looked upon as exclusively a stored and reserve secretion. Substances can diffuse out of the cqlloid without changes in colloid volume and with no visible changes in the cell wall (Williams, 1944). When colloid is reduced in volume, the reduction is probahly accomplished by diffusion through the cell wall. By the same token, it is conceivable that substances .could diffuse into the colloid without active participation of the cells. Iodine operates to reduce cell volume, increase colloid, and decrease its viscosity, but the response to iodine is not uniform in all follicles. Thyrotropic hormone increases cell volume and decreases colloid, but in some follicles it may cause increase in colloid without cell changes. in 50microcurie doses in rabbits produces no change in structure of grafts, It is concentrated in the colloid, and autographs of living grafts can be made with 12-day exposures. The autographs so made demonstrate nothing except that iodine is concentrated by grafts of thyroid. The film must of necessity be too far removed from the radioactive source to permit the useful kind csf autographs that can be made from sections. Fifty microcuries of I I 3 l , given 24 hours before grafting, completely

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ROY G. WILLIAMS

eliminates the initial stimulus to vascular growth that is otherwise so prominent a feature with thyroid grafts. All follicular structure is quickly lost. However, many thyroid cells remain alive and new follicles form after about five weeks. Thereafter, their appearance and behavior is the same as in any untreated thyroid autografts. Thiouracil in doses of 100 mg. daily causes loss of colloid, increase in cell volume, and increase in vascularity. With prolonged use, small follicles are completely eliminated, the drug apparently acting chronically as a specific poison. The number of follicles per unit of volume is thereby reduced by as much as 35%. With thiouracil, large follicles at the periphery of grafts may lose most of their colloid and regain it within two weeks, and then repeat the loss and gain in the following two weeks, in each filled stage retaining their large size. These large follicles are generally thought to be less active than the smaller ones, but they are certainly not inactive. New follicles form in grafts, but the process is not active in untreated animals. No instance of new follicle formation by budding from prcexisting follicles have been seen in many hundreds of follicles. New follicles form from interfollicular cells, some of which are cells remaining after dissolution of follicles. Anything interfering with the integrity of the connective tissue capsule of a follicle results in its dissolution, but some of the cells remain alive and capable of forming new follicles if they gain the proper relationship to each other and to the surrounding connective tissue (Williams, 1937, 1939a, b, 1941, 1944). Autografts of adrenal cortex were first made in chambers by Hou (1929). These did not survive long, and no studies of the cells were made. With improved methods, grafts of zona glomerulosa and capsule survive indefinitely in the animal’s lifetime. The cells are clearly visible with high magnification. Their cytoplasm is filled with granules that appear and disappear according to a sequence that has been determined (Williams, 1945). Survival of zona glomerulosa is not regulated by body need for adrenal secretion. With the main glands in place, zona fasciculata generally does not form from zona glomerulosa grafts, although it may if grafts are old ones. When the main glands are removed, new zona fasciculata forms rapidly from glomerulosa. If two glomerulosa grafts are so located that one is separated from the only available artery by another graft, the former will be supplied with blood by a portal circulation. This has nothing to do with adrenal as such but with the location of grafts with respect to arteries. The first vessels in any graft are always capillaries. Within the capillary plexus, Some vessels ordinarily develop into arteries and others into venules, but arteries

STUDIES WITH TRANSPARENT CHAMBER METHODS

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will not develop unless at some point or points an already formed artery sends blood into the plexus. If only veins carry blood to a plexus, no arteries develop and the circulation is portal. This finding would indicate, among other things, that it is possible to alter experimentally the vascular gradient in a tissue surviving as an autograft. The vascular gradient normally in adrenal cortex has been adduced to explain the differences in cells in the various zones. I n the case of the zona glomerulosa grafts mentioned, one graft occupied a position in a vascular gradient comparable to the zona fasciculata in whole glands. This altered position had no effect on the cells as far as could be determined. Zona fasciculata transplanted alone behaves quite differently from zona glomerulosa. The transplants have no power to stimulate growth of endothelium, neither that grafted nor that of the host region. The cells gradually become aligned along the host vessels, chiefly along veins, and slowly disappear over many months by a process which in some exes may be holocrine secretion. The cytoplasm of fasciculata cells is filled with minute granules that obscure the nucleus. They coalesce and become larger and highly refractile. These droplets then change in consistency, take a position in contact with the cell membrane, and rather rapidly decrease in size until they disappear, presumably by diffusion through the cell wall. The results from studies of grafts of adrenal cortex support the concept that cells arise at the periphery and are moved through the gIand toward the medulla where their existence ends. In grafts, the zona fasciculata has no regenerative capacity. The cells are largely post mitotics (Williams,

1945, 1947).

