The Pulmonary Vascular Pathology of Experimental Radiation Pneumonitis David 0. Slauson, DVM, PhD, Fletcher F. Hahn, DVM, PhD, and Thomas L. Chiffelle, MD
Dogs exposed by inhalation to an aerosol of fused aluminosilicate particles containing the radionuclide yttrium 90 developed radiation pneumonitis. The aerosol had a mean aerodynamic diameter of 0.8 to 1.2 p with a og of 1.6 to 1.9. The 36 dogs included in this report received initial lung burdens of 590 to 5200 sCi "Y/kg body weight and died at 7.5 to 237 days after exposure with total cumulative radiation doses to lung of 9300 to 70,000 rads. Vascular lesions in the lungs were marked. Early changes included edema of vessel wails with leukocytic infiltration, dilation of perivascular lymphatic channels, and occasional periarterial lymphangiectasia. Splitting and reduplication of the elastica were occasionally visible. The most striking inflammatory vascular changes were vasculitis and fibrinoid necrosis, which involved bronchial and pulmonary vessels at somewhat different times. Such lesions were often segmental and included fibrinoid necrosis and a variable leukocytic infiltrate in and around the actively involved lesions. Vasculitis was most commonly seen in small muscular arterioles, but veins and venules also occasionally exhibited similar inflammatory lesions. Progressive vascular inflammation led to extensive intimal proliferative lesions and fibromuscular hypertrophy with eventual fibrous accumulation around blood vessels, obliterative intimal and medial thickening, and luminal narrowing. Such changes eventually formed the morphologic basis for increased pulmonary vascular resistance and the development of cardiac dilatation and hypertrophy reflecting pulmonary hypertension. (Am J Pathol 88:635-654, 1977)
PULMONARY VASCULAR LESIONS have long been noted to occur in the lung injured bv radiation, although relatively little attention has been given to their pathogenesis or sequence of development. Radiation-induced injury to small vessels and to endothelial cells is well documented in both man 1-3 and experimental animals.4 7 Large and medium-sized arteries are apparently less sensitive to radiation injury, although pathologic changes can occur.2'8- Acute damage with necrosis and arterial rupture has been reported,11 but relativelyr few examples of chronic changes leading to arterial narrowing and occlusion have been described.10 Warren and Spencer3 emphasized the diagnostic value of vascular lesions in the later stages of chronic radiation pneumonitis. From the Department of Pathology. Cornell University College of Veterinary Medicine. Ithaca. New York, and the Lovelace Foundation for Medical Education and Research. Albuquerque. New Mexico Supported by Contract E(29-2)-013 from the US Energy Research and Development Administration. This research was conducted in facilities full! accredited b\- the American Association for Accreditation of Laboratorv Animal Care. Accepted for publication April 22. 1977. Address reprint requests to Dr. David 0 Slauson. Department of Pathology. College of Veterinary Medicine, Cornell Universitv, Ithaca, NY 14853 635
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Marked hyaline fibrosis and vascular alterations with increased alveolar septal thickening and resultant obliteration of the alveolar capillaries as well as distinct diminution of the lumina of larger vessels were noted. Edema of vessel walls along with endothelial swelling and vacuolation, coarsening and reduplication of the elastica, and hyaline swelling of both veins and arteries in radiation pneumonitis were described by Warren and Gates,2 although leukocytic infiltration was not a prominent feature. Dogs given 4200 to 8400 rads external thoracic radiation fractionated over a 5week period developed occasional occlusive vascular changes with fibromuscular and intimal hyperplasia, reduplication of the internal elastic membrane, and rare thrombosis.12 Lesions were more prominent in larger arteries than in smaller arterioles. Hopewell 13 also described late-occurring segmental occlusive lesions in irradiated vessels and suggested a relationship to the development of hypertension. Pulmonary vascular lesions were described by Jennings and Arden 14 in rats given external thoracic x-irradiation in either single 3000 R-exposures or fractionated doses of 600 R/week for 5 weeks. Marked vascular thickening, observed at 60 days after exposure in the fractionated group, was characterized by prominent subintimal hyalinization. Rats exposed to between 500 and 3000 rads of external x-irradiation and examined for ultrastructural changes in heart, skeletal muscle, lung, and kidney revealed that the common site of damage in all four organs was the vascular endothelium.15 Constrictive changes have also been described in the pulmonary arterioles of rats 1 to 2 weeks after exposure to 3600 rads of single external thoracic x-irradiation, with thickening and degenerative changes in the media.16 Jennings and Arden 17 summarized the histopathologic findings in 173 cases of radiation pneumonitis secondary to thoracic radiotherapy (dose, 500 to 6000 R) and noted striking fibrin deposits but relatively infrequent obliterative endoarteritis in arterioles; only 14% of their cases exhibited significant vascular pathologic changes. Fried and Goldberg 18 studied postirradiation changes in the lung and thorax and called attention to hypertrophy of the right ventricle, which they attributed to narrowing of the alveolar capillaries with increased resistance throughout the lung. They proposed a terminal state of mediastinal fibrosis with stenosis of the pulmonary veins, hyperemia of the right ventricle, and eventual congestive failure. Late-occurring vascular lesions in man caused by radiation have often been attributed to damage to the internal elastic lamina or to the vasa vasorum,10 a suggestion which has been further emphasized by experimental work.19 In an additional study, Jacobsen 20 indicated that irradiated arteries and arterioles underwent obliteration by intimal thickening, and recent thrombi were found that had undergone recanalization.
