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Ultrastructural injury following X-irradiation of rat parotid gland acinar cells
ArchsoralBiol.Vol. 17,pp. 1177-1186, 1972. Pergamon Press. Printed in Great Britain.
ULTRASTRUCTURAL INJURY FOLLOWING X-IRRADIATION OF RAT PAROTID GLAND ACINAR CELLS N.
E.
PRATT
and M.
SODICOFF
Department of Anatomy, Temple University, School of Medicine, Philadelphia, Pennsylvania, U.S.A. Summary-Following X-irradiation with 1600 rad to the head and neck area of rats, parotid glands were examined at 1, 3 and 5 hr, 1, 2, 3, 47 and 8 days for evidence of ultrastructural damage of acinar cells. Damage was evident as early as 3 hours with maximum destruction seen at 2 days. Ultrastructural damage took the form of cytolytic bodies composed of damaged cell organelles and structures called light bodies which may represent autophagic vacuoles to be finally extruded from the cell or inflammatory cells which have invaded the cell. At the 7- and 8-day interval, the most prominent alteration of cellular architecture was the presence of many empty vacuoles. The study demonstrates the early onset of observable damage with doses as low as 1600 rad. INTRODUCTION
changes in irradiated salivary glands have been reported in both the older and more recent literature (ENGLISH, 1955; CHERRY and GLUCKSMANN, 1959; ELZAY, LEVITT and SWEENEY, 1969; KASHIMA, KIRKHAM and ANDREW& 1965; PHILLIPS, 1970). Reports of radiation damage to rodent parotid gland acinar cells have varied widely with respect to time of earliest appearance and X-ray dose required to induce damage; the earliest changes reported occurred at 24 hr after doses of near 4000 rad (PHILLIPS, 1970). All previous studies examined irradiated parotid gland tissue using the light microscope. The present report is believed to be the first to use electron microscopy to detect early stages of radiation damage to salivary glands. Understanding of damage caused by X-irradiation to the parotid gland is of importance since the gland is frequently irradiated, with both low and high doses, in the course of the clinical management of many diseases and the well-known complication of “dry-mouth” is often a troublesome consequence of radiation therapy to the oropharynx. It is felt that ultrastructural examination of irradiated tissue will add yet another dimension to the analysis of the radiosensitivity of this organ. HISTOPATHOLOGIC
MATERIALS
AND
METHODS
Rats to be irradiated were restrained, unanaesthetized, in a ventilated lucite tube, which was shielded with 3 mm of lead so that only the head and neck regions received the full radiation exposure. Animals received 1600 rad as a single exposure delivered by a 300 kVp X-ray machine, using 300 kV, 20 mA, with added tiltration of 0.5 mm Cu and 1.04 mm Al (HVL 1.57 mm Cu), STD 50 cm, at an exposure rate of about 187 rad/min. All irradiation took place between 8 and 10 a.m. Radiation exposures were calibrated prior to each exposure with a 250 R Victoreen ionization chamber. 1177 Irradiation.
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Electron microscopy. Female Sprague-Dawley rats (180 & 10 g) were seria!ly killed, 2 to 4 at each time interval, at 1, 3 and 5 hr; and at 1, 2, 3,4, 7 and 8 days following irradiation. The fixative of choice was half-strength Karnovsky’s combination of formaldehyde and glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 (KARNOVSKY,1965). Following a 15-min perfusion through the ascending aorta with room temperature fixative, the parotid gland tissue was removed, diced and fixed for an additional 5 hr in the same fixative. The tissue was then washed for 12 hr in several changes of cold (3-4C) 0.1 M phosphate buffer and post-fixed for 2 hr in cold 1 per cent osmium tetroxide buffered with 0.1 M collidine (pH 7.4). Embedding in Epon 812 followed routine dehydration of the tissue. Thin sections were cut on an MT-2 Porter-Blum ultramicrotome and placed on bare 300 mesh copper grids. The sections were stained with uranyl acetate and lead citrate and examined and photographed in an RCA EMU-4 electron microscope at an accelerating voltage of 50 kV. The original plate magnifications ranged between 3000 and 48,000 times. RESULTS 1 hour.
At this time, the acinar cells showed no obvious disturbances in morphology. Normal parotid morphology (Fig. 1) was similar to that described by SCOTT and PEASE (1959), PARKS (1961, 1962) and RUTBERG (1961). 3 hours. The appearance of most acinar cells was normal. However, in a very few of the cells, cytolytic bodies (a term used here to describe areas of cellular degeneration) were now found. Membranes around these areas of cytoplasmic degeneration were either absent or only partially complete. Figure 2 shows a typical cytolytic body within an acinar cell. The size of these structures varied from that of a nucleus to that of a secretory droplet. Appearances of cytolytic bodies also varied. Some were areas of cytoplasmic density with embedded secretory droplets, while others contained large amounts of rough endoplasmic reticulum, remnants of mitochondria and amorphous dense bodies. In addition to cytolytic bodies, other complex structures, here called light bodies, were also present. These structures were only sometimes membranebound. The light bodies had the following characteristics: (a) multiple nuclear fragments, each of which had a complete nuclear envelope, and the chromatin segregated so that the heterochromatin was separated from the euchromatin, frequently resulting in a crescent-form appearance; (b) rough endoplasmic reticulum arranged not only as parallel cisternae but also as concentric rings or whorls; and (c) ground cytoplasm which was often lighter than that of the acinar cell and contained many free ribosomes. Not all light bodies, however, displayed all the characteristics. Figure 3 shows a light body which is identified primarily by the occurrence of multiple nuclear fragments having crescent-form appearance. It is not enclosed by a membrane and appears to be free in the cytoplasm of the acinar cell. 5 hours. Although most acinar cells were normal, focal areas of acinar cell damage were present. Such areas were more numerous than at 3 hr, but the cytolytic bodies were similar in appearance. Light bodies were also present more frequently than before. Figure 4 shows a light body containing multiple crescent form nuclear fragments, each with an intact nuclear envelope and rough endoplasmic reticulum arranged as parallel cisternae and concentric rings. In Fig. 4, the light body is membrane bound and is completely within an acinar cell near its base. However, other light bodies appeared to be bulging from the basal surface of acinar cells but were not clearly separated from them. From the appearance of the light bodies seen at the 3 and 5 hr interval, it is not clear whether they represent separate cell types within an acinar cell,