Pieces of seminiferous tubule and islands of interstitial cells survive readily as autografts in chambers. Testicular interstitial cells arise from cells indistinguishable from connective tissue. They acquire cytoplasmic granules, reach a certain size, at which time they have a characteristic appearance, and then undergo regression. The process of granulation and d e g r ad a t i o n is not cyclic. They are fixed post mitotics with a life span of about nine months. This is the first instance in which information concerning the length of life of a highly differentiated cell has been obtained in a living animal, and it illustrates an important potentiality of the method, since such data can be obtained, at present, by no other means. Pieces of seminiferous tubule become closed vesicles. The spermatogenic cells rapidly disappear or never progress beyond the stage of secondary spermatocytes. They tend to separate from the wall and lie free in the lumen. Sertoli cells persist. They do not constitute a syncytium. They produce droplets that are discharged into the lumen and slowly disappear.

384

ROY G . WILLIAMS

Luteinizing hormone stimulates excessive droplet production by Sertoli cells, and the process is repeatable at fairly frequent intervals. Droplets appearing in the lumen arise as the result of apocrine secretion by Sertoli cells. The inferences from the foregoing are that the pituitary controls discharge of sperm cells into the tubules and that secretion pressure of Sertoli cells provides the means for moving material along the tubule (Williams, 195Oa). Leydig cells do not arise at random from the connective tissue of grafts. They arise only near tubules or other interstitial cells. A piece of tubule without interstitial cells near it becomes fibrotic with great increase in the collagenous fibers of the tunica propria and shrinkage of Sertoli cells. Interstitial cells are largest when located near tubules. These and other findings suggest that there is a reciprocal relationship between interstitial cells and tubules. With interstitial cells missing, the tubules resemble those seen in the aged. Whatever relationships there may be between the two types of cells are of a local nature and not produced through the blood stream, since the animals studied were young and had no testicular deficiency. The inferences from these findings are several, one of which is that testicular age changes may not be primary results of age as such but secondary to closure of tubule outlet or failure of interstitial cells or both ( Willianis, 195%). The vasculature in and around islands of interstitial cells resembles one form of simple hemangioma. Similar vascular arrangements were not found in whole testis by injection methods, Interstitial cell grafts produced a spreading factor and did so without themselves degenerating. These findings give a lead toward the experimental production of some hemangiomas, a condition about which knowledge has not progressed much since the time of Ribbert (Williams, 1949). The spleen in autografts duplicates the structure of the organ as described by Mollier (1910). With the spleen removed, grafts may increase threefold in size, but with the spleen in place, they do not increase. Malpighian body transplanted alone does not survive. Red pulp taken at random reconstitutes itself to a splenic lobule complete, except for nerves, smooth muscle, and sheathed arteries, with a new Malpighian body, penicillus arteries, ampullae of Thoma, and an intermediate sinusoidal zone demonstrating the structural characteristics as described by Mollier ( 1910). Various experiments concerning functions of spleen grafts substantiate many of the prevailing ideas obtained by other methods. Intravascular phagocytosis occurs and also intravascular fragmentation of protoplasmic masses which may be platelet formation. Information has been obtained