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Relatively little attention has been paid to the development of lesions in the pulmonarv vasculature in radiation pneumonitis caused by inhaled internal emitters, although vascular lesions have been described.21-23 The present study details the sequential development of pulmonarv vascular lesions in 35 dogs with radiation pneumonitis caused bv the pulmonarv deposition of a beta-emitting radionuclide, yttrium 90, in relatively insoluble fused aluminosilicate particles. The clinicopathologic features of radiation pneumonitis following 9Y inhalation in dogs and the overall histopathology of the lung in this disease have been reported.21'23'24 The present report is focused onlv on the pulmonarv vascular changes. Materials and Methods -I
-
The animals used in this studv were drawn from one of a large number of inhalation studies involving the beagle dog being conducted at the Inhalation Toxicology Research Institute, Lovelace Foundation for Medical Education and Research. The experimental approach used in these studies is directed toward evaluating the influence of radiation dose rate and total radiation dose on dose-response relationships.2 The initial lung burdens used were predicted to result in early deaths (to 1 * ear after exposure) due to inflammatory pulmonary changes; in deaths at later times (after 1 vear) due to moderate to marked pathologic changes such as pulmonary fibrosis at intermediate dose levels; and in more subtle and late-occurring changes several years after exposure, including pulmonary neoplasia at lower initial lung burden levels. For the preparation of this report. 36 dogs which died or were sacrificed during the first year after exposure to relatively high initial lung burden levels of "Y inhaled in aluminosilicate particles were used. Detailed descriptions of the aerosol apparatus, exposure methods, isotope and aerosol preparation, and other experimental variables have been previously reported. 5 "
Expermtal Procedures and Desin All dogs used in these studies were derived from our ow-n closed breeding colony of beagle dogs,"930 and were exposed as young adults at 12 to 14 months of age. Each dog was exposed individually using an apparatus previously described.' For preparation of the aerosol, "*Y was separated from its parent isotope, strontium 90, by the selective formation of colloidal hydrogen phosphate at a pH of 5.0.31 The separated 9Y was transferred into montmorillonite clav by ion exchange. The clay was then filtered, resuspended into distilled water, and placed in a nebulizer type aerosol generator.2 The nebulized aerosol was passed through a column heated at 1100 C and then cooled by the addition of diluting air before delivery to the dog for inhalation. The resultant fused aluminosilicate particles containing "Y were spherical in shape, had a size distribution curve that was similar to a normal logarithmic curve, and had an activity median aerodynamic diameter ranging from 0.8 to 1.2 A and geometric standard deviations of 1.6 to 1.9. Further details on the experimental procedures have been reported.25 Yttrium 90 was selected for study- because it represents a typical short-lived high-energy (Em,,a = 2.28 MeV) beta emitter with a physical half-life of about 64.2 hours and because it is one of the fission-product radionuclides found in significant quantities in a nuclear reactor inventorv after a sustained period of operation. The retention and tissue distribution pattern of "Y inhaled in fused aluminosilicate particles is that which would be anticipated for an insoluble material of the particular size being studied.' The respiratorx tract is, therefore, the primary organ system irradiated by the beta particles from 'Y. The
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retention half-life of "Y particles in the lung approximates the physical half-life of MY; that is, 90% of the infinite dose is delivered in about 9 days and the irradiation is essentially complete by 28'days after deposition. Certain of the early biologic effects of inhaled M°Y in the dog,21 23 and its effects on pulmonary function and related parameters,24 have been reported. The present report is based on histopathologic study of dogs with radiation pneumonitis which died during the first year after 90Y exposure and includes 35 animals that received initial lung burdens ranging from 590 to 5200,uCi/kg body weight and died at 7.5 to 237 days after exposure, with total cumulative radiation doses to the lung ranging from 9,300 to 70,000 rads. Dose and Dose Rate
The methods of dose calculation have been previously reported.2" The total radiation dose to lung was basically determined by the nature of the radionuclide and its decay characteristics, its physical half-life, the retention of the particles in the lung (biologic or effective half-life), and the duration of radiation exposure or time to'death. In essence, the relatively insoluble 90Y particles were inhaled, a portion was deposited deep in the lung, and was not cleared or cleared slowly; hence, the particles decayed and irradiated lung tissue at a relatively high but rapidly decreasing dose rate (Text-figure 1). Because 'Y has a high energy beta emission, the lung is irradiated in a relatively uniform fashion. Pathology A complete necropsy was performed on each animal at the time of death or after euthanasia performed because death was imminent. Tissues for histopathologic study were routinely fixed in 10% neutral buffered formalin. The lungs were fixed by bronchial perfusion and reexpanded to their approximate dimensions in an inflated state. After routine processing and paraffin embedding, sections of tissue were cut at 5 ,s and routinely stained with hematoxylin and eosin. Selected tissues were also stained with a modified elastic Masson's trichrome procedure, by routine elastic Van Gieson and Masson's trichrome methods, and by the phosphotungstic acid-hematoxylin procedure. The lungs of many dogs were cultured for viruses, bacteria, and fungi at necropsy. No significant pathogens were isolated from these lungs by routine microbiologic methods."1 n-
0.241 -J 0.204o
Y16200
4000
A
-j
0
0
800 E
O 0.16(OE
minosilicate particles (calculated for an initial lung burden [ILB] of 1.0 mCi). a The initial dose rate to lung for a 10-kg dog was calculated ~~~~~~~~~400i to be approximately 0.23 rads/minute/mCi and the infinite cumulative dose to be approximately 1200 rads/
1._c E
"'aa w
11 4 cr
to cn w
0.121
0
0.081
0-
I-
w U) 0
TEXT-FIGURE 1-Radiaation dose and dose rate to lung in the beagle after inhalation of 90Y in fused alu-
.4
4
0)
80
160
320 240 TIME (hrs)
400
480
mCi.