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or rather represent areas of acinar cell degeneration in the form of autophagic vacuoles. Inflammatory cell types such as lymphocytes and macrophages were found degenerating within the acinus outside the acinar cell, but within the confines of the basement membrane (Fig. 5). 1 day. Evidence of cytoplasmic damage was abundant and widespread, although focally distributed (Fig. 6). Typical areas of acinar cells containing cytolytic bodies such as those seen at 3 and 5 hr were present. Membranes usually were not present around these structures. Necrotic cellular debris having some resemblance to light bodies was found within the acinar grouping. However, the advanced state of the degeneration made identification of the specific cell type difficult. Many acini were found wherein the entire cell cluster was in an advanced state of necrosis (Fig. 7). As seen at earlier times, typical light bodies were found, although some of these structures were found to contain secretory droplets (Fig. 8). In certain instances secretory droplets were found in the intercellular space between the light body, which already contained other secretory droplets, and the acinar cell, suggesting some form of transfer mechanism. Many light bodies appeared to be separate entities separated from acinar cells by an intercellular space (Fig. 8), but similar structures were also found within acinar cells and not surrounded by a continuous membrane (Fig. 9). Many lymphocytes and macrophages were found within the acini, covered by the basement membrane. 2 days. Areas of acinar cell destruction were widespread with large areas of necrosis and whole acinar cell clusters frequently degenerating. In cells showing less severe cytoplasmic damage cytolytic bodies similar to those seen at earlier periods were found. In addition, many light bodies of varied appearance were present within acini (Figs. 10 and 11). In some cases these light bodies seemed to resemble lymphocytes and macrophages. In Fig. 10, a light body resembling a lymphocyte but containing a crescent-form nucleus and abundant rough endoplasmic reticulum is nearly engulfed by a macrophage. In Fig. 11, the light body contains crescent-form nuclei and lymphocyte-like cytoplasm. Other light bodies were large, being at least the size of acinar cells. The overall general impression of this tissue was that it was greatly disorganized due to the shrunken and atrophic appearance of many, but by no means all, acinar groups. Intercellular spaces between acinar cells were frequently widened with cells appearing shrunken and angular. 3 and 4 days. Fewer cytolytic bodies were found within acinar cells at this time, although many unrecognizable degenerating cells resembling large cytolytic bodies were frequently found. Light bodies and inflammatory cells were found very infrequently at this time. This period was characterized by the initial appearance of large vacuoles, focally distributed, in many of the acinar cells. The vacuoles were similar to those seen at 7 and 8 days (Fig. 12) and varied considerably in size, as did the cytolytic bodies at this and earlier time intervals. Some vacuoles were larger than the nucleus. The vacuoles were frequently membrane-bound and their contents varied,
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some appearing empty, some containing a light flocculent material, and others containing thin filmy membranes. 7 and 8 days. One notable difference at this interval, as compared to earlier times, is that the observed changes were widespread and not focal. The most obvious acinar cell derangement was the presence of multiple clear vacuoles in almost all of the acinar cells (Fig. 12). Vacuoles of various sizes were round and usually membrane bound. They were usually completely empty, but occasionally contained some filmy membraneous debris. Rarely were acinar cells found containing cytolytic bodies, nor were light bodies present. Acinar cells were often shrunken and gave the appearance of being angular. In summary, acinar cell damage was first detectable 3 hr following irradiation by the appearance of cytolytic and light bodies. The frequency of occurrence of these forms of cell damage increased with time and, by 2 days, tissue disorganization and atrophy of acinar groups was maximum. By 3 days, the cytolytic and light bodies were found less frequently and intracellular vacuoles began to be present. At one week following irradiation, the major indication of damage was the widespread appearance of severe vacuolization. DISCUSSION
From studies based upon light microscopic examination of paraffin sections, it has been stated that following irradiation, acinar cells of the rat parotid gland do not show the acute histopathological changes seen in the human parotid (ENGLISH, 1955; VAN DEN BRENK et al., 1969; SHAFER, 1953). Yet, more recently, atrophy, necrosis, degenerative nuclear changes and functional alterations have been described one day following irradiation with 4000 rad (PHILLIPS, 1970). Indeed, when paraffin sections of parotid tissue from the present study were examined by light microscopy, intracellular inclusions were easily observable by day 3 following irradiation, although more subtle evidence of damage was thought to be seen as early as 1 day. The inclusions were often of similar form to those seen by electron microscopy but stained with haematoxylin. They were, however, not distinguishable into cytolytic or light bodies and often gave the appearance of pyknotic nuclei. At 7 days, the acinar cells were obviously vacuolated. Previous investigators have not described such degenerative changes in the parotid so soon after such a low dose as 1600 rad. In the present study, which utilized the electron microscope to examine and assess the effects of radiation on the parotid gland, signs of acinar cell damage were noted as early as 3 hr following irradiation with only 1600 rad, making it appear that the response of the rat and human parotids may be more alike than previously thought. The present study did not concern itself with very subtle alterations in cellular organelles, but rather focused attention on more obvious signs of cytoplasmic and nuclear degeneration. It would therefore seem likely that more subtle changes may indeed be present at even earlier times following irradiation. Following 1600 rad, acinar cell damage seemed to take two forms: the cytolytic body and the so-called light body. The cytolytic body is a focal area of damaged
