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Pathologic changes in the lung following single and multi-fraction irradiation
Inf. J. Radiotion
Oncobgy
Biol.
Phys.,
1977,
Vol. 2. pp. 475-490.
Pergamon Press.
Printed in tbe U.S.A
0 Original Con tribu tion PATHOLOGIC CHANGES IN THE LUNG FOLLOWING SINGLE AND MULTI-FRACTION IRRADIATIONt ELIZABETH L. TRAVIS, Ph.D.S, RUSSELL A. HARLEY, M.D.“, JIMMY 0. FENN, M.S., CHRISTOPHER J. KLOBUKOWSKI, M.S. and HENRY B. HARGROVE, B.S. Department of Radiology, Division of Basic Radiation Sciences and, “Department of Pathology, Medical University of South Carolina, Charleston, SC 29401, U.S.A. The lbniting factor in the treatment of malignant disease with irradiation is the tolerante of normal tissue irradiated. In the present study the right lungs of rats were exposed to single doses of 2000 rad of X-radiation, to 10 x 200 rad, or to 5 X 400 rad. Anhnals from each group were sacriiiced monthly for 6 months post exposure. Sections of lung were examined by light microscopy (LM) and by scanning or transmission electron microscopy (SEM and TEM). A focal exudative lesion was seen at 2 months after the single dose; it progressed to a proliferative and then reparative, fibrotic lesion by 6 months. Changes in epithelial lung components, particularly the presence of Type 11 pneumocytes, were found with both LM and TM. VascuIar changes were less pronounced. A strilchtg Sndiig was the presence of mast cells in the alveolar wak. Neither of the multi-fraction schedules produced any of these changes, except hyperplasia of Type 11 celIs following 5 X 400 rad. The possible bnplication of Type 11 and mast cells in radiation pneumonitis and fibrosis is discussed. Radiation,
Lung, Multiple fractions,
Late injury, Mast cells, Type 11 pneumocytes.
INTRODUCTION The parenchymal cells of the lung may be considered relatively radioresistant, but the lung, as an organ, is radioresponsive and, in radiotherapy terminology, does not tolerate substantial doses of irradiation. In the treatment of many malignant neoplastic diseases such as Hodgkin’s disease and esophageal carcinoma, the normal lung is often the doselimiting factor in the radiotherapy treatment plan. Radiation pneumonitis is a clinical entity that has been described often; it may progress to radiation fibrosis, resulting in tPresented in part at the 1975 Annual Meeting of the American Society of Therapeutic Radiologists, San Francisco, Calif. (1975). $Present address: Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex 6HA 2RN, England. Reprint requests to: Mr. J. 0. Fenn, Directer,
patient morbidity and/or mortality.3.~‘1.26,33 For this reason, delineation of those factors which play a participating role in this response are important. Studies in patients and animals have resulted in extensive descriptions of the pathologit changes in irradiated lungs. Warren and review of Spencer ,33 in a comprehensive patients who had received thoracic irradiation, emphasized hyaline membrane formation as a primary feature of radiation peneumonitis and proposed the alveolar epithelium and capillary endothelium as the Basic Radiologie Sciences Division, Medical University of South Carolina, Charleston, SC 29401, U.S.A. Acknowledgements-The authors express their appreciation to Mrs. E. Setser and Mr. W. B. Greene for assistance with electron microscopy. 475
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primary sites of injury. Subsequent studies only, excluding the mediastinum, using a generally have focused on one of these areas single anterior field measuring 4 x 4 cm. The resulting in numerous theories concerning the remainder of the body was shielded with a primary mechanism(s) of radiation injury in cerro-safe shield measuring 10 x 12 x 2 cm the lung, including vascular damage,‘,‘2,18,19,23.33with a 4 x 4 cm cut-out in one side to allow damage to Type 1 and 11 pneumocytes3.6.32 irradiation of the right lung only. This techinvolvement of lymphatic channels draining nique allowed the left lung to serve as an internal control in each animal for pulmonary the lungs,3.28 and an immune response.‘* infection. Only one animal was excluded for However, the site of the radiation induced lesion stil1 remains controversial. this reason. Positioning of the radiation portal In the present 6 months study, the right was checked periodically with radiographs. Irradiation was administered using a GE lungs of rats were exposed either to a single Maxitron deep therapy unit, having a half dose of radiation which produced a progressive, fibrosing lung lesion or to two multivalue layer (HVL) of 1.6 Cu, operated at fraction schedules with daily fraction sizes 250 kV, 30 mA, with added Thoraeus 111filter at a target skin distance of 30 cm. Output of comparable to those used in radiation therapy. It was hoped that these methods the unit under these conditions was 195 rad would assist in identifying those common per min. Utilizing depth dose tables, back factors between fibrosing and non-fibrosing scatter factor, and the appropriate roentgen irradiated lungs as wel1 as those factors to rad conversion factor, the dose rate in rad which differ between the two. to the lung tissue at midline was calculated at 186rad per min. METHODS AND MATERIALS Immediately following irradiation, al1 animals were monitored carefully for recovery Animals from anesthesia. The animals used throughout this study were male Sprague Dawley rats (Zivic Miller Autopsy Laboratories, Inc., Allison Park, PA) Two animals from each irradiation group weighing between 175 and 200g. They were and one control animal were killed monthly kept in a controlled environment and fed for 6 months by ether anesthesia. While the standard rat chow and water ad libitum. animal was stil1 breathing, the thorax was opened and the lungs perfused through the Irradiation procedure trachea with a 2% gluteraldehyde solution The animals were divided randomly into buffered to pH 7.2 with 0.1 molar (M) four groups according to the following radiFollowing permonosodium cacodylate. ation schedule: fusion, the trachea was tied, the lungs subsequently removed from the thorax and fixed Group 1: 2000 rad; delivered in a single overnight in 10% neutral buffered formalin. dose; Sections from the right and left hing were Group 2: 2000rad; delivered in 400rad fixed by the appropriate technics and exdaily fractions for 5 consecutive amined by light microscopy (LM), transdays: mission electron microscopy (TEM), and Group 3: 2000 rad; delivered in 200rad scanning electron microscopy (SEM). Al1 daily fractions for 10 consecutive tissues excluding those for TEM were stored days, excluding weekend; in 10% buffered formalin. Group 4: Sham irradiation. At autopsy both lungs were carefully exAl1 animals were anesthetized prior to ir- amined for gross pathologie changes sugradiation with an intraperitoneal injection of gestive of pulmonary infection. When an sodium pentobarbital (Elkins-Sinn, Inc.) at a infection was suspected, sections were reconcentration of 45 mglkg. Each animal was moved from this and uninvolved areas and carefully examined microscopically. Any irradiated individually to the right thorax
Radiation induced pathology of lung 0 E. L. TRAVIS et a/.
animal found to have a pulmonary infection in either the right or left lung was excluded from the study. The criteria used was the presence of leukocytes in the irradiated and/or unirradiated lung; only one animal was excluded. Tissue preparation
Tissues fixed overnight in formalin were embedded in paraffin and 5 p sections cut with a microtome. Al1 sections were evaluated using the following stains: hematoxylin and eosin, Gordon and Sweet’s method for reticulin, and Masson’s trichrome for the demonstration of collagen. Scanning
electron microscopy
Tissues fixed in formalin were cut into 3 x 3 mm tubes, dehydrated in graded alcohols, critical point dried, mounted on stubs, gold coated in a vacuum evaporater and examined using a Hitachi Scan Scope.TM Transmission
electron microscopy
Specimens for TEM were removed from the lung immediately after intratracheal perfusion with gluteraldehyde and prior to formalin fixation of the lungs. The tissue was minced quickly in cold 2% gluteraldehyde buffered to pH 7.2 with 0.1 M monosodium cacodylate solution. The tissue was fixed in the refrigerator for 16-18 hr in this same solution and subsequently rinsed thoroughly with sucrose buffer. Tissues were post-fixed for 1 hr in 2% osmium tetroxide buffered to pH 7.2 with 0.1 M monosodium cacodylate, and subsequently embedded in Epon. Thick sections were stained with toluidine blue, examined under the light microscope, and areas selected for thin sectioning. Ultra-thin sections were doubly stained with uranyl acetate and lead citrate and examined with an AEITM Electron Microscope. Areas examined on al1 sections included those within, adjacent to, and distant from the radiationinduced lesion. RESULTS Controls
The gross appearance of both the right and left lung was normal in al1 animals except one
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in which focal, patchy, green nodules were noted on both lungs. Light microscopy revealed the presence of an acute necrotizing pneumonia in this animal. Normal histology and morphology was observed in the right and left lungs of the remaining control animals with al1 technics. An occasional ultrastructural finding in blood vessels of the normal lung was vesiculated capillary endothelium. 2000 Rad Gross changes in the right lung were not evident prior to 2 months post-exposure. At two months the right irradiated lung exhibited focal, pale, patchy areas, but was not resistant to expansion. These changes progressed with time, the irradiated lungs becoming shrunken, firm, and resistant to expansion by 6 months. In al1 cases, the non-irradiated left lung was grossly normal except for compensatory hypertrophy at 6 months. Light microscopy revealed minima1 focal perivasculitis and perivascular edema at 1 month post-exposure. Tissues examined at 2 months post-exposure revealed several discrete, subpleural, exudative lesions exhibiting an eosinophilic, proteinaceous material in the alveolar spaces, mild interstitial edema, and an intra-alveolar infiltrate of monocytic cells (Figs. 1A and B). The cellular infiltrate consisted of two populations by staining characteristics; one population exhibited a large basophilic nucleus with extensively vacuolated eosinophilic cytoplasm, the second group, although similar to the one previously described, exhibited a basophilic cytoplasm with smaller, less numerous vacuoles. Vascular alterations were less pronounced, primarily consisting of adventitial edema. An increased area of the lung was involved by the exudative lesion at 3 months, the response now becoming multi-focal in nature. The histologie characteristics of these lesions were similar to those observed at 2 months with the exception of an increased number of cells in the alveolar spaces. Alterations were present in other lung constituents as well. The interstitial area was loosened and widened, exhibiting a mild cellular infiltrate of plasma cells. Large, atypical Type 11 pneu-
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Fig. 1. (A) Rat lung exposed to 2000rad and autopsied at 3 months post-exposure exhibiting the focal nature of the early lesion and the exudative, desquamative histologie characteristic with marked intra-alveolar edema. Hematoxylin and eosin, A: x60.