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The 35 dogs in this study died of radiation pneumonitis at intervals ranging from 7.5 to 237 days after exposure. The time of death after inhalation of 90Y in fused aluminosilicate particles correlated well with the total cumulative radiation dose to lung (Text-figure 2). Thus, animals receiving in excess of 20,000 rads total dose to lung did not survive bevond 90 days after exposure, whereas dogs receiving somewhat lower total radiation doses did not die of acute pneumonitis but lived longer after exposure and hence were included in the group at risk for the development of more chronic pulmonary lesions including vascular lesions. Puary Vascuiar Pa
Virtually all of the dogs in this studs developed pulmonarx vascular lesions which characteristically had one of several histologic patterns, as described below. It is important to note, however, that the time sequence of appearance of the vascular changes described here varied, and adequate histopathologic evaluation can come only from studying as many sections of tissue as possible. Various lesions were seen within vessels from the same lung, since not all of the pulmonarv vessels in a single case had progressed to the same stage of abnormality at the same time. Thus we frequently encountered the situation where some muscular pulmonarv arteries in a dog known to have progressed to a more advanced level of 7G _
~60 0
TE-xTr-FICURE 2-Sur-
a o vival time after inhalation exposure of dogs to "Y in Fo 40 fused aluminosilicate par- 0, \ ticles as a function of radiation dose to lung. There I is reasonabl good correlation between survival time O0 a 20 and dose. L.J
o0 s
0
o
0
o0 o
>
X
O 0~~~~~~~~~~~~~
SURVIVAL TIME (DAYS)
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vascular disease still showed early changes. For this reason, one animal may demonstrate the histologic features of several different degrees and forms of pulmonary vascular injury. Hence, in this discussion and in the figure legends, the degree of damage relates only to the particular vessel shown and not necessarily to the animal as a whole. Where possible, we have indicated the time sequence for the development of various forms of vascular lesions. Edema and Vascular Infiltration
A commonly encountered early vascular event in the lung injured by radiation was the presence of edema within vessel walls accompanied by leukocytic infiltration into the edematous media and adventitia as well as perivascular leukocytic accumulation. Dilation of perivascular lymphatic chanhiels and occasional periarterial lymphangiectasia were also noted. In Qontrast to neutrophils, which were the major infiltrating cells in lesions characterized by fibrinoid necrosis with arteritis, the leukocytes most commonly encountered in these early edematous vessel lesions were generally lymphocytes, with only scattered neutrophil polymorphonuclear le'ukocytes present (Figure 1). Such changes were encountered most commonly within and about small thin-walled pulmonary arterioles but were also seen in pulmonary veins (Figure 2). While such changes were often more commonly seen in the acute phases of lung injury (to 60 days after exposure), they were also encountered in the lungs of animals dying at later times after exposure, at a time when pulmonary fibrosis and occlusive vascular lesions were well developed. Vasculitis and Fibnnoid Vascular Necrosis
Lesions characteristic of fibrinoid vascular necrosis were seen in many of the dogs. These lesions were typically seen at two different time frames in the course of events after exposure and involved both the pulmonary and systemic (bronchial) arteries. At early times after exposure (less than 1 month), fibrinoid necrosis primarily involved the bronchial arteries (Figure 3); similar changes were less commonly encountered in the muscular pulmonary arteries and arterioles. Conversely, the second time-frame for the appearance of fibrinoid necrosis was late in the progression of the disease (over 150 days after exposure), when the lesions almost exclusively involved muscular pulmonary arteries and arterioles, with sparing of the bronchial arteries (Figure 4). Although some variation was seen in this two-phase appearance of fibrinoid vascular necrosis in the two circulations, the pattern generally held true. These lesions characteristically occurred in medium-sized and small
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muscular arterioles and included necrosis and swelling of the media, which often presented a glossy or smudgy appearance, with loss of nuclei, and usually took the staining reactions for fibrin. In some vessels, the medial necrosis was accompanied by an intense, reactive acute inflammatory response around and within the necrotic vessel wall with prominent accumulation of neutrophil leukocytes. There was often loss or disruption of the elastic laminae in affected vessels, and the lesions in general were either segmental or uniform in their involvement of the vessel wall. Hemorrhage into the media was also encountered. Occasionally vessels exhibited changes suggestive of a healing phase of previous vascular necrosis. Such arteries were characterized by the appearance of granulation tissue within the media or by intensely staining hemosiderin or hematoidin deposits within the vessel walls, reflecting prior hemorrhage. Sometimes small capillaries ramified through such granulating lesions, making distinction from healing thrombosis with recanalization difficult. Chronic vasculitis with intense medial lymphocytic infiltrations and luminal reduction was also sometimes seen, and suggested a healing phase of previous acute arteritis (Figure 5). Fibromuscular hypertrophy of the vascular media was a characteristic feature of the middle and later stages of the development of chronic radiation pneumonitis. It was often seen in conjunction with intimal proliferation, although it sometimes appeared to have preceded changes in the intima. Fibromuscular hypertrophy of the media was sometimes accompanied by medial elastosis with thickening of the elastic laminae and a marked increase in the amount of elastic fibers within the media. Fibrosis of the media between the elastic fibers and muscular bundles and fibrous replacement of the muscle bundles was also sometimes encountered. In general, medial changes were less commonly encountered than were intimal changes, and when medial hypertrophy was present it was often accompanied by intimal proliferation which in combination sometimes served to virtually occlude the vascular lumen (Figure 6). hvn Pioaon an Thk_rrg
Lesions affecting the intima of vessels were commonly encountered. It was often difficult to place such lesions in a time frame, as proliferative intimal lesions (early changes) were often seen in the same lungs which exhibited occlusive and relatively acellular (chronic) intimal sclerotic changes. Thus, it seemed that the process of intimal thickening was a continuing one, with variations in severity, extension, and histologic