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cellular organelles, most often composed primarily of damaged rough endoplasmic reticulum, but often including mitochondria, secretory droplets and unidentifiable dense bodies. The appearance of these structures is often badly distorted and recognition of specific structures is sometimes difficult, especially by 3 and 4 days. Membranes enclosing these structures are seldom complete. These cytolytic bodies are similar to those found in other irradiated tissues (GHIDONI and CAMPBELL,1969; WELLMAN,VOLK and LEWITAN,1966; HUGON and BORGERS,1966) which have been shown to be acid phosphatase positive (HUGON and BORGERS,1966). The cause of such cytoplasmic lesions could be the result of (a) direct damage to an organelle (WILLS, 1970) such as the endoplasmic reticulum, mitochondria, etc., followed by subsequent fusion with lysosomes, or (b) a primary insult to the lysosome, thus releasing hydrolytic enzymes (RENE,DARDENand PARKER,1971) to the cytoplasm for autodigestion. The second form of cytoplasmic damage consists of a structure referred to as the light body, which is more complex and more difficult to understand than the cytolytic body. In its most complex form, such cytoplasmic lesions are completely enclosed within an acinar cell and membrane bound. Nuclear material is fragmented, forming 2 to 4 micronuclei, each with an intact nuclear envelope and the nuclear contents segregated into dense and light chromatin, often giving a crescent appearance. Rough endoplasmic reticulum is well preserved, but often forms circles and concentric rings in fingerprint-like patterns. The preservation of the rough endoplasmic reticulum is often so good that its relationship to the nuclear envelope is quite clear. The background cytoplasm stains less intensely and is more abundant than that of the acinar cell and sometimes includes secretory droplets. Such structures within an acinar cell can be much greater in size than the nucleus itself. In its least complex form, the light cell may be a crescent-form nuclear fragment free in the cytoplasm of an acinar cell or a less dense cytoplasmic area with rings of rough endoplasmic reticulum. A possible interpretation is that these smaller structures will enlarge and/or fuse to form the larger and more complex bodies. Structures similar to the most complex form of the light body are frequently seen completely outside the acinar cell but the physical separation between acinar cell and light body is often not clear, consequently giving the impression that the light body is being extruded from the base of an acinar cell. Light bodies, even when completely separated from acinar cells, are rarely outside the basement membrane of the acinus. This proposed sequence of events, leading from the formation to the extrusion of an autophagic vacuole is similar to that proposed by GHIDONIand CAMPBELL(1969), but might actually be incorrect. Another interpretation of the sequence is that some other cell type, such as an inflammatory cell, has invaded the acinus and migrated inside the acinar cell finally to be digested (HUGONand BORGERS,1966), leaving behind the empty vacuoles seen at 7 and 8 days. Credence is lent to this latter hypothesis by the occurrence of some cell types which bear a resemblance to lymphocytes and macrophages by virtue of their size, shape and cytoplasmic contents. Yet these cells have the crescent-form nuclear material and abundant rough endoplasmic reticulum as do the light bodies. Indeed, multiple crescent-form nuclear fragments (LUCAS and PEAKMAN,1969; JORDAN,1967) and
1182
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increased amounts of rough endoplasmic reticulum (SPANGLER and CASSEN,1967) have been shown in irradiated lymphocytes. Truly, since only the head and neck were irradiated, we do not know which, if any, of the inflammatory cells were actually irradiated. Since the light bodies were usually only found within the acinus, it would seem less likely that these changes were caused by irradiation, but rather might have occurred in response to acinar cell damage. In summary, the structures called light bodies are puzzling and may represent the extrusion of autophagic vacuoles from acinar cells or the migration of inflammatory cells into acinar cells. Nuclear damage was seen only associated with light bodies. Nuclei of acinar cells always appeared normal even when large or numerous cytolytic or light bodies were present. Since the mitotic rate of acinar cells is extremely low (NOVI and BASERGA, 1971), it would seem unlikely that mitotically related nuclear damage would occur. However, since the origin of the nuclei of the light bodies remains questionable, the possibility of acinar cell nuclear damage cannot be excluded. The cytolytic bodies and light bodies made their first appearance at 3 hr and reached their greatest frequency of occurrence at 2 days. By the 3rd and 4th day, they were very infrequent and not present at all in the 7th and 8th day. Their distribution throughout the parotid tissue was spotty and never uniformly distributed. It is interesting to speculate that the empty and near empty vacuoles at 7 and 8 days may represent the end result of degradation and digestion of these inclusion bodies. However, the uniform distribution of these vacuoles throughout the tissue as compared to the focal distribution of the cytolytic bodies makes this explanation questionable and leaves us with the further speculation that the vacuole formation may be a separate event unrelated to the cytolytic bodies. Another possibility explaining the disappearance of cytolytic and light bodies is that those acinar cells with them totally degenerate and are finally cleared away by macrophages. From the available information, we cannot distinguish which of the possible mechanisms of resolution is occurring. Acknowledgement-We would like to express our thanks to Miss RUHDEAN SCOTTfor her excellent technical assistance. This research was supported by General Research Support Grant 5SOl-FR05417-10 and NIH Grant 1 R23 DE03256. R&sum&-Aprts irradiation par rayons X, a la dose de 1600 R au niveau de la tete et de la nuque de rats, les glandes parotides ont et& Btudi&es 1,2, 3,4, 7 et 8 jours apres pour Ctudier les lesions ultrastructurales des cellules acineuses. Des alterations sont dvidentes dts 3 heures, avec un maximum de destruction a 2 jours. Les lesions ultrastructurales prennent la forme de corps cytolytiques, constitur5es d’organelles cellulaires endommages et par des structures appelees corps claires qui peuvent rep&enter des vacuoles autophagiques, tinalement rejetees de lacellule, ou des cellules inflammatoires qui ont envahi la cellule. Au bout de 7 et 8 iours. l’alteration la nlus nette de l’architecture cellulaire est la presence de nombreusds vacuoles vides. Cette etude confirme la precocite des lesions a des doses aussi faibles que 1600 r. Zusammenfassung-Nach Rontgenbestrahlung mit 1600r auf Kopf- und Nackenpartie von Ratten wurden die Parotisdriisen 1,3, 5 Stunden und 1,2,3,4,7 und 8 Tage spater auf Anzeichen ultrastruktureller Schlden an den Azinizellen untersucht. Eine Schldigung war 3 Stunden nach Bestrahlung erkennbar, die maximale Destruktion wurde nach 2 Tagen beobachtet. Die ultrastrukturelle Schldigung trat in Form zytolytischer Korper
EFFECTSOF IRRADIATION OF PAROTIDGLANDACINARCELLS
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aus zerstorten Zellorganellen und unter dem Bild von Struckturen, die als “light bodies” bezeichnet wurden, auf, wobei letztere Autophagen-Vacuolen darstellen, die schliel3lich von der Zelle abgestoaen werden oder mit entziindlichen Zellprozessen zusammenhlngen. Nach 7 bis 8 Tagen ist die am meisten vorherrschende zellulare Verlnderung das Vorhandensein zahlreicher leerer Vakuolen. Die Untersuchung demonstriert die frtihe Nachweisbarkeit von Schlden bei so niedrigen Strahlendosen wie 1600 r.