STUDIES W I T H TRANSPARENT CHAMBER METHODS

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about spleen as a source of cellular additions to the blood stream (Williams, 195Ob). Autografts of omentum, in which the original omental vessels were clearly visible before and after transplanting, demonstrated conclusively that, in that tissue, the original vessels survive and constitute a major factor in revascularization. The original vessels are subject to realignment in response to hemodynamics in the same way that vessels respond in intact parts. Conway ef al. (1951a, b, c, 1952a, b) studied autografts and homografts of skin in mice. They found that, shortly after placing of grafts, a plasmatic circulation enabled them to survive during the first week. Blood vessels then developed by capillary budding, and the vascularization was complete by about fifteen days. While capillary budding (sprouting is a more descriptive term) as a process of vascularization of grafts undoubtedly occurs, nevertheless some of their data are open to question. I n none of the fields as shown in the photographs is it possible to see capillary sprouts, because the magnifications are too low. Capillary sprouting cannot be seen without using the compound microscope at magnifications of about 200 >(: or more. The process of vascularization as given in their diagrams explaining the photographs is not the full story as it purports to be but only the end stage ; the stage of venule development and all vessels shown have the arrangement characteristic of venules. The parallelism of external vessels related to grafts as shown in the diagrams is a common occurrence and has no special or important significance. In one diagram, it is said that a large vessel is growing into the graft. I t can be stated categorically that large vessels do not grow into grafts but form from pre-existing capillaries. In incised wounds, there is some suggestive evidence that adjacent formed vessels are concerned with the repair, in addition to vascular sprouting, but that possibility does not apply in the vascularization of grafts. The times of vascularization as given in the articles quoted have no significance because the authors could determine nothing about the first stages of the process, since the magnifications used were inadequate to see the extremely delicate sprouts with which vascularization begins. While capillary sprouts do form from venules, it is doubtful if those shown in the diagrams were capillary buds, and certainly no sprouts show in the photographs. It can be stated with assurance that in normal, uninfected tissue, endotlielial sprouting occurs only within the graft and from the area on which it is placed and that the stimulus to such formation does not extend beyond its lateral margins. Vessels do not grow toward a normal graft, from outside its lateral limits, but they do receive blood drainage from it. Conway, J o s h , ef al. (1952b) could find no evidence that corticotropin regulated

386

ROY G. WILLIAMS

the survival time of skin homografts and with this finding there is no disagreement. 2. Honaologozts Grafts

.

The results with homografts hereinafter discussed summarizes the studies made with chamber methods, and no attempt will be made to quote or reconcile the contradictory statements with which the extensive general literature abounds. As can be seen from Table I, survival of homografts is not the rule. Except for the V p carcinoma, the only homografts that survived were from choroid plexus and ciliary body. It is generally agreed that homografts survive longest in the anterior chamber of the eye or in the brain. Therefore, it was thought that the cells probably producing the fluid in those regions should themselves survive as homografts. Choroid plexus grafts develop a series of temporary fluid-filled vesicles of various sizes. The fluid slowly disappears and reappears in other places. Ciliary process survives as a mass of deeply pigmented cells. No vesicles formed in these grafts. Tissue taken from iris alone does not survive. Corticotropin, cortisone, and deoxycorticosterone have no effect in prolonging survival of thyroid, adrenal fasciculata, or spleen, and thiouracil fed to the host for many weeks before homografting of spleen has no effect in prolonging survival of that tissue. A striking characteristic of most autografts is their ability to stimulate the growth of endothelium, both their own and that of the vessels with which they are in contact. Homografts generally have no such ability, or if they do, the vessels cannot be maintained. I n the same tissue in different animals, transplanted endothelium may grow well for a time in homografts, or it may not grow at all, or its growth may be limited. The same variety of response applies to the host endothelium. In spleen, for example, the transplanted endothelium may grow vigorously for the first 24 to 48 hours, but no connections are made with host vessels and the blood never circulates in the newly formed plexus. The blood becomes laked and the grafts rapidly disappear. In thyroid, a fully circulating plexus may develop and the follicles appear perfectly normal. This may continue for several weeks or even months, and then the blood flow stops, the blood becomes laked, and the grafts rapidly disappear. In other thyroid grafts, no vessels form and the grafts disappear within a few days despite the fact that they were sufficiently small so that they could have been supplied by diffusions from the host vessels with which they were in contact. Unlike autografts, one cannot predict what the vascular response in a homograft will be or for how many days the graft will survive. The only thing about