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appearance being continijally present. It was necessary, therefore, to try to reconstruct the sequence of appearance of various intimal changes as they related to individual vessels rather than to the lung as a whole or to the time after radiation injury at which those lungs were examined. For purposes of this report, we have considered that the cellular intimal proliferative changes (especially endothelial proliferation) represented early events, whereas changes in elastic tissue, collagen, reticulin, and acellular fibrosis of the intima were late-occurring changes within any one individual vessel. Intimal lesions encountered in the dogs in this study were often focal and were best appreciated when a vessel was sectioned in a nearly longitudinal plane. Proliferative intimal lesions were encountered as early as 24 days after exposure and as late as 220 days after exposure. They generally consisted of either a loose proliferation of endothelial elements or of accumulations of several cell layers between the internal elastic membrane and the endothelium. In more advanced lesions, the marked cellular proliferation was gradually replaced by a preponderance of collagen and reticulin fibers with marked luminal reduction (Figure 7). Admixed elastic fibers were sometimes encountered, and there often was an outer circular fibrous layer with elastosis and an inner, more cellular layer, suggesting different phases in the process of intimal fibrosis. Severe intimal proliferative changes were sometimes seen in larger, elastic arteries as well as in the smaller, muscular walled vessels (Figure 8). Chronic Obliterative Vascular Lesions
Lesions which partially or totally occluded the vascular lumina were seen with increasing frequency related to the length of time after exposure. Thus, lungs from dogs surviving to 150 days after exposure almost always exhibited some chronic obliterative vascular lesions, and the longest surviving dogs in this experiment had the most severe lesions. In general, it was difficult to reconstruct the histologic events that led to fibrous obliteration of the vasculature. The lesions were often more pronounced in medium-sized and smaller vessels than in larger muscular arteries or elastic arteries and were generally widespread within all lobes of the lung. Bronchial arteries were not spared by the obliterative process although the changes were often more pronounced in branch pulmonary arterioles in the lung parenchyma itself. Within some larger vessels, the luminal obliteration was more cellular and proliferative, with fibroplasia and peripheral recanalization particularly prominent (Figure 9). Around smaller muscular arterioles in the lung parenchyma where obliterative changes were most pronounced, a dense collar of collagenous tissue often
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surrounded the adventitial aspects of the obliterated vessels (Figure 10). When sectioned longitudinally, such scarred and totally obliterated vessel tracts were difficult to define as vascular structures. Obliterative changes were also seen in the smallest identifiable pulmonary arterioles, and capillary level obliterative changes were often seen in association with the process of interstitial fibrosis. NVonpuKAry Cardiovasr Parameters
Right ventricular hvpertrophy, often with marked dilatation, was seen in many of the dogs at necropsy. Since we did not directly quantitate pulmonarv hypertension in this group of dogs by measuring pulmonary arterv mean pressure and pulmonary vascular resistance, comparisons of cardiac weights were made between the exposed group of dogs and a similar number of age-matched and sex-matched control beagle dogs which had been sacrificed for other studies. The mean age of the control group was not different from that of the dogs with pneumonitis when tested statisticallv (P = 1.65). None of the control dogs exhibited pulmonarv or cardiac lesions. Comparisons of heart weight/body weight ratios were not useful, as many of the dogs with pneumonitis exhibited significant weight loss, which unfairly biased the data. We therefore compared only total heart weight between the exposed and control groups as an index of the cardiac manifestations of pulmonary vascular disease. As shown in Table 1, a highly significant difference (P < 0.001) was found between the means of the heart weights even though there was considerable overlap in the ranges for the two groups. This difference in part reflects a substantial increase in heart weight for dogs dying of pneumonitis at longer times after exposure when occlusive pulmonary vascular lesions were well developed, rather than a uniform or generalized change throughout the exposed group. Table 1-Heart Weight in Dogs Wih Radiation Pneumonitis Compared to Sex and Age Matched Controls Pneumonitis Controls No.ofdogs 34 29 Male 17 13 Female 17 16 Mean age ± SE (Days)* 448 27 509 10 Heart weight (g) Mean ± 1 SDt 82.9 ± 18.6 61.2 ± 15.5 SEM 3.2 2.6 Range 43.3-121.5 45.3-99.3 Mean age + SE in days; the means are not statistically different (P = 1.65). t Mean heart weight ± 1 SD; the means are statistically different (P < 0.001) as dtermined by a two-tailed t test.
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Discussion
The pulmonary vascular lesions described here were prominent histopathologic features of radiation pneumonitis as produced experimentally in the dog by inhalation exposure to aluminosilicate particles containing the radionuclide "Y. Although the vascular component of this disease was prominent, it was not unique, as similar vascular lesions have been reported in dogs that inhaled 144Ce in aluminosilicate particles 22 as well as in animals and man that received external radiation.2'3 The pathogenesis of the pulmonary vascular lesions seen in radiation pneumonitis is poorly understood. We believe that there are four major factors involved in the development of these lesions: direct radiation injury, local chronic hypoxia, interstitial fibrosis, and the development of pulmonary hypertension.