mocytes were attached to the alveolar walls. The nature of the vascular alterations were consistent with those observed at 2 months although more vessels were involved. In addition, perivascular plasma cel1 cuffing was prominent. A second proliferative component of the lesion was demonstrated at this time. Reticulin stain revealed increased reticulin
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Fig. 1. (B) High magnification of area in box in A. Large vacuolated cells are clearly visible in the alveolar spaces. Hematoxylin and eosin, x300.
focally deposited throughout those alveolar walls involved by the exudative lesion. Collagen was not demonstrated. The histologie characteristics of the response changed between 4 and 6 months postexposure. Although bizarre giant cells were present in the alveolar space, the exudative lesion became less dominant (Fig. 2). Multifocal interstitial areas of the lung exhibited progressive loosening and widening in ad-
Fig. 2. At 6 months following a single dose of 2000 rad the lesion exhibits loosened, widened alveolar walls and patchy, fibrotic areas. Hematoxylin and eosin, ~96.
Radiation induced pathology of hg
dition to a marked cellular infiltrate (Fig. 3). The primary component of the infiltrate consisted of plasma cells with lymphocytes occasionally observed. Fibroblasts, not readily identifiable in the interstitial areas of normal lung, were observed frequently in those interstitial areas exhibiting alterations. Although normal appearing Type 11 cells were attached to the alveolar walls, bizarre, hypertrophied Type 11 cells were evident. In
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areas exhibiting atelectasis, the alveolar walls were denuded of epithelial cells. Vascular alterations consisting of prominent adventitial edema and marked vessel dilatation were evident in the lesion and adjacent areas from 3 to 6 months. Leukocytic pavementing was observed occasionally in relatively few blood vessels. Reticulin proliferation progressed, becoming more diffusely distributed throughout the lung (Fig. 4). Col-
Fig. 3. Another area of the 6 month lesion exhibiting marked interstitial edema and chronic inflammatory cel1 infiltrate. Hematoxylin and eosin, x300.
Fig. 4. Reticulin stain points out the proliferative nature of the 6 month lesion with increased reticulin present in the alveolar walls (hollow arrow). This also illustrates edema of the adventitial layer of a blood vessel (solid arrow). Gordon & Sweet’s reticulin, x300.
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lagen was also observed in focal areas of the alveolar walls at 4 months, becoming more diff usely located throughout the alveolar walls and pleura with increasing post-exposure times. Normal lung architecture was obliterated in multi-focal areas at 6 months post-exposure, while other areas appeared relatively normal. The alveolar walls in these latter areas were partially denuded of epithelial cells, ischemic, and appeared fragile, as evidenced by the presence of fragmented, tortuous alveoli. As at previous post-exbosure times, the entire lung was not involved with these latter phases of the response and normal lung tissue remained. The response of the lung at 6 months can best be described as a patchy, multi-focal proliferative response with patchy fibrosïs. The left unirradiated lungs appeared normal and were consistent with controls throughout the study (Fig. 5). NO animals exhibited microscopic evidente of pulmonary infection. SEM revealed the focal nature of the early lesion with fibrin deposition in the alveolar spaces and cells entrapped in this fibrin framework (Fig. 6B and C). At 6 months, the lesion was multi-focal and consolidated, exhibiting thick fibrin branches with large
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numbers of cells entrapped in the fibrin (Fig. 6D). TEM confirmed the alterations observed with light microscopy. At 3 months postexposure, vascular changes in the lesion consisted primarily of marked dilatation, this change becoming less obvious in areas adjacent to the lesion with normal vasculature observed in areas distant from the lesion. Moderate basement membrane edema was observed and the endothelium, although edematous, was attached to the basement membrane with no vesiculation. Many areas of the lesion exhibited patent capillaries; no attempt to count the patent capillaries was made (Fig. 7A). Edema of the alveolar walls was striking and increased interstitial collagen was observed. Although Type 11 cells appeared decreased in number, the number of Type 1 cells appeared unchanged. A particularly interesting observation was the presence of mast cells in the alveolar walls, which were often found in intimate association with interstitial cells containing a large amount of rough endoplasmic reticulum (RER) therefore resembling fibroblasts (Fig. 7B). Two populations of cells were observed in the alveolar spaces consistent with the light microscopic findings. The predominant cel1
Fig. 5. Left lung of experimental animal exhibiting histology animal. Hematoxylin and eosin,
consistent
x120.
with that of a control
Radiation induced pathology of lung 0 E. L. TRAVISet al.
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0
--.
(Dl
Fig. 6. Scanning electron micrographs of control and irradiated rat lungs. (A) normal lung. (B and C) 2000 rad, 3 months post-exposure, exhibiting fibrillar material in the alveolar spaces and cells with villous projections resembling microvilli. These cells are compatible with the SEM appearance of foamy macrophages. (D) 200rad, 6 months post-exposure showing proliferation of the fibrillar material in the alveolar spaces and complete obliteration of normal lung architecture. A: x240, B and C x 600, D: x 1200.
Fig. 7. (A) A patent capillary with intact endothelium (arrow) and containing an erythrocyte (E) seen in the rat lung exposed to a single dose of 2OOOrad and autopsied at 3 months post-exposure. UALC, x 10800.
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Fig. 7. (B) Edematous alveolar wal1 from an animal exposed to 2000rad at 3 months post-exposure with a clear aveolar space on one side (top) and debris filled alveolar space on the other (bottorn). A mast cel1(smal1arrow) is present in the interstitial area associated with a cel1 containing prominent RER (large arrow). A clear vascular space (B.V.) is present directly above the mast cel1 with normal endothelium. UALC, ~5274. was large, with numerous microvilli and lipid and glycogen-containing cytoplasmic vacuoles. The other cel1 observed, although morphologically similar to the previously described cell, exhibited larger vacuoles and myelin bodies in the cytoplasm. Occasionally this cel1 appeared to be in the process of detaching from the alveolar wall. Morphological changes indicative of degeneration were observed in both cells. The alveolar spaces were filled with an electron dense material. Al1 of the above described changes were less evident in areas adjacent to the lesion. Lung architecture was normal in areas distant from the lesion. At 6 months post-exposure normal lung architecture was obliterated in multi-focal areas. Vascular changes in the lesion were not consistent, ranging from severe basement membrane edema and sloughing of the ento mild basement membrane dothelium, edema with intact endothelium. Vessels often were markedly dilated but occlusion was not found. As at three months, vascular changes were less striking than alterations in other lung components; patent capillaries were present in the involved areas (Fig. 8). Alveolar walls were markedly widened with increased interstitial collagen. Parti-
cularly striking was atypical hypertrophy observed in both types of alveolar epithelial cells. The nucleus of the hypertrophic Type 1 cells was large with a prominent nucleolus (Fig. 9A). Hypertrophic Type 11 cells exhibited cytoplasmic lamellar bodies (Fig. 9B). Increased fibrin formation was observed in the alveolar spaces with foamy macrophages phagocytosing this material. The active interstitial cells noted at 3 months post-exposure were now bizarre, exhibiting a large amount of RER, a large, sometimes more than one, nucleus, and a prominent nucleolus (Fig. 10). These cells may represent the atypical “radiation fibroblasts” often described at the light microscopic level. The most consistent unusual finding in this tissue was the large numbers of mast cells in the alveolar walls. Very often these cells were found in intimate association with the previously described atypical fibroblastoid cell. Increased collagen formation often was evident in this same area (Fig. 11). The left unirradiated lung was normal with the exception of occasional vascular alterations consisting of endothelial blebs and swelling. 400 Rad x 5 Grossly the lungs of these
animals
ap-
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Fig. 8. Patent capillary (B.V.) in the lung of a rat exposed to 2000rad at 6 months post-exposure. Endothelial lining (arrow) is intact as is the epithelium facing an alveolar space (AS). Cel1 in the capillary is unidentified. UALC, x10350.
Fig. 9. (A) Atypical hypertrophic Type 1 cel1 attached to the alveolar wal1 of an animal exposed to 2000 rad at 6 months post-exposure. Note the large prominent nucleus and smal1 amount of cytoplasm. UALC, x6690. peared normal. Light microscopic examination of the tissue revealed no lesions and no increased collagen or reticulin. SEM failed to
reveal any lesions. TEM revealed subtle changes in lung morphology not evident with the other technies utilized, involving both alveolar pneumocytes and vasculature. A striking change at 3 months post-exposure was hyperplasia (“twinning”) of Type 11 pneumocytes (Fig.