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which one can be certain is that for the tissues studied (Table I), with the exceptions noted, survival time will not compare favorably with autografts. On first thought it might be supposed that the vascular differences between autografts and homografts are important factors in the failure of homograft survival. However, they may be effects of something else rather than causes. Autografts of adrenal fasciculata have no ability to stimulate growth of endothelium, but many of them survive for months. II3' temporarily suppresses the growth of endothelium in autografts of thyroid, which then resemble homografts of the gland. But some cells survive and after a time reconstitute follicular structure and become vascularized so that one cannot tell that vascularization had been delayed. It would seem likely, therefore, that whether or not a graft can stimulate formation of a new vascular plexus is not the deciding factor in its survival. The failure of homo- and heterografts to survive consistently or at all has been ascribed to anaphylaxis, genetic factors, and other things. The body undoubtedly has defenses against foreign invasions of any sort. A piece of glass, for example, becomes etched by the corrosive action of tissue fluids. But there is an additional factor that may play a part and which has not heretofore been emphasized. Each surviving graft of whatever sort creates its own internal environment. Failure to survive may therefore be related in the first instance to inability of transplanted cells to function adequately or at all in a foreign region or another animal, thus preventing maintenance or renewal of their protective intercellular environment. It is doubtful if host lymphocytes or other white cells are important in the survival of homografts, at least insofar as their presence locally is concerned. As homografts disappear, the region is sometimes infiltrated with white cells, but not in excessive numbers, such as would be produced by an infection. In many instances, no more white cells are present than would be there normally. There may or may not be sticking of leukocytes to the walls of host vessels in the neighborhood, a delicate sign of vascular injury that might be expected near dying cells. These variations in white cell response indicate that the circumstances surrounding disappearance of a graft are not always the same. Many homografts appear to dissolve and fade away rather than die and have the remains removed by phagocytes. The differences noted apply to the same tissue in successive hosts as well as to different tissues. 3. Tuntors Tumor grafts are generally homografts. Tumor might survive as autografts, but none of .the accounts encountered in chamber literature were concerned with them. A striking difference between a tumor homograft

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and a homograft of normal cells in which the tumor was primary is that the former commonly survives but the latter does not. The tumor graft behaves in that respect like an autograft. The tissue differentials (an expression used by Loeb, 1945), or whatever it is that ordinarily prevents survival of most homografts in most regions, seems to be missing or modified in cancer cells, although even tumors tend to be species specific. However, by serial transplantation in the brains of rats and other animals (Greene, 1951), the species factor can be overcome and heterografts be made to survive. Vascular reaction to transplanted tumor has attracted investigators and was the first phase of the cancer problem to be studied with chamber methods. Ide et al. (193%) studied the Brown-Pearce rabbit epithelioma. They used the first method of transplanting as described above, wherein the transplant was made when the chamber was installed and before it was vascularized. Vessels appeared from three to eight days later. Tumor growth, as seen grossly, coincided with the onset of vascular growth and progressed steadily until the chamber was completely filled. The tumors then suffered complete or partial resorption. The authors stated that, since the rapidly growing tumor was able to initiate in an unprepared site an adequate blood supply which was characteristic and not observed in the controls or in injury repair sites, it was probable that the tumor elaborated a vessel-growth-stimulating substance. While it cannot be denied that the tumor may have produced such a substance, that possibility does not automatically follow from the fact that its vascularization was different from the controls or from that in injured sites, for if it did, one would have to suppose that there was something common about the vascularization of any tissue and with the process of injury repair. But, aside from the general mechanism of endothelial sprouting, that is not necessarily the case. The final form of the vasculature is a function of each tissue and varies from tissue to tissue, depending upon the internal environment and other factors that each provides. Any surviving graft acquires a vasculature characteristic for that tissue, and the fact that it may be different from that produced by another type of tissue does not prove the presence or absence of a vessel-growth-stimulating substance, for there are many other factors involved. It should be pointed out that the authors only expressed a probability and did not claim to have proved the matter. The vascularization of a malignant tumor was also studied by Algire (1943b), who used a melanoma implanted in transparent chambers installed in dorsal skin flaps on mice. In this case, vascularization did not begin for twenty days, and during that time there was little tumor growth. After vascularization began, migration and proliferation of tumor cells