Direct radiation-induced injury to the lung and its vasculature certainly is a primary cause of damage. Direct vascular injury is marked in radiation pneumonitis and probably initiates an inflammation and repair sequence that leads to more chronic changes as sequelae of the tissue repair process. Some of the early necrotizing changes in bronchial arteries, for example, may represent direct radiation-induced changes. The progressive development of changes in the pulmonary vasculature as detailed here, however, suggests that processes other than those associated with the initial injury are necessary to the development and pathogenesis of the more chronic lesions. This is particularly evident when one considers that the lesions persist and progress at much later times after exposure than the 9 days required to deliver 90% of the infinite dose from inhaled 90Y particles, and also because lesions evidencing various developmental stages were often found in the same lung. Thus, while direct radiation-induced injury may initiate the inflammatory process, the persistance and progression of the vascular lesions suggest that continuing radiation is not necessary for the maintenance and amplification of the injury process. In addition to the repair processes activated after initial vascular injury, changes secondary to chronic hypoxia, interstitial fibrosis, and the development of pulmonary Iypertension as sequelae to the initial insult seem implicated in the pathogenesis of the progressive vascular lesions. Chronic hypoxia can lead to pulmonary vascular disease 32,33 and may well be a contributing cause in the pathogenesis of the vascular changes seen in these dogs with radiation pneumonitis. The pathophysiologic features in dogs have been previously detailed.2'24 Pulmonary vascular changes have also been observed in a variety of other situations in which chronic hypoxia is present, including progressive muscular dystrophy with shallow breathing,34 damage to the respiratory center,35 simple obesity,36
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and various other disease states.32 The hypoxia in such situations is due to inadequate intake of ambient air of normal oxygen content and leads to carbon dioxide retention. Similar changes have been observed in patients with kyphoscoliosis who develop "heart failure of the hunchback.'"37 Chronic hypoxia can also result during normal breathing or even hvperventilation of air with a low oxygen content, such as encountered at high altitudes. Such a situation can lead to right ventricular hypertrophy, increased pulmonary vascular resistance and pulmonarv hvpertension, and marked medial hypertrophy of small muscular pulmonarv arteries.3' Similar observations have been made in animals living at high altitude41 or under conditions simulating high altitude.42 Chronic hvpoxia can also lead to pulmonary arterial hypertension in experimental animals," but the mechanism of its development is uncertain. It mav be brought about and maintained by constriction of the pulmonarv vascular tree. Acetylcholine administration can lower the elevated arterial pressure," although the autonomic nervous svstem plavs a questionable role, since pulmonary hypertension is uninfluenced by sympathectomy or atropine. Hypoxia itself may act directly on the muscular pulmonarv arteries causing them to constrict and hypertrophv.' Pulmonary fibrosis commonly develops in chronic radiation pneumonitis and may contribute to the pathogenesis of the vascular changes in the lung. Alveolar capillaries become compressed, distorted, and eventually obliterated by the excess collagenous tissue response. The greatly thickened and relatively avascular alveolar walls thus greatlv handicap the diffusion of oxygen from the alveolar spaces into the remaining pulmonarv capillaries. When interstitial fibrosis develops, as it does in radiation pneumonitis," the consequences to the pulmonarv vasculature can be great. The widespread obstruction and loss of the capillary bed of the lung at the alveolar level in interstitial fibrosis can lead to pulmonarv arterial hypertension, which in turn can produce hypertensive changes in the muscular pulmonary arteries and arterioles. In addition, severe injurv to the respiratory bronchioles in radiation pneumonitis 3 can lead to fibrous obliteration of these structures and when this occurs in the lung already compromised by interstitial fibrosis, a situation analogous in manv ways to "honeycomb lung" emerges. In addition to radiation, many other diseases can lead to interstitial fibrosis and honeycombing, with its attendant loss of capillaries, alveolar-capillary block, chronic hvpoxia due to reduced local ventilation, and the development of increased pulmonarv vascular resistance and pulmonar hypertension. Finally, the development of pulmonarv hvpertension itself can be a major contributor to the development of progressive vascular changes in the pulmonary vessels. A slight initial rise in pressure as a consequence of
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acute vascular damage, including edema of the vessel walls, can induce medial hypertrophy which can potentiate the further development of pulmonary vascular resistance. Medial hypertrophy as well as secondary intimal changes which narrow the arterial lumen can, therefore, cause further elevation of the pulmonary arterial pressure. A vicious circle can thus become established in which the initial vascular changes induce increased vascular resistance, which subsequently leads to pulmonary hypertension and its morphologic sequelae. A wide variety of pulmonary vascular lesions have been described in pulmonary hypertension, including medial hypertrophy, atherosclerosis, fibrosis of the intima, elastosis, plexiform lesions, dilated thin-walled arterial branches, fibrinoid necrosis of the media, and thrombosis with recanalization. While many of these changes were encountered in our dogs with radiation pneumonitis, we believe that the key lesion suggesting pulmonary hypertension was the development of fibrinoid vascular necrosis at times far removed from the period over which the radiation dose was delivered. The relatively high incidence of fibrinoid necrosis or necrotizing arteritis in idiopathic pulmonary hypertension tends to single this lesion out from other lesions seen in hypertensive pulmonary vascular disease.47 Thus, necrotizing arteritis when present is characteristic but not pathognomonic of pulmonary hypertension. Some authors 36 suggest that necrosis of the media suggests that the pulmonary hypertension was of rather sudden onset and was exaggerated to extreme heights. In our series of dogs, fibrinoid necrosis was seen relatively soon after exposure, largely in bronchial arteries, and as a late-occurring lesion in the pulmonary arterioles of dogs with welldeveloped radiation pneumonitis, pulmonar'y fibrosis, and obliterative vascular lesions. We believe that the former lesions may represent direct radiation effects while the latter, appearing well after the radiation dose was delivered, are manifestations of pulmonary hypertension. The cardiac hypertrophy seen in affected dogs tends to support this proposition. The pulmonary vascular lesions that developed in these dogs with radiation pneumonitis constitute an important part of the overall disease process. Pulmonary fibrosis also can occur in radiation pneumonitis; however, fibrous scar tissue in the lung eventually becomes neither ventilated nor perfused and hence can be considered physiologically resected. The development of pulmonary vascular lesions, on the other hand, can lead to self-perpetuating sequelae such as chronic hypoxia and hypertension, which increase rather than decrease in severity and importance as the disease progresses. References 1. Warren S: Effects of radiation on normal tissues. VI. Effects of radiation on the cardiovascular system. Arch Pathol 34:1070-1084, 1942