12). NO cel1 membrane was observed between these cells, but more than one nucleus was present. Although Type 11 cells were diminished in numbers, no changes were observed in Type 1 pneumocytes. The endothelium and basement membrane of blood vessels were intact with minima1 swelling observed. However, cytoplasmic vacuolation was observed occasionally in endothelial cells of a few blood vessels (Fig. 13A).
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Fig. 9. (B) Atypical Type 11 pneumocyte (hollow arrow) attached to an alveolar wal1 containing a mast cel1 (solid arrow) in the interstitial area in close relationship to collagen (Co). Dose-2000 rad; Timemonths post-exposure. UALC, x3150.
Fig. 10. Transmission electron micrograph of atypical bizarre fibroblastoid cel1 often observed in the alveolar walls of animals exposed to 2000 rad at 6 months post-exposure exhibiting a large nucleus with prominent nucleolus. The round cel1 is a lymphocyte. UALC, x6336.
At 6 months post-exposure, a diminished number of Type 11 cells were observed as compared to both control lungs and lungs examined at earlier post-exposure times. An occasional foamy macrophage was observed in the alveolar spaces. Edema fluid was observed in blood vessels at this time with the previously described changes in endothelial
cells also observed. It must be pointed out that the blood vessel changes were not as consistent or as frequent as the changes in Type 11 pneumocytes. Mast cells were observed only in their normal location, i.e. in the pleura and in the adventitial layer of blood vessels and bronchioles, and never were in the alveolar walls
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Fig. ll. A mast cel1 intimately associated with an atypical fibroblastoid cel1 in the interstitial area of lung after a dose of 2000 rad at 6 months post irradiation. An erythrocyte (E), collagen (Co), and partially occluded vascular space (VS) are also present. UALC, x4800.
Fig. 12. Hyperplastic
Type 11 cells observed at 6 months following multi-fraction schedule. UALC, x.5400.
in animals exposed to this multi-fraction schedule during the time of the study. The appearance of the left unirradiated lung was consistent with that in both control animals and the left lung of animals exposed to the single high dose. The vascular changes previously described in the left lung also were noted in the left lungs of these animals with the same frequency.
the 400rad X 5
200 Rad x 10 Minima1 changes were observed in the lungs of animals exposed to this dose fractionation schedule. Grossly, the lungs appeared normal and no lesions were observed by LM or SEM throughout the time of the study. At no time was any evidente of fibrosis observed. Particular attention was directed to the
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(Bl Fig. 13. Patent capillaries with intact endothelium in lungs of animals exposed to the 400 rad x 5 (A) and 2000 rad x 10 (B) multi-fraction schedules at 6 months after irradiation; clear alveolar spaces are present in both. (A) UALC, x7200; (B) UALC, x12600.
morphology of Type 1 and 11 pneumocytes and blood vessels using TEM. Although no changes were observed in the alveolar epithelial cells at any time, an occasional finding at 6 months post-exposure was swelling of the basement membrane of blood vessels and cytoplasmic vacuolation of endothelial cells (Fig. 13B). These findings also were observed in the left, unirradiated lung with the same frequency.
Mast cells were not observed in the alveolar walls of either the right or left lung during the time of this study. DISCUSSION Numerous studies have been performed investigating the response of the lungs following single doses of radiation which produce a progressive pneumonitis culminating in fibrosis. One of the difficulties en-
Radiation induced pathology of lung 0 E. L. TRAVIS et al.
countered using such doses is that al1 components of the lung exhibit profound changes, therefore rendering interpretation of these changes as they relate to the pathogenesis of radiation pneumonitis difficult at best. The single fraction used in this study produced three phases of response in the lung: exudative (early), proliferative (intermediate), and reparative (late), occurring at 2-4 months, 3-6 months and 4-6 months respectively. NO definitive time marked the progression of the response from one phase to another; rather, a gradual change was observed with histologie features of the preceding phase becoming less dominant as the response progressed to the next phase. This progressive response was consistent with that reported by others following this same single but dose,‘9V*3*‘ 2 contradictory to the findings of one author who reported resolution of the early pneumonitis phase within four months following a single dose of 2000 rad to the right thorax.35 In many ways, the histologie characteristics of al1 phases of the response were consistent with other studies of the response of the lungs following irradiation, i.e. interstitial and intra-alveolar edema, bizarre epithelial cells, alterations in reticulin and increased interstitial collagen. These are non-specific findings and have been observed in the lungs following bleomycin,zX17 busulfan,‘] and paraquat5.13 administration, al1 agents which produce interstitial pneumonia and pulmonary fibrosis. One significant histologic differente in our study as compared to others was the infrequent observation of uccluded blood vessels and the presence of patent capillaries in areas of the irradiated lung exhibiting histologie changes indicative of damage. These findings suggest that vascular damage was less in our study than that reported by some authors’,23,33 but consistent with the observations of others.6.1’.12.30.32 Our findings are also consistent with the response of the lungs following other non-infectieus insults.2,‘7,2’ Although vascular alterations varied throughout the specimen with the severity of the lesion, in general, vascular changes were not as striking as the changes seen in other lung constituents, even in those areas exhibiting severe lesions.
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Ultrastructural examination of lungs exposed to the single fraction of radiation in this study supported the light microscopic observations, with vascular changes the less profound. Patent capillaries were often observed in edematous and fibrosing alveolar Although endothelial cells were walls. hypertrophic and basement membrane edema was present, vascular occulusion was not a common finding. In the two multifraction schedules tested in this study, there was minima1 ultrastructural evidente of vascular damage, consisting of mild basement membrane disruption. The observation of these same vascular changes in the left lungs of al1 experimental and control animals raises the question of whether the vascular changes noted, particularly in the right lung of animals exposed to both fractionation schedules, are indeed radiation-induced. These vascular lesions are consistent with those in lungs of animals sacrificed with ether, the method of sacrifice employed in this study.’ For this reason the cause of the vascular lesions in animals exposed to both fractionation schedules cannot, be attributed to radiation with certainty. The increased frequency of vascular lesions in animals exposed to the single fraction clearly suggests that they are radiation-induced; however, the characteristics of these lesions were not significantly different from those observed in the lungs of animals that were exposed to either multi-fraction schedule. Although vascular damage is suggested most often as the primary pathogenesis of radiation injury in the lung, our study did not support this theory fully. Alternate potential mediators of radiation pneumonitis suggested by the present study are the epithelial lung components, i.e. Type 1 (membranous) and Type 11 (granular) pneumocytes. The normal turnover time of these cells is 8 days for Type 1 pneumocytes and 27 days for Type II.4~27~29 For this reason, these cells may manifest radiation injury after exposure to a single fraction as used in this study and may reflect changes following the higher fractionation schedule. In the present study, the number of Type 11 cells were greatly diminished in the single fraction following the group, slightly decreased Y
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400 rad multi-fraction schedule, and unaffected by the 200 rad fractionation schedule. The loss of Type 11 cells in the single fraction group increased with increasing post-irradiation time. The observation of large cells with numerous vacuoles containing glycolipid materials and the presence of electron dense material in the alveolar spaces suggested that Type 11 cells were in the process of desquamation from the alveolar wal1 with subsequent degeneration in the alveolar spaces. However, the morphology of the cells present in the alveolar spaces also was suggestive of foamy macrophages, particularly since these cells wil1 phagocytose cellular debris. Although foamy macrophages are not an uncommon finding in rat lungs, particularly as the animal ages, these cells only occasionally were observed in the lungs of control animals, in the left unirradiated lung of experimental animals, and in the right lungs of animals exposed to the 200rad fractionation schedule. They were most predominant in the single fraction group and frequently observed in the group exposed to the 400 rad multi-fraction schedule. A common factor between lungs exposed to a single and fractionated dose of radiation was hyperplasia of Type 11 cells. This phenomenon is not unique to radiation injury of the lung and has been reported following other lung insults, including nitrogen dioxide36 and oxygen’3.‘4*25 exposure and carbon tetrachloride’ and monocrotaline’5 administration. It has been postulated that following severe injury which denudes the alveolar walls of both types of pneumocytes, proliferation of Type 11 pneumocytes functions as a regenerative response in an effort to repopulate the naked alveolar walls with epithelium.* Because of their role in synthesizing surfactant, the material in the lung which stabilizes the alveoli, a loss of these cells or changes in their normal morphology could alter lung function profoundly.‘6’2 Depopulation of Type 11 cells could result in a loss of surfactant leading to transudation of fluids across the alveolar walls and subsequent atelectasis, a response in the lung generally attributed to vascular injury. Morphologic abnormalities in Type 11 cells could result in