REFERENCES CHERRY,C. P. and GLUCKSMANN,A. 1959. Injury and repair following irradiation of salivary glands in male rats. Br. J. Radiol. 32. 596-607. ELZAY, R. P., LEVITT, S. H. ~~~‘SWEENEY,W. T. 1969. Histologic effect of fractionated doses of selectively applied megavoltage irradiation on the major salivary glands of the albino rat. Radiology 93, 146-152. ENGLISH,J. A. 1955. Morphologic effects of irradiation on the salivary glands of rats. J. dent. Rex 34,4-II. GHIDONI, J. J. and CAMPBELL,M. M. 1969. Karyolytic bodies. Archs Path. 88, 480488. HUGON, J. alId BORGERS,M. 1966. Ultrastructural and cytochemical studies in the epithelium of the duodenal crypts of whole body X-irradiated mice. Lab. Invest. 15, 1528-1543. JORDAN,S. W. 1967. Ultrastructural studies of spleen after whole body irradiation in mice. Exptl molec. Path. 6, 156-171. KASHIMA,H. K., KIRKHAM,W. R. and ANDREWS, J. R. 1965. Postirradiation sialadenitis. Am. J. Roent. 94, 271-291. KARNOVSKY,M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. cell. Biol. 27, 137-338A. LUCAS, D. R. and PEAKMAN,E. M. 1969. Ultrastructural changes in lymphocytes in lymph nodes, spleen and thymus after sublethal and supralethal doses of X-rays. J. Path. 99, 163-169. NOVI, A. M. and BASERGA,R. 1971. Association of hypertrophy and DNA synthesis in mouse salivary glands after chronic administration of isopreteranol. Am. J. Path. 62, 295-308. PARKS,H. F. 1961. On the fine structure of the parotid gland of the mouse and rat. Am. J. Anat. 108, 303-329. PARKS,H. F. 1962. Morphological study of the extrusion of secretory materials by the parotid glands of mouse and rat. J. ultrastruct. Res. 6,449-465. PHILLIPS, R. M. 1970. X-ray-induced changes in function and structure of the rat parotid gland. J. oral Surg. 28, 432-437. RENE, A. A., DARDEN, J. H. and PARKER, J. L. 1971. Radiation-induced ultrastructural and biochemical changes in lysosomes. Lab. Inuest. 25, 230-239. RUTBERG,U. 1961. Ultrastructure and secretory mechanism of the parotid gland. Acta odont. stand. 19, Suppl. 30, l-69. SCOTT,B. L. and PEASE,D. C. 1959. Electron microscopy of the salivary gland and lacrimal glands of the rat. Am. J. Anat. 104,115-161. SHAFER,W. G. 1953. The effect of single and fractionated doses of selectively applied X-ray irradiation on the histologic structure of the major salivary glands of the rat. J. dent. Res. 32,796806. SPANGLER,G. and CASSEN,B. 1967. Electrophoretic mobility, size distribution and electron micrograph responses of lymphocytes to radiation. Rad. Res. 30,22-37, VAN DEN BRENK, H. A. S., HURLEY,R. A., GOMEZ. C. and RICHTER.W. 1969. Serum amvlase as a measure of salivary gland radiation damage. Br; J. Radiol. 42, 688-700. WELLMAN.K. F.. VOLK. B. W. and LEWITAN.A. 1966. The effect of radiation on the fine structure and enzymecontent of the dog pancreas.‘Lab. Invest. 15, 1007-1023. WILLS, E. D. 1970. Effects of irradiation on sub-cellular components-I. Lipid peroxide formation in the endoplasmic reticulum. ht. J. Radiat. Biol. 17, 217-228.
N.
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E. PRATT AND M.
PLATE
SODICOFF
1
FIG. 1. Typical parotid acinar cell from a normal rat showing the characteristic basally located flattened rough endoplasmic reticulum and apically located dense secretory droplets. x 8800
FIG. 2. Three hours post-irradiation. A typical cytolytic body contains damaged rough endoplasmic reticulum (arrow), dense bodies (D) and other membraneous material (M). x25,000
FIG.
3. Three hours post-irradiation. Simple form of a light body containing crescentform nuclear fragments (N) and rough endoplasmic reticulum arranged in parallel stacks. This structure appears only partially membrane bound. x 27,000
FIG. 4. Five hours post-irradiation. A complex form of a membrane-bound light body is present within an acinar cell. Rough endoplasmic reticulum is arranged in both stacks and whorls, and multiple crescent-form nuclear fragments (N) are present. Cytolytic bodies can be seen both within the light body (large arrow) and within the acinar cell (small arrow) where it contains a secretory droplet (S). x 12,800
A.O.H.
I’l.ATl I I183
f.p.
PLATE 2
N. E. PRATTAND M. SODICOFF
1185
PLATE2 FIG. 5. Five hours post-irradiation.
A degenerating lymphocyte cells within an acinus. x 13,300
is seen between acinar
FIG. 6. One day post-irradiation.
A thick section (1 pm) showing the focally distributed damage as seen by the light microscope. The arrows are pointing to areas of damage which resemble those structures called cytolytic and light bodies as seen with the electron microscope. x 1000 FIG. 7. One day post-irradiation. An acinar cell cluster in an advanced degeneration. x 8200 FIG. 8. One day post-irradiation.
stage of
Typical light bodies, containing nuclear fragments (N), whorls of rough endoplasmic reticulum and secretory granules (S) are seen within an acinus both separated from (large arrows) as well as within (small arrows) acinar cells. x 7900
A.O.B.
17/8--e
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PLATE3 Fro. 9. One day post-irradiation. A typical light body having no membraneous enclosure is seen. A cytolytic body (C) also is present in this acinar cell. x 15,000 FIG. 10. Two days post-irradiation. Within an acinus, a lymphocyte-lie containing a crescent-form nucleus (N) and abundant rough endoplasmic nearly engulfed by a macrophage (M). x 11,600
light body reticulum is
FIG. 11. Two days post-irradiation. This light body contains multiple crescent-form nuclei (IV) and lymphocyte-like cytoplasm. x 21,700 FIG. 12. Eight days post-irradiation.
Multiple vacuoles (V) are found in these acinar cells. x 6700
A.O.B.
PLATE 3 I_p. 1186
ULTRASTRUCTURAL INJURY FOLLOWING X-IRRADIATION OF RAT PAROTID GLAND ACINAR CELLS N.