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2. Warren S. Gates 0: Radiation pneumonitis: Experimental and pathologic observations. Arch Pathol .30:440-460. 1940 3. Warren S, Spencer J: Radiation reaction in the lung. Am J Roentgenol 43:682-701, 1940
4. Jennings FL, Turner RA: Radiosensitivity of epithelium and endothelium in the lungs. Radiat Res 22:201. 1964 (Abstr) 3. Mladrazo A. Suzuki Y. Churg J: Radiation pneumonitis: Ultrastructural changes in the pulmonary alveoli following high doses of radiation. Arch Pathol 96:262-268, 1973 6. Phillips TL; An ultrastructural study of the development of radiation injury in the lung. Radiology 87:49-54, 1966 7. Reinhold HS, Buisman GH: Radiosensitivity of capillary endothelium. Br J Radiol 46:54-57, 1973 8. Benson EP: Radiation injury to large arteries. Radiology 106:195-197, 1973 9. Haywvard RH: Arteriosclerosis induced by radiation. Surg Clin North Am 52:359-366, 1972 10. Louis EL. NcLoughlin MJ. Wortzman G: Chronic damage to medium and large arteries following irradiation. J Can Assoc Radiol 25:94-104, 1974 11. Poon TP, Kanshepolsky J, Tehertkoff V: Rupture of the aorta due to radiation injury: Report of a case and electron microscopic study. JANIA 205:875-878, 1968 12. Stetson CG. Boland J: Experimental radiation pneumonitis: Radiographic and pathologic correlation. Cancer 20:2170-2183. 1967 1:3. Hopewvell JW: The late vascular effects of radiation. Br J Radiol 47:157-158. 1974 14. Jennings FL. Arden A: Development of experimental radiation pneumonitis. Arch Pathol 71:437-446. 1961 13. Phillips TL Benak S. Ross G: Ultrastructural and cellular effects of ionizing radiation. Front Radiat Ther Oncol 6:21-43. 1972 16. Kurohara S. Casarett GW: Effects of single thoracic x-ray exposure in rats. Radiat Res 52:263-290, 1972 17. Jennings FL. Arden A: Development of radiation pneumonitis: Time and dose factors. Arch Pathol 74:351-360 1962 1 8. Freid JR, Goldberg H: Post-irradiation changes in the lungs and thorax: The clinical. roentgenological, and pathological study with emphasis on late and terminal stages. Am J Roentgenol 43:877-895, 1940 19. Hampton JC. Rosario B: Permeability of arterial internal elastic laminae in irradiated mice. Exp Mol Pathol 17:307-316, 1972 20. Jacobsen %'C: The deleterious effects of deep roentgen irradiation on lung structure and function. Am J Roentgenol 44:2.35-249. 1940 21. Hobbs CH, Barnes JE. NMcClellan RO, Chiffelle TL Jones RK, Lundgren DL. \Mauderly JL. Pickrell JA. Rypka EW': Toxicity in the dog of inhaled 90Y in fused clay particles: Early biological effects. Radiat Res 49:4.30-460, 1972 22. \cClellan RO. Barnes JE. Boecker BB, Chiffelle TL, Hobbs CH, Jones RK. Mauderly JL. Pickrell JA. Redman HC: Toxicity of beta-emitting radionuclides inhaled in fused clay particles: An experimental approach. Morphology of Experimental Respiratory Carcinogenesis. Edited by P Nettesheim, MG Hanna Jr, JW Deatherage. AEC Symposium Series 21. Springfield. 'Va.. US Department of Commerce. 1970. pp 395-415 23. Slauson DO. Hahn FF. Benjamin SA, Chiffelle TL. Jones RK: Inflammatory se(iiences in acute pulmonary radiation injury. Am J Pathol 82:549-572. 1972 24. Mauderly JL. Pickrell JA. Hobbs CH, Benjamin SA. Hahn FF, Jones RK. Barnes JE: The effects of inhaled 9Y fused clay aerosol on pulmonary function and related parameters of the beagle dog. Radiat Res 56:8.3-96, 1973 25. Bames JE. McClellan RO. Hobbs CH. Kanapilly GM1: Toxicity in the dog of
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inhaled 90Y in fused clay particles: Distribution, retention, kinetics and dosimetry. Radiat Res 49:416-429, 1972 Boecker BB, Aguilar FL, Mercer TT: A canine inhalation exposure apparatus utilizing a whole-body plethysmograph. Health Phys 10:1077-1089, 1064 Cuddihy RG, Browstein DG, Raabe OG, Kanapilly GM: Respiratory tract deposition of inhaled polydisperse aerosols in beagle dogs. J Aerosol Sci 4:35-43, 1973 Raabe OG: Generation and characterization of aerosols. Inhalation Carcinogenesis. AEC Symposium Series 18. Edited by MG Hanna Jr, P Netesheim, JR Gilbert. Springfield, Va., US Department of Commerce, 1970, pp 123-172 Bielfelt SW, Wilson AJ, Redman HC, McCellan RO, Rosenblatt LS: A breeding program for the establishment and maintenance of a stable gene pool in a beagle dog colony to be utilized for long-term experiments. Am J Vet Res 30:2221-2229, 1969 Redman HC, Wilson AJ, Biefelt SW, McClellan RO: Beagle dog production experience at the Fission Product Inhalation Program (1961-1968). Lab Anim Care 20:61-68, 1970
31. Kanapilly GM, Newton GJ: A new method for the separation of multicurie (quantities of 90Y from 'SR. Int J Appl Radiat Isot 22:567-575, 1971 32. Hasleton PS, Heath D, Brewer DB: Hypertensive pulmonary vascular disease in states of chronic hypoxia. J Pathol Bacteriol 95:431-440, 1968 33. Heath D: Hvpoxic hypertensive pulmonary vascular disease. Prog Resp Res 5:13-16, 1970 34. De Fraiture DWH: Een zeldzame oorzaak van chronisch cor pulmonale. Ned T (Geneesk 101:399-401, 1957 35. Naeve RL: Alveolar hypoventilation and cor pulmonade secondary to damage to the respiratory center. Am J Cardiol 8:416-419, 1961 36. Wagenvoort CA, Heath D, Edwards JE: Pathology of the Pulmonary Vasculature. Springfield, Ill., Charles C Thomas, Publishers, 1964 37. Naeye RL: Kyphoscoliosis and cor pulmonale: A study of the pulmonary vascular hed. Am J Pathol 38:561-573, 1961 38. Arias-Stella J, Recavarren S: Right ventricular hypertrophy in native children living at high altitude. Am J Pathol 41:54-64, 1962 39. Naeye RL: Hypoxemia and pulmonary hypertension: A study of the pulmonary vasculatuire. Arch Pathol 71:447-452, 1961 40. Pefialoza D, Sime F, Banchero N, Gamboa R: Pulmonary hypertension in healthy man horn and living at high altitudes. Med Thorac 19:449-460, 1962 41. Will DH, Alexander AF, Reeves JT, Grover RF: High altitude-induced pulmonary hypertension in normal cattle. Cire Res 10:172-177, 1962 42. Valdivia E: Right ventricular hypertrophy in guinea pigs exposed to simulated high altituide. Circ Res 5:612-616, 1957 43. Dagher IK, Mishalanv HG, Simeone FA: Mechanisms of pulmonary hypertension in acuite hypoxia. J Thorac Cardiovase Surg 42:743-754, 1961 44. Harris P: Influence of acetylcholine on the pulmonary arterial pressure. Br Heart J 19:272-278. 1957
45. Pickrell JA, Harris DV, Mauderly JL, Hahn FF: Altered collagen metabolism in radiation-induced interstitial pulmonary fibrosis. Chest 69(Suppl):311-316, 1976 46. Heppleston AG: The pathology of honeycomb lung. Thorax 11:77-93, 1956 47. Heath D, Whitaker W, Brown JW: Idiopathic pulmonary hypertension. Br Heart J 19:8.3-92, 1958
Acknowledgments The authors greatly appreciate the efforts of the staff of the Inhalation Toxicology Research Institute, Lovelace Foundation for Medical Education and Research, in the conduct of these experiments. Without the input of manv individuals with diverse skills, this research could not have been performed. Dr. C. R. Crain and Laurel Southard were particularly helpful with the statistic analyses.