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abnormal surfactant with the same sequentia1 changes. Faulkner and Connolly” described changes in the ultrastructure of Type 11 cells consisting of large lamellar bodies containing dense, coarse material and abundant rough endoplasmic reticulum, giving these cells an immature appearance. Because the lamellar bodies store the surfactant prior to extrusion from the Type 11 pneumocyte into the alveolar spaces, changes such as these could reflect altered surfactant production. Indeed, in a study of pulmonary lipids following lung irradiation, Pflegler et aLz2 and Pickerell et aLz defined alterations in the lipid composition of surfactant which potentially support the role of Type 11 pneumocytes in the pathogenesis of radiation pneumonitis. The finding of bizarre, atypical Type 11 pneumocytes in the lungs of animals exposed to the single fraction used in this study as wel1 as the diminished numbers and hyperplasia of these cells in animals exposed to the single fraction and the 400 rad fractionation schedule, coupled with the lack of vascular changes in the fractionated group, further suggest that Type 11 pneumocytes, if not fully responsible for radiation pneumonitis, certainly contribute to the pathogenesis of this response. Changes in Type 11 pneumocytes have been consistent findings in both experimental and clinical radiation pneumonitis with the exception of one study.’ The multi-focal nature of the observed lesions may also be attributable to the response of Type 11 pneumocytes. Although generally attributed to variations in response of the vasculature, the idea also must be entertained that this lesion is a response of the epithelial components of the lung and that the multi-focal characteristic is a reflection of a perturbation of the kinetics of this population of cells following irradiation. Although the entire irradiated lung was not compromised at any time by any phase of the response, it cannot be stated with certainty whether the reparative response wil1 proin diffuse pulmonary gress, culminating fibrosis. The single, most distinguishing feature in lungs undergoing fibrosis as compared to nonfibrosing lungs in this study was the presence
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of mast cells in the alveolar walls. These cells have been reported by others in irradiated lungs and it has been suggested that they are involved in the reparative phenomenon. Phillipsz3 observed an increased mast cel1 infiltrate from 3 to 6 months following a single dose of 2OOOrad to the lung whereas Watanabe et aL3’ only observed this response following a dose of 3000rad. Faulkner and Connolly” also observed mast cells in the interstitial areas of the lung adjacent to collagen. These studies, in addition to the present one in which a mast cel1 infiltrate was noted only in those irradiated lungs which culminated in fibrosis, suggest the role of the mast cel1 in radiation fibrosis. Noted both at 3 and 6 months post-exposure during the proliferative and reparative phase in the present study, further studies indicate the involvement of these cells in al1 phases of the response.3’ In addition, the mast cel1 infiltrate has been found to be time and dose dependent with increasing numbers of cells noted with increasing dose and at increasing postexposure times.3’ The observed intimate association of the mast cel1 with an active fibroblastoid cel1 in this study is suggestive of a participation of these two cel1 types in the fibrotic process. The atypical fibroblasts observed in lungs following irradiation also have been reported in pulmonary fibrosis
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resulting from other types of trauma.5.34 Further support for a cooperation between these two cells is derived from studies in which mast cells have been identified in the fibrotic process occurring in other organs.*” These, plus the present findings, indicate that the mast cel1 may be a vita1 factor in the fibrotic process. At this time, the pathogenesis of radiation pneumonitis and fibrosis remains elusive and controversial. Although a number of theories have been proposed, most propose that damage to one lung component is the primary factor in this response. However, because of conflicting results, it certainly is possible that radiation pneumonitis and fibrosis may be related to two or more separate factors. Damage to the vascular system previously has been considered the primary site of the radiation induced lesion in the lung. However, it is not at al1 unlikely that Type 11 pneumocytes also may play a role in the development of this response mediated by an alteration in surfactant production leading to lung atelectasis. It als0 is interesting to speculate on the role of the mast cel1 in radiation fibrosis. Its association with the fibroblast and the ensuing pulmonary fibrosis is suggestive of a participation of these two cel1 types in the fibrotic process.
REFERENCES structure of 6oCoradiation pneumonitis in rats. A pathway to pulmonary fibrosis: an ultraLab. Invest. 28: 543-553, 1973. structural study of mouse and rat following 7. Finlay-Jones, J.M., Robertson, T.A., Walter, radiation to the whole body and hemithorax. M.N.: The effect of anesthetics on pulmonary
1. Adamson, I.Y.R., Bowden, D.H., Wyatt, J.P.:
Am. J. Path. 58: 481-498, 1970. 2. Bedrossian, W.M., Luna, M.A., Mackay, B., Lightiger, B.: Ultrastructure of pulmonary bleomycin toxicity. Cancer 32: 44-51, 1973. 3. Bennett, D.E., Million, R.R., Ackerman, L.V.: Bilateral radiation pneumonitis, a complication of the radiotherapy of bronchogenic carcinema. Cancer 23: 1001-1004, 1969. 4. Bertalanffy, F.D., Leblond, C.P.: The continuous renewal of the two types of alveolar cells in the lung of the rat. Anat. Rec. 115: 515-541, 1953. 5. Brooks, E.: Ultrastructure of lung lesions produced by ingested chemicals-1. Effect of the herbicide paraquat on mouse lung. Lab. Invest. 25: 536-545, 1971. 6. Faulkner, C.S., Connolly, K.S.: The ultra-
ultrastructure. Pathol. 3: 181-190, 1971. 8. Gould, E., Smuckler, E.A.: Alveolar injury in acute carbon tetrachloride intoxication. Arch. Idem. Med. 128: lOs117, 1971. 9. Groover, T.A., Christie, A.G., Merritt, E.A.: Intrathoracic changes following roentgen treatment of breast carcinoma. Am. J. Roentgenol. 10: 471476, 1923. 10. Hines, L.E.: Fibrosis of the lung following roentgen-ray treatment for tumors. J. Am. Med. Assoc. 79: 720-722, 1922. ll. Jennings, F.L., Arden, A.: Development of experimental radiation pneumonitis. Arch. Pathol. 71: 437-446, 1961. 12. Jennings, F.L., Arden, A.: Development of radiation pneumonitis. Time and dose factors. Arch. Pathol. 74: 351-359, 1962.
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13. Kapanci, Y., Weibel, E.R., Kaplan, H.D., Robinson, F.R.: Pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys-11. Ultrastructural and morphometric studies. Lab. Invest. 20: 101-106, 1969. 14. Kaplan, H.P., Robinson F.R., Kapanci, Y., Weibel, E.R.: Pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys-1. Clinical and light microscopic studies. Lab. Invest. 20: 94-100, 1969. 15. Kay, J.M., Smith, P., Heath, D.: Electron microscopy of Crotalaria pulmonary hypertension. Thorax 24: 511-526, 1969. 16. King, R.J.: The surfactant system of the lung. Fed. Proc. 33: 2238-2247, 1974. 17. Luna, M.A., Bedrossian, W.M., Lightiger, B., Salem, P.A.: Interstitial pneumonitis associated with bleomycin therapy. Am. J. Clin. Path. 58: 501-510, 1972. 18. Madrozo, A., Suzuki, Y., Ching, J.: Radiation pneumonitis: Ultrastructural changes in the pulmonary alveoli following high doses of radiation. Arch. Puthol. 96: 262-268, 1973. 19. Maisin, J.R.: The ultrastructure of the lung of mice exposed to a supra-lethal dose of ionizing radiation on the thorax. Radiat. Res. 44: 545564, 1970. 20. Murata, K., Okudaira, M., Adashio, N.: Mast cells in human liver tissue. Acta Dermato. Venereol. 53: 157-166, 1974. 21. Padall, L.N., Winkler, S.S.: Busulfan lung. Am. J. Roentgenol. Radium Ther. Nucl. Med. 120: 151-156, 1974. 22. Pflegler, R.C., Boecker, B.B., Redman, H.C., Pickerell, J.A., Mauderly, J.L., Jones, R.K., Benjamin, S.A., McClellan, R.O.: Biological alterations resulting from chronic lung irradiation-1. The pulmonary lipid composition, physiology, and pathology after inhalation by clay beagle dogs of ‘44Ce-labeled fused aerosols. Radiat. Res. 63: 275-298, 1975. 23. Phillips, T.L.: An ultrastructural study of the development of radiation injury in the lung. Radiology 87: 49-54, 1966. 24. Pickerell, J.A., Harris, D.V., Pflegler, R.C., Benjamin, S.A., Belasich, J.J., Jones, R.K.,
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McClellan, R.O.: Biological alterations resulting from chronic lung irradiation-11. Connective tissue alterations following inhalation of ‘“Ce-labeled fused clay aerosol in beagle dogs. Radiat. Res. 63: 299-309, 1975. Rosenbaum, R.N., Wittner, M., Lenger, M.: Mitochondrial and other ultrastructural changes in great alveolar cells of oxygen adapted and poisoned rats. Lab. Invest. 20: 516-528, 1969 Rubin, P., Casarett, G.W.: Clinical Radiation Pathology. Vol. 1. Philadelphia, W.B. Saunders, 1968. Shorter, R.G., Titus, J.L., Divertie, M.B.: Cytodynamics in the respiratory tract of the rat. Thorax 21: 32-37, 1966. Smith, J.C.: Radiation pneumonitis, case report of bilateral reaction after unilateral irradiation. Am. Rev. Resp. Dis. 89: 264-269, 1964. Spencer, H., Shorter, R.G.: Cel1 turnover in pulmonary tissues. Nature 194: 880, 1962. Stetson, C.G., Boland, J.: Experimental radiation pneumonitis. Cancer 20: 2170-2183, 1967. Travis, E.L.: Mast cel1 response in irradiated lung (Abstract). Br. J. Rad. 50: 237-238, 1977. Van den Brenk, H.S.: Radiation effects on the pulmonary system. In, Puthology of Irradiation. ed. by Berdjis, C. Baltimore, Williams & Wilkins, 1971, pp. 569-591. Warren, S., Spencer, J.: Radiation reaction in the lung. Am. J. Roentgenol, Radium Ther.