E.
PRATT
and M.
SODICOFF
Department of Anatomy, Temple University, School of Medicine, Philadelphia, Pennsylvania, U.S.A. Summary-Following X-irradiation with 1600 rad to the head and neck area of rats, parotid glands were examined at 1, 3 and 5 hr, 1, 2, 3, 47 and 8 days for evidence of ultrastructural damage of acinar cells. Damage was evident as early as 3 hours with maximum destruction seen at 2 days. Ultrastructural damage took the form of cytolytic bodies composed of damaged cell organelles and structures called light bodies which may represent autophagic vacuoles to be finally extruded from the cell or inflammatory cells which have invaded the cell. At the 7- and 8-day interval, the most prominent alteration of cellular architecture was the presence of many empty vacuoles. The study demonstrates the early onset of observable damage with doses as low as 1600 rad. INTRODUCTION
changes in irradiated salivary glands have been reported in both the older and more recent literature (ENGLISH, 1955; CHERRY and GLUCKSMANN, 1959; ELZAY, LEVITT and SWEENEY, 1969; KASHIMA, KIRKHAM and ANDREW& 1965; PHILLIPS, 1970). Reports of radiation damage to rodent parotid gland acinar cells have varied widely with respect to time of earliest appearance and X-ray dose required to induce damage; the earliest changes reported occurred at 24 hr after doses of near 4000 rad (PHILLIPS, 1970). All previous studies examined irradiated parotid gland tissue using the light microscope. The present report is believed to be the first to use electron microscopy to detect early stages of radiation damage to salivary glands. Understanding of damage caused by X-irradiation to the parotid gland is of importance since the gland is frequently irradiated, with both low and high doses, in the course of the clinical management of many diseases and the well-known complication of “dry-mouth” is often a troublesome consequence of radiation therapy to the oropharynx. It is felt that ultrastructural examination of irradiated tissue will add yet another dimension to the analysis of the radiosensitivity of this organ. HISTOPATHOLOGIC
MATERIALS
AND
METHODS
Rats to be irradiated were restrained, unanaesthetized, in a ventilated lucite tube, which was shielded with 3 mm of lead so that only the head and neck regions received the full radiation exposure. Animals received 1600 rad as a single exposure delivered by a 300 kVp X-ray machine, using 300 kV, 20 mA, with added tiltration of 0.5 mm Cu and 1.04 mm Al (HVL 1.57 mm Cu), STD 50 cm, at an exposure rate of about 187 rad/min. All irradiation took place between 8 and 10 a.m. Radiation exposures were calibrated prior to each exposure with a 250 R Victoreen ionization chamber. 1177 Irradiation.
1178
N. E. Pa~rr
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M. SODICOFF
Electron microscopy. Female Sprague-Dawley rats (180 & 10 g) were seria!ly killed, 2 to 4 at each time interval, at 1, 3 and 5 hr; and at 1, 2, 3,4, 7 and 8 days following irradiation. The fixative of choice was half-strength Karnovsky’s combination of formaldehyde and glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 (KARNOVSKY,1965). Following a 15-min perfusion through the ascending aorta with room temperature fixative, the parotid gland tissue was removed, diced and fixed for an additional 5 hr in the same fixative. The tissue was then washed for 12 hr in several changes of cold (3-4C) 0.1 M phosphate buffer and post-fixed for 2 hr in cold 1 per cent osmium tetroxide buffered with 0.1 M collidine (pH 7.4). Embedding in Epon 812 followed routine dehydration of the tissue. Thin sections were cut on an MT-2 Porter-Blum ultramicrotome and placed on bare 300 mesh copper grids. The sections were stained with uranyl acetate and lead citrate and examined and photographed in an RCA EMU-4 electron microscope at an accelerating voltage of 50 kV. The original plate magnifications ranged between 3000 and 48,000 times. RESULTS 1 hour.
At this time, the acinar cells showed no obvious disturbances in morphology. Normal parotid morphology (Fig. 1) was similar to that described by SCOTT and PEASE (1959), PARKS (1961, 1962) and RUTBERG (1961). 3 hours. The appearance of most acinar cells was normal. However, in a very few of the cells, cytolytic bodies (a term used here to describe areas of cellular degeneration) were now found. Membranes around these areas of cytoplasmic degeneration were either absent or only partially complete. Figure 2 shows a typical cytolytic body within an acinar cell. The size of these structures varied from that of a nucleus to that of a secretory droplet. Appearances of cytolytic bodies also varied. Some were areas of cytoplasmic density with embedded secretory droplets, while others contained large amounts of rough endoplasmic reticulum, remnants of mitochondria and amorphous dense bodies. In addition to cytolytic bodies, other complex structures, here called light bodies, were also present. These structures were only sometimes membranebound. The light bodies had the following characteristics: (a) multiple nuclear fragments, each of which had a complete nuclear envelope, and the chromatin segregated so that the heterochromatin was separated from the euchromatin, frequently resulting in a crescent-form appearance; (b) rough endoplasmic reticulum arranged not only as parallel cisternae but also as concentric rings or whorls; and (c) ground cytoplasm which was often lighter than that of the acinar cell and contained many free ribosomes. Not all light bodies, however, displayed all the characteristics. Figure 3 shows a light body which is identified primarily by the occurrence of multiple nuclear fragments having crescent-form appearance. It is not enclosed by a membrane and appears to be free in the cytoplasm of the acinar cell. 5 hours. Although most acinar cells were normal, focal areas of acinar cell damage were present. Such areas were more numerous than at 3 hr, but the cytolytic bodies were similar in appearance. Light bodies were also present more frequently than before. Figure 4 shows a light body containing multiple crescent form nuclear fragments, each with an intact nuclear envelope and rough endoplasmic reticulum arranged as parallel cisternae and concentric rings. In Fig. 4, the light body is membrane bound and is completely within an acinar cell near its base. However, other light bodies appeared to be bulging from the basal surface of acinar cells but were not clearly separated from them. From the appearance of the light bodies seen at the 3 and 5 hr interval, it is not clear whether they represent separate cell types within an acinar cell,
EFFECTS
OF IRRADIATION
OF PAROTID
GLAND
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CELLS
1179