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accelerated. Algire and Chalkley (1945) carried the study further, using sarcomas and mammary gland carcinomas, and also investigated the repair of wounds. They found that capillaries arose from the host and that endothelial proliferation appeared as early as three days after implantation, whereas in wounds it did not begin for six days. Despite the fact that the tumors became well vascularized, differentiation of vessels into arterioles and venules was not evident. The authors believe that an outstanding characteristic of the tumor cell is its capacity to elicit continued growth of new capillary endothelium from the host. “This characteristic of the tumor cell, rather than some hypothetical capacity for autonomous growth inherent within the cell, is, from the standpoint of the host, an important expression of neoplastic change.” This generalization implies that growth potentialities in the tumor cells themselves may be secondary in importance to the endothelial proliferation which the tumor can induce, or, stated otherwise, that the tumor grows because it can cause proliferation of blood vessels. There are those, of course, who think just the opposite, namely, that vessels proliferate because the tumor itself has the capacity for continued growth. This is the sort of thing that can lead to endless argument and is similar in its futility to the question, which came first, the chicken or the egg. However, it is clear that the suggested generalization of Algire and Chalkley does not apply to all malignancy, although it cannot be denied that it may apply to some. The Vz carcinoma, an epidermoid carcinoma of rabbits, has no ability to stimulate the growth of vessels (Williams, 1951). It infiltrates among the host vessels, causing changes in them, and finally eliminates all vessels centrally located by external pressure upon them, thus guaranteeing that the tumor center will be necrotic. This tumor is invasive and rapidly fatal, although it does not metastasize freely. The only viable part of it, after the first few days following transplantation, is a narrow peripheral rim where host vessels are available. The vascular response to the Vz carcinoma is very similar to that in normal growing epidermis, which suggests that a malignant tumor may retain, to some degree at least, the ability to influence its vasculature in the same manner as do the normal cells in which it was primary. Vessels near growing normal epidermis are wide, thin-walled, sinusoidal channels, and this is characteristic of those among the growing cells in V t carcinoma. Algire and Chalkley (1945) and Ide et al. (1939b) found large, thin-walled vessels consisting of endothelium only in sarcomas, niarnmary gland Carcinomas, and the Brown-Pearce epithelioma. They noted that arterioles and venules did not develop in the plexus. In growing mouse melanomas, capillaries were smaller and there was a greater tendency

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for arterioles and venules to differentiate (Algire and Legallais, 1948j . Since arterioles and venules normally develop within a capillary plexus in response to hemodynamic factors, it is curious that they sometimes do not develop in tumors, since if there is blood flow, there must be pressure and other differences in vessels. However, to have the formation of arterioles in a capillary plexus, there must be already formed arterioles sending blood into the plexus, and vessels in the plexus must not be of too large caliber. For example, an ampulla of Thoma in the spleen is an abruptly enlarged cone-shaped venous extension of an arteriole. As the spleen becomes vascularized, arteriolar formation does not extend beyond the ampulla. In some forms of hemangiomas, the vasculature is sinusoidal and arterioles do not develop in them. The size of capillaries and venules in a part may be an expression of the nature of the ground substance. If it affords little support for the walls, those composed chiefly of endothelium could be expected to dilate even under normal capillary pressure (Williams, 1949). The walls of vessels in Brown-Pearce tumors are so fragile that they are easily ruptured, and it would seem to be an easy matter for small tumor fragments to be dislodged and swept into the ruptured vessels and hence into the general circulation { Ide et al., 1939). But the authors found no metastases in over 105 cases. They believe that a study of the local defenses of body organs, particularly the lungs, against tumor fragments should be of considerable interest, as indeed it would. But the sweeping into the general circulation of tumor fragments through traumatic openings in vessels may not be so common. When a vessel ruptures, blood is extravasated until the pressure outside the vessel is locally equal to that inside or until the rupture heals or the hemorrhage stops for some other reason. To have tumor cells enter the blood stream, the rent in the vessel would have to stay open and a loose tumor fragment be forced in by external pressure. I t would, presumably, not often be sucked or swept in by the moving blood stream, since if there were any cell movement in the extravascular regions, it would tend to be away from the vessels because of the pressure of extravasation. There is no clear-cut evidence in chamber literature, or if there is it hasn’t been located, as to how metastases begin. Since the vessels in tumors are frequently so thin-walled and large and the tumor cells so actively growing and invasive, it is easily conceivable that tumor could grow into a vessel as a tuft and then be dislodged by the moving blood. Tumor cell masses in vessels have been described in microscopic sections, but they appear not to have been seen by direct observation in the living. The effect of roentgen irradiation on transplants of Brown-Pearce rabbit