Nucl. Med. 43: 682-701, 1940. 34. Wasan, S.M., McElligott, T.F.:
An electron microscopic study of experimentally induced interstitial pulmonary fibrosis. Am. Reu. Resp. Dis. 105: 276-282, 1972. 35. Watanabe, S., Watanabe K., Ohishi, T., Aiba, M., Kageyama, K.: Mast cells in the rat alveolar septa undergoing fibrosis after ionizing radiation. Lab. Invest. 31: 555-567, 1974. 36. Yuen, T.G.H., Sherwin, R.P.: Hyperplasia of type 2 pneumocytes and nitrogen dioxide (10 ppm) exposure. Arch. Environ. Hlth 22: 178-188, 1971.
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0 Original Con tribu tion PATHOLOGIC CHANGES IN THE LUNG FOLLOWING SINGLE AND MULTI-FRACTION IRRADIATIONt ELIZABETH L. TRAVIS, Ph.D.S, RUSSELL A. HARLEY, M.D.“, JIMMY 0. FENN, M.S., CHRISTOPHER J. KLOBUKOWSKI, M.S. and HENRY B. HARGROVE, B.S. Department of Radiology, Division of Basic Radiation Sciences and, “Department of Pathology, Medical University of South Carolina, Charleston, SC 29401, U.S.A. The lbniting factor in the treatment of malignant disease with irradiation is the tolerante of normal tissue irradiated. In the present study the right lungs of rats were exposed to single doses of 2000 rad of X-radiation, to 10 x 200 rad, or to 5 X 400 rad. Anhnals from each group were sacriiiced monthly for 6 months post exposure. Sections of lung were examined by light microscopy (LM) and by scanning or transmission electron microscopy (SEM and TEM). A focal exudative lesion was seen at 2 months after the single dose; it progressed to a proliferative and then reparative, fibrotic lesion by 6 months. Changes in epithelial lung components, particularly the presence of Type 11 pneumocytes, were found with both LM and TM. VascuIar changes were less pronounced. A strilchtg Sndiig was the presence of mast cells in the alveolar wak. Neither of the multi-fraction schedules produced any of these changes, except hyperplasia of Type 11 celIs following 5 X 400 rad. The possible bnplication of Type 11 and mast cells in radiation pneumonitis and fibrosis is discussed. Radiation,
Lung, Multiple fractions,
Late injury, Mast cells, Type 11 pneumocytes.
INTRODUCTION The parenchymal cells of the lung may be considered relatively radioresistant, but the lung, as an organ, is radioresponsive and, in radiotherapy terminology, does not tolerate substantial doses of irradiation. In the treatment of many malignant neoplastic diseases such as Hodgkin’s disease and esophageal carcinoma, the normal lung is often the doselimiting factor in the radiotherapy treatment plan. Radiation pneumonitis is a clinical entity that has been described often; it may progress to radiation fibrosis, resulting in tPresented in part at the 1975 Annual Meeting of the American Society of Therapeutic Radiologists, San Francisco, Calif. (1975). $Present address: Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex 6HA 2RN, England. Reprint requests to: Mr. J. 0. Fenn, Directer,
patient morbidity and/or mortality.3.~‘1.26,33 For this reason, delineation of those factors which play a participating role in this response are important. Studies in patients and animals have resulted in extensive descriptions of the pathologit changes in irradiated lungs. Warren and review of Spencer ,33 in a comprehensive patients who had received thoracic irradiation, emphasized hyaline membrane formation as a primary feature of radiation peneumonitis and proposed the alveolar epithelium and capillary endothelium as the Basic Radiologie Sciences Division, Medical University of South Carolina, Charleston, SC 29401, U.S.A. Acknowledgements-The authors express their appreciation to Mrs. E. Setser and Mr. W. B. Greene for assistance with electron microscopy. 475
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primary sites of injury. Subsequent studies only, excluding the mediastinum, using a generally have focused on one of these areas single anterior field measuring 4 x 4 cm. The resulting in numerous theories concerning the remainder of the body was shielded with a primary mechanism(s) of radiation injury in cerro-safe shield measuring 10 x 12 x 2 cm the lung, including vascular damage,‘,‘2,18,19,23.33with a 4 x 4 cm cut-out in one side to allow damage to Type 1 and 11 pneumocytes3.6.32 irradiation of the right lung only. This techinvolvement of lymphatic channels draining nique allowed the left lung to serve as an internal control in each animal for pulmonary the lungs,3.28 and an immune response.‘* infection. Only one animal was excluded for However, the site of the radiation induced lesion stil1 remains controversial. this reason. Positioning of the radiation portal In the present 6 months study, the right was checked periodically with radiographs. Irradiation was administered using a GE lungs of rats were exposed either to a single Maxitron deep therapy unit, having a half dose of radiation which produced a progressive, fibrosing lung lesion or to two multivalue layer (HVL) of 1.6 Cu, operated at fraction schedules with daily fraction sizes 250 kV, 30 mA, with added Thoraeus 111filter at a target skin distance of 30 cm. Output of comparable to those used in radiation therapy. It was hoped that these methods the unit under these conditions was 195 rad would assist in identifying those common per min. Utilizing depth dose tables, back factors between fibrosing and non-fibrosing scatter factor, and the appropriate roentgen irradiated lungs as wel1 as those factors to rad conversion factor, the dose rate in rad which differ between the two. to the lung tissue at midline was calculated at 186rad per min. METHODS AND MATERIALS Immediately following irradiation, al1 animals were monitored carefully for recovery Animals from anesthesia. The animals used throughout this study were male Sprague Dawley rats (Zivic Miller Autopsy Laboratories, Inc., Allison Park, PA) Two animals from each irradiation group weighing between 175 and 200g. They were and one control animal were killed monthly kept in a controlled environment and fed for 6 months by ether anesthesia. While the standard rat chow and water ad libitum. animal was stil1 breathing, the thorax was opened and the lungs perfused through the Irradiation procedure trachea with a 2% gluteraldehyde solution The animals were divided randomly into buffered to pH 7.2 with 0.1 molar (M) four groups according to the following radiFollowing permonosodium cacodylate. ation schedule: fusion, the trachea was tied, the lungs subsequently removed from the thorax and fixed Group 1: 2000 rad; delivered in a single overnight in 10% neutral buffered formalin. dose; Sections from the right and left hing were Group 2: 2000rad; delivered in 400rad fixed by the appropriate technics and exdaily fractions for 5 consecutive amined by light microscopy (LM), transdays: mission electron microscopy (TEM), and Group 3: 2000 rad; delivered in 200rad scanning electron microscopy (SEM). Al1 daily fractions for 10 consecutive tissues excluding those for TEM were stored days, excluding weekend; in 10% buffered formalin. Group 4: Sham irradiation. At autopsy both lungs were carefully exAl1 animals were anesthetized prior to ir- amined for gross pathologie changes sugradiation with an intraperitoneal injection of gestive of pulmonary infection. When an sodium pentobarbital (Elkins-Sinn, Inc.) at a infection was suspected, sections were reconcentration of 45 mglkg. Each animal was moved from this and uninvolved areas and carefully examined microscopically. Any irradiated individually to the right thorax
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animal found to have a pulmonary infection in either the right or left lung was excluded from the study. The criteria used was the presence of leukocytes in the irradiated and/or unirradiated lung; only one animal was excluded. Tissue preparation
Tissues fixed overnight in formalin were embedded in paraffin and 5 p sections cut with a microtome. Al1 sections were evaluated using the following stains: hematoxylin and eosin, Gordon and Sweet’s method for reticulin, and Masson’s trichrome for the demonstration of collagen. Scanning
electron microscopy
Tissues fixed in formalin were cut into 3 x 3 mm tubes, dehydrated in graded alcohols, critical point dried, mounted on stubs, gold coated in a vacuum evaporater and examined using a Hitachi Scan Scope.TM Transmission
electron microscopy
Specimens for TEM were removed from the lung immediately after intratracheal perfusion with gluteraldehyde and prior to formalin fixation of the lungs. The tissue was minced quickly in cold 2% gluteraldehyde buffered to pH 7.2 with 0.1 M monosodium cacodylate solution. The tissue was fixed in the refrigerator for 16-18 hr in this same solution and subsequently rinsed thoroughly with sucrose buffer. Tissues were post-fixed for 1 hr in 2% osmium tetroxide buffered to pH 7.2 with 0.1 M monosodium cacodylate, and subsequently embedded in Epon. Thick sections were stained with toluidine blue, examined under the light microscope, and areas selected for thin sectioning. Ultra-thin sections were doubly stained with uranyl acetate and lead citrate and examined with an AEITM Electron Microscope. Areas examined on al1 sections included those within, adjacent to, and distant from the radiationinduced lesion. RESULTS Controls
The gross appearance of both the right and left lung was normal in al1 animals except one
477
in which focal, patchy, green nodules were noted on both lungs. Light microscopy revealed the presence of an acute necrotizing pneumonia in this animal. Normal histology and morphology was observed in the right and left lungs of the remaining control animals with al1 technics. An occasional ultrastructural finding in blood vessels of the normal lung was vesiculated capillary endothelium. 2000 Rad Gross changes in the right lung were not evident prior to 2 months post-exposure. At two months the right irradiated lung exhibited focal, pale, patchy areas, but was not resistant to expansion. These changes progressed with time, the irradiated lungs becoming shrunken, firm, and resistant to expansion by 6 months. In al1 cases, the non-irradiated left lung was grossly normal except for compensatory hypertrophy at 6 months. Light microscopy revealed minima1 focal perivasculitis and perivascular edema at 1 month post-exposure. Tissues examined at 2 months post-exposure revealed several discrete, subpleural, exudative lesions exhibiting an eosinophilic, proteinaceous material in the alveolar spaces, mild interstitial edema, and an intra-alveolar infiltrate of monocytic cells (Figs. 1A and B). The cellular infiltrate consisted of two populations by staining characteristics; one population exhibited a large basophilic nucleus with extensively vacuolated eosinophilic cytoplasm, the second group, although similar to the one previously described, exhibited a basophilic cytoplasm with smaller, less numerous vacuoles. Vascular alterations were less pronounced, primarily consisting of adventitial edema. An increased area of the lung was involved by the exudative lesion at 3 months, the response now becoming multi-focal in nature. The histologie characteristics of these lesions were similar to those observed at 2 months with the exception of an increased number of cells in the alveolar spaces. Alterations were present in other lung constituents as well. The interstitial area was loosened and widened, exhibiting a mild cellular infiltrate of plasma cells. Large, atypical Type 11 pneu-
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Fig. 1. (A) Rat lung exposed to 2000rad and autopsied at 3 months post-exposure exhibiting the focal nature of the early lesion and the exudative, desquamative histologie characteristic with marked intra-alveolar edema. Hematoxylin and eosin, A: x60.