or rather represent areas of acinar cell degeneration in the form of autophagic vacuoles. Inflammatory cell types such as lymphocytes and macrophages were found degenerating within the acinus outside the acinar cell, but within the confines of the basement membrane (Fig. 5). 1 day. Evidence of cytoplasmic damage was abundant and widespread, although focally distributed (Fig. 6). Typical areas of acinar cells containing cytolytic bodies such as those seen at 3 and 5 hr were present. Membranes usually were not present around these structures. Necrotic cellular debris having some resemblance to light bodies was found within the acinar grouping. However, the advanced state of the degeneration made identification of the specific cell type difficult. Many acini were found wherein the entire cell cluster was in an advanced state of necrosis (Fig. 7). As seen at earlier times, typical light bodies were found, although some of these structures were found to contain secretory droplets (Fig. 8). In certain instances secretory droplets were found in the intercellular space between the light body, which already contained other secretory droplets, and the acinar cell, suggesting some form of transfer mechanism. Many light bodies appeared to be separate entities separated from acinar cells by an intercellular space (Fig. 8), but similar structures were also found within acinar cells and not surrounded by a continuous membrane (Fig. 9). Many lymphocytes and macrophages were found within the acini, covered by the basement membrane. 2 days. Areas of acinar cell destruction were widespread with large areas of necrosis and whole acinar cell clusters frequently degenerating. In cells showing less severe cytoplasmic damage cytolytic bodies similar to those seen at earlier periods were found. In addition, many light bodies of varied appearance were present within acini (Figs. 10 and 11). In some cases these light bodies seemed to resemble lymphocytes and macrophages. In Fig. 10, a light body resembling a lymphocyte but containing a crescent-form nucleus and abundant rough endoplasmic reticulum is nearly engulfed by a macrophage. In Fig. 11, the light body contains crescent-form nuclei and lymphocyte-like cytoplasm. Other light bodies were large, being at least the size of acinar cells. The overall general impression of this tissue was that it was greatly disorganized due to the shrunken and atrophic appearance of many, but by no means all, acinar groups. Intercellular spaces between acinar cells were frequently widened with cells appearing shrunken and angular. 3 and 4 days. Fewer cytolytic bodies were found within acinar cells at this time, although many unrecognizable degenerating cells resembling large cytolytic bodies were frequently found. Light bodies and inflammatory cells were found very infrequently at this time. This period was characterized by the initial appearance of large vacuoles, focally distributed, in many of the acinar cells. The vacuoles were similar to those seen at 7 and 8 days (Fig. 12) and varied considerably in size, as did the cytolytic bodies at this and earlier time intervals. Some vacuoles were larger than the nucleus. The vacuoles were frequently membrane-bound and their contents varied,
1180
N. E. PRATT AND M. SODICOFF
some appearing empty, some containing a light flocculent material, and others containing thin filmy membranes. 7 and 8 days. One notable difference at this interval, as compared to earlier times, is that the observed changes were widespread and not focal. The most obvious acinar cell derangement was the presence of multiple clear vacuoles in almost all of the acinar cells (Fig. 12). Vacuoles of various sizes were round and usually membrane bound. They were usually completely empty, but occasionally contained some filmy membraneous debris. Rarely were acinar cells found containing cytolytic bodies, nor were light bodies present. Acinar cells were often shrunken and gave the appearance of being angular. In summary, acinar cell damage was first detectable 3 hr following irradiation by the appearance of cytolytic and light bodies. The frequency of occurrence of these forms of cell damage increased with time and, by 2 days, tissue disorganization and atrophy of acinar groups was maximum. By 3 days, the cytolytic and light bodies were found less frequently and intracellular vacuoles began to be present. At one week following irradiation, the major indication of damage was the widespread appearance of severe vacuolization. DISCUSSION
From studies based upon light microscopic examination of paraffin sections, it has been stated that following irradiation, acinar cells of the rat parotid gland do not show the acute histopathological changes seen in the human parotid (ENGLISH, 1955; VAN DEN BRENK et al., 1969; SHAFER, 1953). Yet, more recently, atrophy, necrosis, degenerative nuclear changes and functional alterations have been described one day following irradiation with 4000 rad (PHILLIPS, 1970). Indeed, when paraffin sections of parotid tissue from the present study were examined by light microscopy, intracellular inclusions were easily observable by day 3 following irradiation, although more subtle evidence of damage was thought to be seen as early as 1 day. The inclusions were often of similar form to those seen by electron microscopy but stained with haematoxylin. They were, however, not distinguishable into cytolytic or light bodies and often gave the appearance of pyknotic nuclei. At 7 days, the acinar cells were obviously vacuolated. Previous investigators have not described such degenerative changes in the parotid so soon after such a low dose as 1600 rad. In the present study, which utilized the electron microscope to examine and assess the effects of radiation on the parotid gland, signs of acinar cell damage were noted as early as 3 hr following irradiation with only 1600 rad, making it appear that the response of the rat and human parotids may be more alike than previously thought. The present study did not concern itself with very subtle alterations in cellular organelles, but rather focused attention on more obvious signs of cytoplasmic and nuclear degeneration. It would therefore seem likely that more subtle changes may indeed be present at even earlier times following irradiation. Following 1600 rad, acinar cell damage seemed to take two forms: the cytolytic body and the so-called light body. The cytolytic body is a focal area of damaged
EFFECTS
OF IRRADIATION
OF PAROTID
GLAND
ACINAR
CELLS
1181