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epithelioma was investigated by Downing cf al. (1940). Within one hour of the 10,000-r. dose, a red haze developed microscopically around the tumor and in control regions, but a gross erythema did not appear until five days later. The tissues were then suffused with extravasated red cells to an extent that made microscopic study impossible. Before that stage, all vessels in the irradiated sites became beaded and irregular in contour, and blood flow was reduced, erratic, or absent. However, normal responses to paling and flushing were almost intact. After five days vascular dissolution began, with slow recovery during the following week. The authors observed that endothelial response to irradiation was the same in both tumor and control areas which, they state in effect, indicates that a tumor does not change endothelium in it as far as the response to irradiation is concerned. The effects of irradiation on mammary tumor implants in mice were studied by Merwin et al. (1950). Dosage of 2000 to 3000 r. produced marked regression followed by regrowth. Tumor vessels narrowed progressively for about one week, but when tumor growth was resumed the vessels enlarged. The growth rate of the tumor after the first effects of irradiation subsided was initially about the same as it had been without treatment hut was later gradually reduced, apparently because after irradiation the growth potentialities of the endothelium were less. Vessels in regrowing tumor foci finally broke down, and the now nonvascularized opaque tissue stimulated the growth of adjacent vessels that had not been irradiated. When this occurred, such tumor as was still viable began to grow at its usual rate. Grafts not themselves irradiated were also made on vessels that had been. As in the previous cases, it was evident that irradiated vessels could not regenerate. I t appeared that anything short of complete tissue destruction by irradiation affected the growth properties of the tumor secondarily by damage to its blood supply. In this connection, it is pertinent to note again that 1131 suppressed the growth of endothelium in autografts of normal thyroid. Peripheral hypotension induced by histamine and in other ways reduces the blood supply in transplanted tumors (Algire and Legallais, 19.51). Reduction of tumor circulation is directly correlated with the duration and degree of peripheral hypotension and does not require toxic or lethal doses of histamine. Tumor necrosis results only after large, but not lethal, doses. Blood pressure in tumor vessels approximates normal venous pressure. “If a hypotensive state were maintained for a sufficiently long period one would expect to find histologic or gross evidence of damage to the tumor tissue from the resultant ischemia.” Bacterial potysaccharide affects vessels in mice, both normal and those

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G. WILLIAMS

in tumors, in the following manner. The blood flow and percentage of functional capillaries is progressively decreased. Stasis and occlusion of capillaries develop, reaching a maximum in 3 to 4 hours with recovery in normal tissues within 18 hours. Hemorrhage and necrosis do not occur in normal tissues. The stasis and occlusion in all vessels seem to be associated with edema, suggesting increase in capillary permeability. In tumor vessels, after various periods, vascular occlusion is followed by the sudden appearance of petechiae throughout the tumor, followed by extensive necrosis. However, some tumor cells survive and retain their ability to stimulate the growth of vessels, and tumor growth is then resumed. There appears to be no primary action of the polysaccharide on the tumor cells. The tumor-necrotizing effect of this agent seems to be brought about by ischemia and circulatory stasis induced by hypotension (Algire, 1 9 6 ; Algire, Legallais, and Park, 1947; Algire and Legallais, 194s; Algire, Legallais, and Anderson, 1952). Mechanical obstruction can duplicate the action of bacterial p l y saccharide in producing hemorrhage and tissue damage in some tumors but not in others (Youngner and Algire, 1949a, b). Vascular reactions of inice to mouse fibroblasts cultivated in vitro and therein treated with methylcholanthrene were investigated by Algire, Chalkley, and Earle ( 1950). Various cell strains were created, depending on the length of time each was exposed to the carcinogen. These cultures were then implanted in mice by means of a chamber method. The vascular reaction to these transplants from tissue cultures was parallel with the capacity of the culture strains to give rise to sarcomas in other animals. Plasma clots used as controls produced only a mild foreign body reaction, indicating that cells must be present for a more vigorous vascular proliferation. With increased time in vitro after removal of the carcinogen, the ability of each cell strain to form sarcomas was reduced. This, the authors suggested, may indicate that growth of cultures in an entirely heterologous media for extended periods may alter cell characteristics so that they are less able to live when transplanted to an animal and less able to give rise to sarcomas. 4 . Parasites and Eggs This heading is included mainly because of the promise of the method in such investigations and not because it has been used extensively in the study of parasites. Only a single reference concerning such work has been found. Hoeppli and Hou (1931) placed various parasite eggs in chambers. The devices they used were not well adapted for transplanting, since the effective techniques now available had not then been developed. They found that Ascaris lumbricoides and FasciolopSis eggs produced little leukocyte infiltration, but it was much increased when the egg shells were