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Figure 1-Small pulmonary arteriole showing edema and mononuclear leukocyte infiltration of the vessel wall. There is irregular interstitial fibrosis in the surrounding lung parenchyma. Dog died 70 days after exposure, with cumulative dose to lung of 29,000 rads. (H&E, x 175) Figure 2-Medium-sized pulmonary vein with marked edema of vessel wall accompanied by a modest mononuclear leukocyte infiltrate. Dog died 64 days after exposure; the cumulative dose to lung was 30,000 rads. (H&E, X 280)
Figure 3-Fibrinoid necrosis in bronchial artery of dog dying 24 days after exposure with cumulative dose to lung of 41,000 rads. Note the smudgy appearance of the media, nuclear pyknosis, and perivascular leukocytic reaction. (H&E, x 200)
Figure 4-Fibrinoid necrosis and vasculitis in lung of dog dying at 214 days after exposure, with total cumulative dose to lung of 11,000 rads. Note the hemorrhage into the vessel wall, nuclear pyknosis, fibrinoid change, and cellular infiltration. (H&E, x 250)
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Dogs exposed by inhalation to an aerosol of fused aluminosilicate particles containing the radionuclide yttrium 90 developed radiation pneumonitis. The aerosol had a mean aerodynamic diameter of 0.8 to 1.2 p with a og of 1.6 to 1.9. The 36 dogs included in this report received initial lung burdens of 590 to 5200 sCi "Y/kg body weight and died at 7.5 to 237 days after exposure with total cumulative radiation doses to lung of 9300 to 70,000 rads. Vascular lesions in the lungs were marked. Early changes included edema of vessel wails with leukocytic infiltration, dilation of perivascular lymphatic channels, and occasional periarterial lymphangiectasia. Splitting and reduplication of the elastica were occasionally visible. The most striking inflammatory vascular changes were vasculitis and fibrinoid necrosis, which involved bronchial and pulmonary vessels at somewhat different times. Such lesions were often segmental and included fibrinoid necrosis and a variable leukocytic infiltrate in and around the actively involved lesions. Vasculitis was most commonly seen in small muscular arterioles, but veins and venules also occasionally exhibited similar inflammatory lesions. Progressive vascular inflammation led to extensive intimal proliferative lesions and fibromuscular hypertrophy with eventual fibrous accumulation around blood vessels, obliterative intimal and medial thickening, and luminal narrowing. Such changes eventually formed the morphologic basis for increased pulmonary vascular resistance and the development of cardiac dilatation and hypertrophy reflecting pulmonary hypertension. (Am J Pathol 88:635-654, 1977)
PULMONARY VASCULAR LESIONS have long been noted to occur in the lung injured bv radiation, although relatively little attention has been given to their pathogenesis or sequence of development. Radiation-induced injury to small vessels and to endothelial cells is well documented in both man 1-3 and experimental animals.4 7 Large and medium-sized arteries are apparently less sensitive to radiation injury, although pathologic changes can occur.2'8- Acute damage with necrosis and arterial rupture has been reported,11 but relativelyr few examples of chronic changes leading to arterial narrowing and occlusion have been described.10 Warren and Spencer3 emphasized the diagnostic value of vascular lesions in the later stages of chronic radiation pneumonitis. From the Department of Pathology. Cornell University College of Veterinary Medicine. Ithaca. New York, and the Lovelace Foundation for Medical Education and Research. Albuquerque. New Mexico Supported by Contract E(29-2)-013 from the US Energy Research and Development Administration. This research was conducted in facilities full! accredited b\- the American Association for Accreditation of Laboratorv Animal Care. Accepted for publication April 22. 1977. Address reprint requests to Dr. David 0 Slauson. Department of Pathology. College of Veterinary Medicine, Cornell Universitv, Ithaca, NY 14853 635
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Marked hyaline fibrosis and vascular alterations with increased alveolar septal thickening and resultant obliteration of the alveolar capillaries as well as distinct diminution of the lumina of larger vessels were noted. Edema of vessel walls along with endothelial swelling and vacuolation, coarsening and reduplication of the elastica, and hyaline swelling of both veins and arteries in radiation pneumonitis were described by Warren and Gates,2 although leukocytic infiltration was not a prominent feature. Dogs given 4200 to 8400 rads external thoracic radiation fractionated over a 5week period developed occasional occlusive vascular changes with fibromuscular and intimal hyperplasia, reduplication of the internal elastic membrane, and rare thrombosis.12 Lesions were more prominent in larger arteries than in smaller arterioles. Hopewell 13 also described late-occurring segmental occlusive lesions in irradiated vessels and suggested a relationship to the development of hypertension. Pulmonary vascular lesions were described by Jennings and Arden 14 in rats given external thoracic x-irradiation in either single 3000 R-exposures or fractionated doses of 600 R/week for 5 weeks. Marked vascular thickening, observed at 60 days after exposure in the fractionated group, was characterized by prominent subintimal hyalinization. Rats exposed to between 500 and 3000 rads of external x-irradiation and examined for ultrastructural changes in heart, skeletal muscle, lung, and kidney revealed that the common site of damage in all four organs was the vascular endothelium.15 Constrictive changes have also been described in the pulmonary arterioles of rats 1 to 2 weeks after exposure to 3600 rads of single external thoracic x-irradiation, with thickening and degenerative changes in the media.16 Jennings and Arden 17 summarized the histopathologic findings in 173 cases of radiation pneumonitis secondary to thoracic radiotherapy (dose, 500 to 6000 R) and noted striking fibrin deposits but relatively infrequent obliterative endoarteritis in arterioles; only 14% of their cases exhibited significant vascular pathologic changes. Fried and Goldberg 18 studied postirradiation changes in the lung and thorax and called attention to hypertrophy of the right ventricle, which they attributed to narrowing of the alveolar capillaries with increased resistance throughout the lung. They proposed a terminal state of mediastinal fibrosis with stenosis of the pulmonary veins, hyperemia of the right ventricle, and eventual congestive failure. Late-occurring vascular lesions in man caused by radiation have often been attributed to damage to the internal elastic lamina or to the vasa vasorum,10 a suggestion which has been further emphasized by experimental work.19 In an additional study, Jacobsen 20 indicated that irradiated arteries and arterioles underwent obliteration by intimal thickening, and recent thrombi were found that had undergone recanalization.