cellular organelles, most often composed primarily of damaged rough endoplasmic reticulum, but often including mitochondria, secretory droplets and unidentifiable dense bodies. The appearance of these structures is often badly distorted and recognition of specific structures is sometimes difficult, especially by 3 and 4 days. Membranes enclosing these structures are seldom complete. These cytolytic bodies are similar to those found in other irradiated tissues (GHIDONI and CAMPBELL,1969; WELLMAN,VOLK and LEWITAN,1966; HUGON and BORGERS,1966) which have been shown to be acid phosphatase positive (HUGON and BORGERS,1966). The cause of such cytoplasmic lesions could be the result of (a) direct damage to an organelle (WILLS, 1970) such as the endoplasmic reticulum, mitochondria, etc., followed by subsequent fusion with lysosomes, or (b) a primary insult to the lysosome, thus releasing hydrolytic enzymes (RENE,DARDENand PARKER,1971) to the cytoplasm for autodigestion. The second form of cytoplasmic damage consists of a structure referred to as the light body, which is more complex and more difficult to understand than the cytolytic body. In its most complex form, such cytoplasmic lesions are completely enclosed within an acinar cell and membrane bound. Nuclear material is fragmented, forming 2 to 4 micronuclei, each with an intact nuclear envelope and the nuclear contents segregated into dense and light chromatin, often giving a crescent appearance. Rough endoplasmic reticulum is well preserved, but often forms circles and concentric rings in fingerprint-like patterns. The preservation of the rough endoplasmic reticulum is often so good that its relationship to the nuclear envelope is quite clear. The background cytoplasm stains less intensely and is more abundant than that of the acinar cell and sometimes includes secretory droplets. Such structures within an acinar cell can be much greater in size than the nucleus itself. In its least complex form, the light cell may be a crescent-form nuclear fragment free in the cytoplasm of an acinar cell or a less dense cytoplasmic area with rings of rough endoplasmic reticulum. A possible interpretation is that these smaller structures will enlarge and/or fuse to form the larger and more complex bodies. Structures similar to the most complex form of the light body are frequently seen completely outside the acinar cell but the physical separation between acinar cell and light body is often not clear, consequently giving the impression that the light body is being extruded from the base of an acinar cell. Light bodies, even when completely separated from acinar cells, are rarely outside the basement membrane of the acinus. This proposed sequence of events, leading from the formation to the extrusion of an autophagic vacuole is similar to that proposed by GHIDONIand CAMPBELL(1969), but might actually be incorrect. Another interpretation of the sequence is that some other cell type, such as an inflammatory cell, has invaded the acinus and migrated inside the acinar cell finally to be digested (HUGONand BORGERS,1966), leaving behind the empty vacuoles seen at 7 and 8 days. Credence is lent to this latter hypothesis by the occurrence of some cell types which bear a resemblance to lymphocytes and macrophages by virtue of their size, shape and cytoplasmic contents. Yet these cells have the crescent-form nuclear material and abundant rough endoplasmic reticulum as do the light bodies. Indeed, multiple crescent-form nuclear fragments (LUCAS and PEAKMAN,1969; JORDAN,1967) and
1182
N.E. PRATTANDM. ~ODICOFF
increased amounts of rough endoplasmic reticulum (SPANGLER and CASSEN,1967) have been shown in irradiated lymphocytes. Truly, since only the head and neck were irradiated, we do not know which, if any, of the inflammatory cells were actually irradiated. Since the light bodies were usually only found within the acinus, it would seem less likely that these changes were caused by irradiation, but rather might have occurred in response to acinar cell damage. In summary, the structures called light bodies are puzzling and may represent the extrusion of autophagic vacuoles from acinar cells or the migration of inflammatory cells into acinar cells. Nuclear damage was seen only associated with light bodies. Nuclei of acinar cells always appeared normal even when large or numerous cytolytic or light bodies were present. Since the mitotic rate of acinar cells is extremely low (NOVI and BASERGA, 1971), it would seem unlikely that mitotically related nuclear damage would occur. However, since the origin of the nuclei of the light bodies remains questionable, the possibility of acinar cell nuclear damage cannot be excluded. The cytolytic bodies and light bodies made their first appearance at 3 hr and reached their greatest frequency of occurrence at 2 days. By the 3rd and 4th day, they were very infrequent and not present at all in the 7th and 8th day. Their distribution throughout the parotid tissue was spotty and never uniformly distributed. It is interesting to speculate that the empty and near empty vacuoles at 7 and 8 days may represent the end result of degradation and digestion of these inclusion bodies. However, the uniform distribution of these vacuoles throughout the tissue as compared to the focal distribution of the cytolytic bodies makes this explanation questionable and leaves us with the further speculation that the vacuole formation may be a separate event unrelated to the cytolytic bodies. Another possibility explaining the disappearance of cytolytic and light bodies is that those acinar cells with them totally degenerate and are finally cleared away by macrophages. From the available information, we cannot distinguish which of the possible mechanisms of resolution is occurring. Acknowledgement-We would like to express our thanks to Miss RUHDEAN SCOTTfor her excellent technical assistance. This research was supported by General Research Support Grant 5SOl-FR05417-10 and NIH Grant 1 R23 DE03256. R&sum&-Aprts irradiation par rayons X, a la dose de 1600 R au niveau de la tete et de la nuque de rats, les glandes parotides ont et& Btudi&es 1,2, 3,4, 7 et 8 jours apres pour Ctudier les lesions ultrastructurales des cellules acineuses. Des alterations sont dvidentes dts 3 heures, avec un maximum de destruction a 2 jours. Les lesions ultrastructurales prennent la forme de corps cytolytiques, constitur5es d’organelles cellulaires endommages et par des structures appelees corps claires qui peuvent rep&enter des vacuoles autophagiques, tinalement rejetees de lacellule, ou des cellules inflammatoires qui ont envahi la cellule. Au bout de 7 et 8 iours. l’alteration la nlus nette de l’architecture cellulaire est la presence de nombreusds vacuoles vides. Cette etude confirme la precocite des lesions a des doses aussi faibles que 1600 r. Zusammenfassung-Nach Rontgenbestrahlung mit 1600r auf Kopf- und Nackenpartie von Ratten wurden die Parotisdriisen 1,3, 5 Stunden und 1,2,3,4,7 und 8 Tage spater auf Anzeichen ultrastruktureller Schlden an den Azinizellen untersucht. Eine Schldigung war 3 Stunden nach Bestrahlung erkennbar, die maximale Destruktion wurde nach 2 Tagen beobachtet. Die ultrastrukturelle Schldigung trat in Form zytolytischer Korper
EFFECTSOF IRRADIATION OF PAROTIDGLANDACINARCELLS
1183
aus zerstorten Zellorganellen und unter dem Bild von Struckturen, die als “light bodies” bezeichnet wurden, auf, wobei letztere Autophagen-Vacuolen darstellen, die schliel3lich von der Zelle abgestoaen werden oder mit entziindlichen Zellprozessen zusammenhlngen. Nach 7 bis 8 Tagen ist die am meisten vorherrschende zellulare Verlnderung das Vorhandensein zahlreicher leerer Vakuolen. Die Untersuchung demonstriert die frtihe Nachweisbarkeit von Schlden bei so niedrigen Strahlendosen wie 1600 r.