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broken. The shell of Fa.~ciolopSis buski eggs was less resistant to dissolution by tissue Auid than was the shell of Asccw-is tumbricoides. They also found that worm capsules of Spirocerca sanguinolenta larvae were very resistant to white cells. Physaloptera clausa from hedgehogs and Enterobius znermicduris from man survived only about two days. Physalojtera produced tissue liquefaction around its anterior end, but no similar changes were observed with Enterobius.

VII. TUBERCULOSIS

The early reaction, nine to twenty-three days, to lung bovine tubercle bacilli in chambers is minimal. However, the late reaction, ten to twentyfour days, is an explosive necrotizing response. As determined in one animal by systematic skin testing with old tuberculin, the beginning of late reaction coincides with the development of skin sensitivity. Tissue destruction followed progressive vascular damage resulting .in venous thrombosis (Ebert, Ahern, and Bloch, 1948). The thrombosis occurred only in small vessels, 30 to 40 p in diameter. It was impossible to tell whether the vascular changes were primary or secondary, but there was little doubt that they played an important part in tissue destruction. Ebert and Barclay (1950) studied the effect of chemotherapy in tuberculous infections. Animals were generally sensitized before inoculation of the chamber with 0.004 mg. of virulent bovine tubercle bacilli. They were then treated for six to eight weeks, some with streptomycin (100 mg. daily), some with p-aminosalicylic acid (0.900 mg. daily), and others with the two combined. Thrombosis of small vessels and infarction occurred as it did in untreated animals. In general, healing and extension of the disease process occurred simultaneously in different parts of tubercles as they do without treatment, but in treated animals there was more extensive healing. Healing and extension were commonly cyclic in that there were times when healing was predominant and others when it was not. When treatment was begun before inoculation, the initial necrotizing response was not inhibited. Cortisone reduces the inflammatory response to tuberculous infection of a hypersensitive animal (Ebert, 1951, 1952). In the presence of cortisone, vascular tone is better maintained, damage to endothelium of arterioles and venules is reduced, and there is less diapedesis of leukocytes and less exudate. The reduction in inflammation causes poorer localization of infection. This recalls studies previously quoted (Ebert and Barclay, 1952) in which it was stated that if an inflammation is useful to the body cortisone may be harmful, but it would not be if the inflammation were useless, as in rheumatoid arthritis.

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As a preliminary to a study of its effect in tuberculous infection, Markham et al. (1951) investigated the behavior in Zrivo of an almost insoluble antibiotic, micrococcin. A fine suspension of the material was forced into chambers through an access hole and also, in other animals, by removing the chamber cover. The micrococcin was taken up by macrophages and retained for many months without apparent damage to them. Vascular endothelium was unaffected. The antibiotic behaved in the body like a bland foreign body, When carbon and micrococcin are injected intravenously, the particles are agglutinated by the platelets and the masses adhere to the walls of vessels in the neighborhood of tuberculous lesions and tend to stay in the vessels. The micrococcin produces no effect on the growth of tubercles even when in the center of necrotic tubercles (Sanders et al. 1951). Micrococcin in Triton WR-1339, a detergent of low toxicity, was tested in tuberculous infection by Heatley et al. (1952). The course of experimental tuberculosis and the nature of the lesions in guinea pigs were not influenced by intravenous injections of the solution.

VIII. CONCLUSION The transparent chamber method is a means for creating a microscopic section, as it were, without the disadvantages of killing the cells, subjecting them to potent chemicals, and converting three dimensions to two. With such preparations, studies need not be limited to descriptive histology. They also reduce the need for inference in determining a vital sequence. The method will continue to be used as it has been because none of the subjects studied has been exhausted. The most likely extensions of its use, with suitable, easily made modifications, would seem to be with polarized light, ultraviolet light, reflecting microscopes, fluorescent microscopy, microdissection, and precise chemical methods adapted to living cells. ACKNOWLEDGMENTS The author is deeply indebted to Miss Patricia Rochford for her invaluable assistance in the preparation of this review, and to Dr. E. R. Clark, who kindly read the manuscript.

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