mocytes were attached to the alveolar walls. The nature of the vascular alterations were consistent with those observed at 2 months although more vessels were involved. In addition, perivascular plasma cel1 cuffing was prominent. A second proliferative component of the lesion was demonstrated at this time. Reticulin stain revealed increased reticulin
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Fig. 1. (B) High magnification of area in box in A. Large vacuolated cells are clearly visible in the alveolar spaces. Hematoxylin and eosin, x300.
focally deposited throughout those alveolar walls involved by the exudative lesion. Collagen was not demonstrated. The histologie characteristics of the response changed between 4 and 6 months postexposure. Although bizarre giant cells were present in the alveolar space, the exudative lesion became less dominant (Fig. 2). Multifocal interstitial areas of the lung exhibited progressive loosening and widening in ad-
Fig. 2. At 6 months following a single dose of 2000 rad the lesion exhibits loosened, widened alveolar walls and patchy, fibrotic areas. Hematoxylin and eosin, ~96.
Radiation induced pathology of hg
dition to a marked cellular infiltrate (Fig. 3). The primary component of the infiltrate consisted of plasma cells with lymphocytes occasionally observed. Fibroblasts, not readily identifiable in the interstitial areas of normal lung, were observed frequently in those interstitial areas exhibiting alterations. Although normal appearing Type 11 cells were attached to the alveolar walls, bizarre, hypertrophied Type 11 cells were evident. In
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areas exhibiting atelectasis, the alveolar walls were denuded of epithelial cells. Vascular alterations consisting of prominent adventitial edema and marked vessel dilatation were evident in the lesion and adjacent areas from 3 to 6 months. Leukocytic pavementing was observed occasionally in relatively few blood vessels. Reticulin proliferation progressed, becoming more diffusely distributed throughout the lung (Fig. 4). Col-
Fig. 3. Another area of the 6 month lesion exhibiting marked interstitial edema and chronic inflammatory cel1 infiltrate. Hematoxylin and eosin, x300.
Fig. 4. Reticulin stain points out the proliferative nature of the 6 month lesion with increased reticulin present in the alveolar walls (hollow arrow). This also illustrates edema of the adventitial layer of a blood vessel (solid arrow). Gordon & Sweet’s reticulin, x300.
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lagen was also observed in focal areas of the alveolar walls at 4 months, becoming more diff usely located throughout the alveolar walls and pleura with increasing post-exposure times. Normal lung architecture was obliterated in multi-focal areas at 6 months post-exposure, while other areas appeared relatively normal. The alveolar walls in these latter areas were partially denuded of epithelial cells, ischemic, and appeared fragile, as evidenced by the presence of fragmented, tortuous alveoli. As at previous post-exbosure times, the entire lung was not involved with these latter phases of the response and normal lung tissue remained. The response of the lung at 6 months can best be described as a patchy, multi-focal proliferative response with patchy fibrosïs. The left unirradiated lungs appeared normal and were consistent with controls throughout the study (Fig. 5). NO animals exhibited microscopic evidente of pulmonary infection. SEM revealed the focal nature of the early lesion with fibrin deposition in the alveolar spaces and cells entrapped in this fibrin framework (Fig. 6B and C). At 6 months, the lesion was multi-focal and consolidated, exhibiting thick fibrin branches with large
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numbers of cells entrapped in the fibrin (Fig. 6D). TEM confirmed the alterations observed with light microscopy. At 3 months postexposure, vascular changes in the lesion consisted primarily of marked dilatation, this change becoming less obvious in areas adjacent to the lesion with normal vasculature observed in areas distant from the lesion. Moderate basement membrane edema was observed and the endothelium, although edematous, was attached to the basement membrane with no vesiculation. Many areas of the lesion exhibited patent capillaries; no attempt to count the patent capillaries was made (Fig. 7A). Edema of the alveolar walls was striking and increased interstitial collagen was observed. Although Type 11 cells appeared decreased in number, the number of Type 1 cells appeared unchanged. A particularly interesting observation was the presence of mast cells in the alveolar walls, which were often found in intimate association with interstitial cells containing a large amount of rough endoplasmic reticulum (RER) therefore resembling fibroblasts (Fig. 7B). Two populations of cells were observed in the alveolar spaces consistent with the light microscopic findings. The predominant cel1
Fig. 5. Left lung of experimental animal exhibiting histology animal. Hematoxylin and eosin,
consistent
x120.
with that of a control
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Fig. 6. Scanning electron micrographs of control and irradiated rat lungs. (A) normal lung. (B and C) 2000 rad, 3 months post-exposure, exhibiting fibrillar material in the alveolar spaces and cells with villous projections resembling microvilli. These cells are compatible with the SEM appearance of foamy macrophages. (D) 200rad, 6 months post-exposure showing proliferation of the fibrillar material in the alveolar spaces and complete obliteration of normal lung architecture. A: x240, B and C x 600, D: x 1200.
Fig. 7. (A) A patent capillary with intact endothelium (arrow) and containing an erythrocyte (E) seen in the rat lung exposed to a single dose of 2OOOrad and autopsied at 3 months post-exposure. UALC, x 10800.
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Fig. 7. (B) Edematous alveolar wal1 from an animal exposed to 2000rad at 3 months post-exposure with a clear aveolar space on one side (top) and debris filled alveolar space on the other (bottorn). A mast cel1(smal1arrow) is present in the interstitial area associated with a cel1 containing prominent RER (large arrow). A clear vascular space (B.V.) is present directly above the mast cel1 with normal endothelium. UALC, ~5274. was large, with numerous microvilli and lipid and glycogen-containing cytoplasmic vacuoles. The other cel1 observed, although morphologically similar to the previously described cell, exhibited larger vacuoles and myelin bodies in the cytoplasm. Occasionally this cel1 appeared to be in the process of detaching from the alveolar wall. Morphological changes indicative of degeneration were observed in both cells. The alveolar spaces were filled with an electron dense material. Al1 of the above described changes were less evident in areas adjacent to the lesion. Lung architecture was normal in areas distant from the lesion. At 6 months post-exposure normal lung architecture was obliterated in multi-focal areas. Vascular changes in the lesion were not consistent, ranging from severe basement membrane edema and sloughing of the ento mild basement membrane dothelium, edema with intact endothelium. Vessels often were markedly dilated but occlusion was not found. As at three months, vascular changes were less striking than alterations in other lung components; patent capillaries were present in the involved areas (Fig. 8). Alveolar walls were markedly widened with increased interstitial collagen. Parti-
cularly striking was atypical hypertrophy observed in both types of alveolar epithelial cells. The nucleus of the hypertrophic Type 1 cells was large with a prominent nucleolus (Fig. 9A). Hypertrophic Type 11 cells exhibited cytoplasmic lamellar bodies (Fig. 9B). Increased fibrin formation was observed in the alveolar spaces with foamy macrophages phagocytosing this material. The active interstitial cells noted at 3 months post-exposure were now bizarre, exhibiting a large amount of RER, a large, sometimes more than one, nucleus, and a prominent nucleolus (Fig. 10). These cells may represent the atypical “radiation fibroblasts” often described at the light microscopic level. The most consistent unusual finding in this tissue was the large numbers of mast cells in the alveolar walls. Very often these cells were found in intimate association with the previously described atypical fibroblastoid cell. Increased collagen formation often was evident in this same area (Fig. 11). The left unirradiated lung was normal with the exception of occasional vascular alterations consisting of endothelial blebs and swelling. 400 Rad x 5 Grossly the lungs of these
animals
ap-
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Fig. 8. Patent capillary (B.V.) in the lung of a rat exposed to 2000rad at 6 months post-exposure. Endothelial lining (arrow) is intact as is the epithelium facing an alveolar space (AS). Cel1 in the capillary is unidentified. UALC, x10350.
Fig. 9. (A) Atypical hypertrophic Type 1 cel1 attached to the alveolar wal1 of an animal exposed to 2000 rad at 6 months post-exposure. Note the large prominent nucleus and smal1 amount of cytoplasm. UALC, x6690. peared normal. Light microscopic examination of the tissue revealed no lesions and no increased collagen or reticulin. SEM failed to
reveal any lesions. TEM revealed subtle changes in lung morphology not evident with the other technies utilized, involving both alveolar pneumocytes and vasculature. A striking change at 3 months post-exposure was hyperplasia (“twinning”) of Type 11 pneumocytes (Fig.