REFERENCES CHERRY,C. P. and GLUCKSMANN,A. 1959. Injury and repair following irradiation of salivary glands in male rats. Br. J. Radiol. 32. 596-607. ELZAY, R. P., LEVITT, S. H. ~~~‘SWEENEY,W. T. 1969. Histologic effect of fractionated doses of selectively applied megavoltage irradiation on the major salivary glands of the albino rat. Radiology 93, 146-152. ENGLISH,J. A. 1955. Morphologic effects of irradiation on the salivary glands of rats. J. dent. Rex 34,4-II. GHIDONI, J. J. and CAMPBELL,M. M. 1969. Karyolytic bodies. Archs Path. 88, 480488. HUGON, J. alId BORGERS,M. 1966. Ultrastructural and cytochemical studies in the epithelium of the duodenal crypts of whole body X-irradiated mice. Lab. Invest. 15, 1528-1543. JORDAN,S. W. 1967. Ultrastructural studies of spleen after whole body irradiation in mice. Exptl molec. Path. 6, 156-171. KASHIMA,H. K., KIRKHAM,W. R. and ANDREWS, J. R. 1965. Postirradiation sialadenitis. Am. J. Roent. 94, 271-291. KARNOVSKY,M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. cell. Biol. 27, 137-338A. LUCAS, D. R. and PEAKMAN,E. M. 1969. Ultrastructural changes in lymphocytes in lymph nodes, spleen and thymus after sublethal and supralethal doses of X-rays. J. Path. 99, 163-169. NOVI, A. M. and BASERGA,R. 1971. Association of hypertrophy and DNA synthesis in mouse salivary glands after chronic administration of isopreteranol. Am. J. Path. 62, 295-308. PARKS,H. F. 1961. On the fine structure of the parotid gland of the mouse and rat. Am. J. Anat. 108, 303-329. PARKS,H. F. 1962. Morphological study of the extrusion of secretory materials by the parotid glands of mouse and rat. J. ultrastruct. Res. 6,449-465. PHILLIPS, R. M. 1970. X-ray-induced changes in function and structure of the rat parotid gland. J. oral Surg. 28, 432-437. RENE, A. A., DARDEN, J. H. and PARKER, J. L. 1971. Radiation-induced ultrastructural and biochemical changes in lysosomes. Lab. Inuest. 25, 230-239. RUTBERG,U. 1961. Ultrastructure and secretory mechanism of the parotid gland. Acta odont. stand. 19, Suppl. 30, l-69. SCOTT,B. L. and PEASE,D. C. 1959. Electron microscopy of the salivary gland and lacrimal glands of the rat. Am. J. Anat. 104,115-161. SHAFER,W. G. 1953. The effect of single and fractionated doses of selectively applied X-ray irradiation on the histologic structure of the major salivary glands of the rat. J. dent. Res. 32,796806. SPANGLER,G. and CASSEN,B. 1967. Electrophoretic mobility, size distribution and electron micrograph responses of lymphocytes to radiation. Rad. Res. 30,22-37, VAN DEN BRENK, H. A. S., HURLEY,R. A., GOMEZ. C. and RICHTER.W. 1969. Serum amvlase as a measure of salivary gland radiation damage. Br; J. Radiol. 42, 688-700. WELLMAN.K. F.. VOLK. B. W. and LEWITAN.A. 1966. The effect of radiation on the fine structure and enzymecontent of the dog pancreas.‘Lab. Invest. 15, 1007-1023. WILLS, E. D. 1970. Effects of irradiation on sub-cellular components-I. Lipid peroxide formation in the endoplasmic reticulum. ht. J. Radiat. Biol. 17, 217-228.
N.
1184
E. PRATT AND M.
PLATE
SODICOFF
1
FIG. 1. Typical parotid acinar cell from a normal rat showing the characteristic basally located flattened rough endoplasmic reticulum and apically located dense secretory droplets. x 8800
FIG. 2. Three hours post-irradiation. A typical cytolytic body contains damaged rough endoplasmic reticulum (arrow), dense bodies (D) and other membraneous material (M). x25,000
FIG.
3. Three hours post-irradiation. Simple form of a light body containing crescentform nuclear fragments (N) and rough endoplasmic reticulum arranged in parallel stacks. This structure appears only partially membrane bound. x 27,000
FIG. 4. Five hours post-irradiation. A complex form of a membrane-bound light body is present within an acinar cell. Rough endoplasmic reticulum is arranged in both stacks and whorls, and multiple crescent-form nuclear fragments (N) are present. Cytolytic bodies can be seen both within the light body (large arrow) and within the acinar cell (small arrow) where it contains a secretory droplet (S). x 12,800
A.O.H.
I’l.ATl I I183
f.p.
PLATE 2
N. E. PRATTAND M. SODICOFF
1185
PLATE2 FIG. 5. Five hours post-irradiation.
A degenerating lymphocyte cells within an acinus. x 13,300
is seen between acinar
FIG. 6. One day post-irradiation.
A thick section (1 pm) showing the focally distributed damage as seen by the light microscope. The arrows are pointing to areas of damage which resemble those structures called cytolytic and light bodies as seen with the electron microscope. x 1000 FIG. 7. One day post-irradiation. An acinar cell cluster in an advanced degeneration. x 8200 FIG. 8. One day post-irradiation.
stage of
Typical light bodies, containing nuclear fragments (N), whorls of rough endoplasmic reticulum and secretory granules (S) are seen within an acinus both separated from (large arrows) as well as within (small arrows) acinar cells. x 7900
A.O.B.
17/8--e
1186
N. E. PRATTAND
M. SODICOFF
PLATE3 Fro. 9. One day post-irradiation. A typical light body having no membraneous enclosure is seen. A cytolytic body (C) also is present in this acinar cell. x 15,000 FIG. 10. Two days post-irradiation. Within an acinus, a lymphocyte-lie containing a crescent-form nucleus (N) and abundant rough endoplasmic nearly engulfed by a macrophage (M). x 11,600
light body reticulum is
FIG. 11. Two days post-irradiation. This light body contains multiple crescent-form nuclei (IV) and lymphocyte-like cytoplasm. x 21,700 FIG. 12. Eight days post-irradiation.
Multiple vacuoles (V) are found in these acinar cells. x 6700
A.O.B.
PLATE 3 I_p. 1186