12). NO cel1 membrane was observed between these cells, but more than one nucleus was present. Although Type 11 cells were diminished in numbers, no changes were observed in Type 1 pneumocytes. The endothelium and basement membrane of blood vessels were intact with minima1 swelling observed. However, cytoplasmic vacuolation was observed occasionally in endothelial cells of a few blood vessels (Fig. 13A).
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Fig. 9. (B) Atypical Type 11 pneumocyte (hollow arrow) attached to an alveolar wal1 containing a mast cel1 (solid arrow) in the interstitial area in close relationship to collagen (Co). Dose-2000 rad; Timemonths post-exposure. UALC, x3150.
Fig. 10. Transmission electron micrograph of atypical bizarre fibroblastoid cel1 often observed in the alveolar walls of animals exposed to 2000 rad at 6 months post-exposure exhibiting a large nucleus with prominent nucleolus. The round cel1 is a lymphocyte. UALC, x6336.
At 6 months post-exposure, a diminished number of Type 11 cells were observed as compared to both control lungs and lungs examined at earlier post-exposure times. An occasional foamy macrophage was observed in the alveolar spaces. Edema fluid was observed in blood vessels at this time with the previously described changes in endothelial
cells also observed. It must be pointed out that the blood vessel changes were not as consistent or as frequent as the changes in Type 11 pneumocytes. Mast cells were observed only in their normal location, i.e. in the pleura and in the adventitial layer of blood vessels and bronchioles, and never were in the alveolar walls
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Fig. ll. A mast cel1 intimately associated with an atypical fibroblastoid cel1 in the interstitial area of lung after a dose of 2000 rad at 6 months post irradiation. An erythrocyte (E), collagen (Co), and partially occluded vascular space (VS) are also present. UALC, x4800.
Fig. 12. Hyperplastic
Type 11 cells observed at 6 months following multi-fraction schedule. UALC, x.5400.
in animals exposed to this multi-fraction schedule during the time of the study. The appearance of the left unirradiated lung was consistent with that in both control animals and the left lung of animals exposed to the single high dose. The vascular changes previously described in the left lung also were noted in the left lungs of these animals with the same frequency.
the 400rad X 5
200 Rad x 10 Minima1 changes were observed in the lungs of animals exposed to this dose fractionation schedule. Grossly, the lungs appeared normal and no lesions were observed by LM or SEM throughout the time of the study. At no time was any evidente of fibrosis observed. Particular attention was directed to the
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(Bl Fig. 13. Patent capillaries with intact endothelium in lungs of animals exposed to the 400 rad x 5 (A) and 2000 rad x 10 (B) multi-fraction schedules at 6 months after irradiation; clear alveolar spaces are present in both. (A) UALC, x7200; (B) UALC, x12600.
morphology of Type 1 and 11 pneumocytes and blood vessels using TEM. Although no changes were observed in the alveolar epithelial cells at any time, an occasional finding at 6 months post-exposure was swelling of the basement membrane of blood vessels and cytoplasmic vacuolation of endothelial cells (Fig. 13B). These findings also were observed in the left, unirradiated lung with the same frequency.
Mast cells were not observed in the alveolar walls of either the right or left lung during the time of this study. DISCUSSION Numerous studies have been performed investigating the response of the lungs following single doses of radiation which produce a progressive pneumonitis culminating in fibrosis. One of the difficulties en-
Radiation induced pathology of lung 0 E. L. TRAVIS et al.
countered using such doses is that al1 components of the lung exhibit profound changes, therefore rendering interpretation of these changes as they relate to the pathogenesis of radiation pneumonitis difficult at best. The single fraction used in this study produced three phases of response in the lung: exudative (early), proliferative (intermediate), and reparative (late), occurring at 2-4 months, 3-6 months and 4-6 months respectively. NO definitive time marked the progression of the response from one phase to another; rather, a gradual change was observed with histologie features of the preceding phase becoming less dominant as the response progressed to the next phase. This progressive response was consistent with that reported by others following this same single but dose,‘9V*3*‘ 2 contradictory to the findings of one author who reported resolution of the early pneumonitis phase within four months following a single dose of 2000 rad to the right thorax.35 In many ways, the histologie characteristics of al1 phases of the response were consistent with other studies of the response of the lungs following irradiation, i.e. interstitial and intra-alveolar edema, bizarre epithelial cells, alterations in reticulin and increased interstitial collagen. These are non-specific findings and have been observed in the lungs following bleomycin,zX17 busulfan,‘] and paraquat5.13 administration, al1 agents which produce interstitial pneumonia and pulmonary fibrosis. One significant histologic differente in our study as compared to others was the infrequent observation of uccluded blood vessels and the presence of patent capillaries in areas of the irradiated lung exhibiting histologie changes indicative of damage. These findings suggest that vascular damage was less in our study than that reported by some authors’,23,33 but consistent with the observations of others.6.1’.12.30.32 Our findings are also consistent with the response of the lungs following other non-infectieus insults.2,‘7,2’ Although vascular alterations varied throughout the specimen with the severity of the lesion, in general, vascular changes were not as striking as the changes seen in other lung constituents, even in those areas exhibiting severe lesions.
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Ultrastructural examination of lungs exposed to the single fraction of radiation in this study supported the light microscopic observations, with vascular changes the less profound. Patent capillaries were often observed in edematous and fibrosing alveolar Although endothelial cells were walls. hypertrophic and basement membrane edema was present, vascular occulusion was not a common finding. In the two multifraction schedules tested in this study, there was minima1 ultrastructural evidente of vascular damage, consisting of mild basement membrane disruption. The observation of these same vascular changes in the left lungs of al1 experimental and control animals raises the question of whether the vascular changes noted, particularly in the right lung of animals exposed to both fractionation schedules, are indeed radiation-induced. These vascular lesions are consistent with those in lungs of animals sacrificed with ether, the method of sacrifice employed in this study.’ For this reason the cause of the vascular lesions in animals exposed to both fractionation schedules cannot, be attributed to radiation with certainty. The increased frequency of vascular lesions in animals exposed to the single fraction clearly suggests that they are radiation-induced; however, the characteristics of these lesions were not significantly different from those observed in the lungs of animals that were exposed to either multi-fraction schedule. Although vascular damage is suggested most often as the primary pathogenesis of radiation injury in the lung, our study did not support this theory fully. Alternate potential mediators of radiation pneumonitis suggested by the present study are the epithelial lung components, i.e. Type 1 (membranous) and Type 11 (granular) pneumocytes. The normal turnover time of these cells is 8 days for Type 1 pneumocytes and 27 days for Type II.4~27~29 For this reason, these cells may manifest radiation injury after exposure to a single fraction as used in this study and may reflect changes following the higher fractionation schedule. In the present study, the number of Type 11 cells were greatly diminished in the single fraction following the group, slightly decreased Y
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400 rad multi-fraction schedule, and unaffected by the 200 rad fractionation schedule. The loss of Type 11 cells in the single fraction group increased with increasing post-irradiation time. The observation of large cells with numerous vacuoles containing glycolipid materials and the presence of electron dense material in the alveolar spaces suggested that Type 11 cells were in the process of desquamation from the alveolar wal1 with subsequent degeneration in the alveolar spaces. However, the morphology of the cells present in the alveolar spaces also was suggestive of foamy macrophages, particularly since these cells wil1 phagocytose cellular debris. Although foamy macrophages are not an uncommon finding in rat lungs, particularly as the animal ages, these cells only occasionally were observed in the lungs of control animals, in the left unirradiated lung of experimental animals, and in the right lungs of animals exposed to the 200rad fractionation schedule. They were most predominant in the single fraction group and frequently observed in the group exposed to the 400 rad multi-fraction schedule. A common factor between lungs exposed to a single and fractionated dose of radiation was hyperplasia of Type 11 cells. This phenomenon is not unique to radiation injury of the lung and has been reported following other lung insults, including nitrogen dioxide36 and oxygen’3.‘4*25 exposure and carbon tetrachloride’ and monocrotaline’5 administration. It has been postulated that following severe injury which denudes the alveolar walls of both types of pneumocytes, proliferation of Type 11 pneumocytes functions as a regenerative response in an effort to repopulate the naked alveolar walls with epithelium.* Because of their role in synthesizing surfactant, the material in the lung which stabilizes the alveoli, a loss of these cells or changes in their normal morphology could alter lung function profoundly.‘6’2 Depopulation of Type 11 cells could result in a loss of surfactant leading to transudation of fluids across the alveolar walls and subsequent atelectasis, a response in the lung generally attributed to vascular injury. Morphologic abnormalities in Type 11 cells could result in
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abnormal surfactant with the same sequentia1 changes. Faulkner and Connolly” described changes in the ultrastructure of Type 11 cells consisting of large lamellar bodies containing dense, coarse material and abundant rough endoplasmic reticulum, giving these cells an immature appearance. Because the lamellar bodies store the surfactant prior to extrusion from the Type 11 pneumocyte into the alveolar spaces, changes such as these could reflect altered surfactant production. Indeed, in a study of pulmonary lipids following lung irradiation, Pflegler et aLz2 and Pickerell et aLz defined alterations in the lipid composition of surfactant which potentially support the role of Type 11 pneumocytes in the pathogenesis of radiation pneumonitis. The finding of bizarre, atypical Type 11 pneumocytes in the lungs of animals exposed to the single fraction used in this study as wel1 as the diminished numbers and hyperplasia of these cells in animals exposed to the single fraction and the 400 rad fractionation schedule, coupled with the lack of vascular changes in the fractionated group, further suggest that Type 11 pneumocytes, if not fully responsible for radiation pneumonitis, certainly contribute to the pathogenesis of this response. Changes in Type 11 pneumocytes have been consistent findings in both experimental and clinical radiation pneumonitis with the exception of one study.’ The multi-focal nature of the observed lesions may also be attributable to the response of Type 11 pneumocytes. Although generally attributed to variations in response of the vasculature, the idea also must be entertained that this lesion is a response of the epithelial components of the lung and that the multi-focal characteristic is a reflection of a perturbation of the kinetics of this population of cells following irradiation. Although the entire irradiated lung was not compromised at any time by any phase of the response, it cannot be stated with certainty whether the reparative response wil1 proin diffuse pulmonary gress, culminating fibrosis. The single, most distinguishing feature in lungs undergoing fibrosis as compared to nonfibrosing lungs in this study was the presence
Radiation induced pathology of iung 0 E. L. TRAVISet al.
of mast cells in the alveolar walls. These cells have been reported by others in irradiated lungs and it has been suggested that they are involved in the reparative phenomenon. Phillipsz3 observed an increased mast cel1 infiltrate from 3 to 6 months following a single dose of 2OOOrad to the lung whereas Watanabe et aL3’ only observed this response following a dose of 3000rad. Faulkner and Connolly” also observed mast cells in the interstitial areas of the lung adjacent to collagen. These studies, in addition to the present one in which a mast cel1 infiltrate was noted only in those irradiated lungs which culminated in fibrosis, suggest the role of the mast cel1 in radiation fibrosis. Noted both at 3 and 6 months post-exposure during the proliferative and reparative phase in the present study, further studies indicate the involvement of these cells in al1 phases of the response.3’ In addition, the mast cel1 infiltrate has been found to be time and dose dependent with increasing numbers of cells noted with increasing dose and at increasing postexposure times.3’ The observed intimate association of the mast cel1 with an active fibroblastoid cel1 in this study is suggestive of a participation of these two cel1 types in the fibrotic process. The atypical fibroblasts observed in lungs following irradiation also have been reported in pulmonary fibrosis
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resulting from other types of trauma.5.34 Further support for a cooperation between these two cells is derived from studies in which mast cells have been identified in the fibrotic process occurring in other organs.*” These, plus the present findings, indicate that the mast cel1 may be a vita1 factor in the fibrotic process. At this time, the pathogenesis of radiation pneumonitis and fibrosis remains elusive and controversial. Although a number of theories have been proposed, most propose that damage to one lung component is the primary factor in this response. However, because of conflicting results, it certainly is possible that radiation pneumonitis and fibrosis may be related to two or more separate factors. Damage to the vascular system previously has been considered the primary site of the radiation induced lesion in the lung. However, it is not at al1 unlikely that Type 11 pneumocytes also may play a role in the development of this response mediated by an alteration in surfactant production leading to lung atelectasis. It als0 is interesting to speculate on the role of the mast cel1 in radiation fibrosis. Its association with the fibroblast and the ensuing pulmonary fibrosis is suggestive of a participation of these two cel1 types in the fibrotic process.
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1. Adamson, I.Y.R., Bowden, D.H., Wyatt, J.P.:
Am. J. Path. 58: 481-498, 1970. 2. Bedrossian, W.M., Luna, M.A., Mackay, B., Lightiger, B.: Ultrastructure of pulmonary bleomycin toxicity. Cancer 32: 44-51, 1973. 3. Bennett, D.E., Million, R.R., Ackerman, L.V.: Bilateral radiation pneumonitis, a complication of the radiotherapy of bronchogenic carcinema. Cancer 23: 1001-1004, 1969. 4. Bertalanffy, F.D., Leblond, C.P.: The continuous renewal of the two types of alveolar cells in the lung of the rat. Anat. Rec. 115: 515-541, 1953. 5. Brooks, E.: Ultrastructure of lung lesions produced by ingested chemicals-1. Effect of the herbicide paraquat on mouse lung. Lab. Invest. 25: 536-545, 1971. 6. Faulkner, C.S., Connolly, K.S.: The ultra-
ultrastructure. Pathol. 3: 181-190, 1971. 8. Gould, E., Smuckler, E.A.: Alveolar injury in acute carbon tetrachloride intoxication. Arch. Idem. Med. 128: lOs117, 1971. 9. Groover, T.A., Christie, A.G., Merritt, E.A.: Intrathoracic changes following roentgen treatment of breast carcinoma. Am. J. Roentgenol. 10: 471476, 1923. 10. Hines, L.E.: Fibrosis of the lung following roentgen-ray treatment for tumors. J. Am. Med. Assoc. 79: 720-722, 1922. ll. Jennings, F.L., Arden, A.: Development of experimental radiation pneumonitis. Arch. Pathol. 71: 437-446, 1961. 12. Jennings, F.L., Arden, A.: Development of radiation pneumonitis. Time and dose factors. Arch. Pathol. 74: 351-359, 1962.
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13. Kapanci, Y., Weibel, E.R., Kaplan, H.D., Robinson, F.R.: Pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys-11. Ultrastructural and morphometric studies. Lab. Invest. 20: 101-106, 1969. 14. Kaplan, H.P., Robinson F.R., Kapanci, Y., Weibel, E.R.: Pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys-1. Clinical and light microscopic studies. Lab. Invest. 20: 94-100, 1969. 15. Kay, J.M., Smith, P., Heath, D.: Electron microscopy of Crotalaria pulmonary hypertension. Thorax 24: 511-526, 1969. 16. King, R.J.: The surfactant system of the lung. Fed. Proc. 33: 2238-2247, 1974. 17. Luna, M.A., Bedrossian, W.M., Lightiger, B., Salem, P.A.: Interstitial pneumonitis associated with bleomycin therapy. Am. J. Clin. Path. 58: 501-510, 1972. 18. Madrozo, A., Suzuki, Y., Ching, J.: Radiation pneumonitis: Ultrastructural changes in the pulmonary alveoli following high doses of radiation. Arch. Puthol. 96: 262-268, 1973. 19. Maisin, J.R.: The ultrastructure of the lung of mice exposed to a supra-lethal dose of ionizing radiation on the thorax. Radiat. Res. 44: 545564, 1970. 20. Murata, K., Okudaira, M., Adashio, N.: Mast cells in human liver tissue. Acta Dermato. Venereol. 53: 157-166, 1974. 21. Padall, L.N., Winkler, S.S.: Busulfan lung. Am. J. Roentgenol. Radium Ther. Nucl. Med. 120: 151-156, 1974. 22. Pflegler, R.C., Boecker, B.B., Redman, H.C., Pickerell, J.A., Mauderly, J.L., Jones, R.K., Benjamin, S.A., McClellan, R.O.: Biological alterations resulting from chronic lung irradiation-1. The pulmonary lipid composition, physiology, and pathology after inhalation by clay beagle dogs of ‘44Ce-labeled fused aerosols. Radiat. Res. 63: 275-298, 1975. 23. Phillips, T.L.: An ultrastructural study of the development of radiation injury in the lung. Radiology 87: 49-54, 1966. 24. Pickerell, J.A., Harris, D.V., Pflegler, R.C., Benjamin, S.A., Belasich, J.J., Jones, R.K.,
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McClellan, R.O.: Biological alterations resulting from chronic lung irradiation-11. Connective tissue alterations following inhalation of ‘“Ce-labeled fused clay aerosol in beagle dogs. Radiat. Res. 63: 299-309, 1975. Rosenbaum, R.N., Wittner, M., Lenger, M.: Mitochondrial and other ultrastructural changes in great alveolar cells of oxygen adapted and poisoned rats. Lab. Invest. 20: 516-528, 1969 Rubin, P., Casarett, G.W.: Clinical Radiation Pathology. Vol. 1. Philadelphia, W.B. Saunders, 1968. Shorter, R.G., Titus, J.L., Divertie, M.B.: Cytodynamics in the respiratory tract of the rat. Thorax 21: 32-37, 1966. Smith, J.C.: Radiation pneumonitis, case report of bilateral reaction after unilateral irradiation. Am. Rev. Resp. Dis. 89: 264-269, 1964. Spencer, H., Shorter, R.G.: Cel1 turnover in pulmonary tissues. Nature 194: 880, 1962. Stetson, C.G., Boland, J.: Experimental radiation pneumonitis. Cancer 20: 2170-2183, 1967. Travis, E.L.: Mast cel1 response in irradiated lung (Abstract). Br. J. Rad. 50: 237-238, 1977. Van den Brenk, H.S.: Radiation effects on the pulmonary system. In, Puthology of Irradiation. ed. by Berdjis, C. Baltimore, Williams & Wilkins, 1971, pp. 569-591. Warren, S., Spencer, J.: Radiation reaction in the lung. Am. J. Roentgenol, Radium Ther.
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An electron microscopic study of experimentally induced interstitial pulmonary fibrosis. Am. Reu. Resp. Dis. 105: 276-282, 1972. 35. Watanabe, S., Watanabe K., Ohishi, T., Aiba, M., Kageyama, K.: Mast cells in the rat alveolar septa undergoing fibrosis after ionizing radiation. Lab. Invest. 31: 555-567, 1974. 36. Yuen, T.G.H., Sherwin, R.P.: Hyperplasia of type 2 pneumocytes and nitrogen dioxide (10 ppm) exposure. Arch. Environ. Hlth 22: 178-188, 1971.