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Relatively little attention has been paid to the development of lesions in the pulmonarv vasculature in radiation pneumonitis caused by inhaled internal emitters, although vascular lesions have been described.21-23 The present study details the sequential development of pulmonarv vascular lesions in 35 dogs with radiation pneumonitis caused bv the pulmonarv deposition of a beta-emitting radionuclide, yttrium 90, in relatively insoluble fused aluminosilicate particles. The clinicopathologic features of radiation pneumonitis following 9Y inhalation in dogs and the overall histopathology of the lung in this disease have been reported.21'23'24 The present report is focused onlv on the pulmonarv vascular changes. Materials and Methods -I
-
The animals used in this studv were drawn from one of a large number of inhalation studies involving the beagle dog being conducted at the Inhalation Toxicology Research Institute, Lovelace Foundation for Medical Education and Research. The experimental approach used in these studies is directed toward evaluating the influence of radiation dose rate and total radiation dose on dose-response relationships.2 The initial lung burdens used were predicted to result in early deaths (to 1 * ear after exposure) due to inflammatory pulmonary changes; in deaths at later times (after 1 vear) due to moderate to marked pathologic changes such as pulmonary fibrosis at intermediate dose levels; and in more subtle and late-occurring changes several years after exposure, including pulmonary neoplasia at lower initial lung burden levels. For the preparation of this report. 36 dogs which died or were sacrificed during the first year after exposure to relatively high initial lung burden levels of "Y inhaled in aluminosilicate particles were used. Detailed descriptions of the aerosol apparatus, exposure methods, isotope and aerosol preparation, and other experimental variables have been previously reported. 5 "
Expermtal Procedures and Desin All dogs used in these studies were derived from our ow-n closed breeding colony of beagle dogs,"930 and were exposed as young adults at 12 to 14 months of age. Each dog was exposed individually using an apparatus previously described.' For preparation of the aerosol, "*Y was separated from its parent isotope, strontium 90, by the selective formation of colloidal hydrogen phosphate at a pH of 5.0.31 The separated 9Y was transferred into montmorillonite clav by ion exchange. The clay was then filtered, resuspended into distilled water, and placed in a nebulizer type aerosol generator.2 The nebulized aerosol was passed through a column heated at 1100 C and then cooled by the addition of diluting air before delivery to the dog for inhalation. The resultant fused aluminosilicate particles containing "Y were spherical in shape, had a size distribution curve that was similar to a normal logarithmic curve, and had an activity median aerodynamic diameter ranging from 0.8 to 1.2 A and geometric standard deviations of 1.6 to 1.9. Further details on the experimental procedures have been reported.25 Yttrium 90 was selected for study- because it represents a typical short-lived high-energy (Em,,a = 2.28 MeV) beta emitter with a physical half-life of about 64.2 hours and because it is one of the fission-product radionuclides found in significant quantities in a nuclear reactor inventorv after a sustained period of operation. The retention and tissue distribution pattern of "Y inhaled in fused aluminosilicate particles is that which would be anticipated for an insoluble material of the particular size being studied.' The respiratorx tract is, therefore, the primary organ system irradiated by the beta particles from 'Y. The
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retention half-life of "Y particles in the lung approximates the physical half-life of MY; that is, 90% of the infinite dose is delivered in about 9 days and the irradiation is essentially complete by 28'days after deposition. Certain of the early biologic effects of inhaled M°Y in the dog,21 23 and its effects on pulmonary function and related parameters,24 have been reported. The present report is based on histopathologic study of dogs with radiation pneumonitis which died during the first year after 90Y exposure and includes 35 animals that received initial lung burdens ranging from 590 to 5200,uCi/kg body weight and died at 7.5 to 237 days after exposure, with total cumulative radiation doses to the lung ranging from 9,300 to 70,000 rads. Dose and Dose Rate
The methods of dose calculation have been previously reported.2" The total radiation dose to lung was basically determined by the nature of the radionuclide and its decay characteristics, its physical half-life, the retention of the particles in the lung (biologic or effective half-life), and the duration of radiation exposure or time to'death. In essence, the relatively insoluble 90Y particles were inhaled, a portion was deposited deep in the lung, and was not cleared or cleared slowly; hence, the particles decayed and irradiated lung tissue at a relatively high but rapidly decreasing dose rate (Text-figure 1). Because 'Y has a high energy beta emission, the lung is irradiated in a relatively uniform fashion. Pathology A complete necropsy was performed on each animal at the time of death or after euthanasia performed because death was imminent. Tissues for histopathologic study were routinely fixed in 10% neutral buffered formalin. The lungs were fixed by bronchial perfusion and reexpanded to their approximate dimensions in an inflated state. After routine processing and paraffin embedding, sections of tissue were cut at 5 ,s and routinely stained with hematoxylin and eosin. Selected tissues were also stained with a modified elastic Masson's trichrome procedure, by routine elastic Van Gieson and Masson's trichrome methods, and by the phosphotungstic acid-hematoxylin procedure. The lungs of many dogs were cultured for viruses, bacteria, and fungi at necropsy. No significant pathogens were isolated from these lungs by routine microbiologic methods."1 n-
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The 35 dogs in this study died of radiation pneumonitis at intervals ranging from 7.5 to 237 days after exposure. The time of death after inhalation of 90Y in fused aluminosilicate particles correlated well with the total cumulative radiation dose to lung (Text-figure 2). Thus, animals receiving in excess of 20,000 rads total dose to lung did not survive bevond 90 days after exposure, whereas dogs receiving somewhat lower total radiation doses did not die of acute pneumonitis but lived longer after exposure and hence were included in the group at risk for the development of more chronic pulmonary lesions including vascular lesions. Puary Vascuiar Pa
Virtually all of the dogs in this studs developed pulmonarx vascular lesions which characteristically had one of several histologic patterns, as described below. It is important to note, however, that the time sequence of appearance of the vascular changes described here varied, and adequate histopathologic evaluation can come only from studying as many sections of tissue as possible. Various lesions were seen within vessels from the same lung, since not all of the pulmonarv vessels in a single case had progressed to the same stage of abnormality at the same time. Thus we frequently encountered the situation where some muscular pulmonarv arteries in a dog known to have progressed to a more advanced level of 7G _
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a o vival time after inhalation exposure of dogs to "Y in Fo 40 fused aluminosilicate par- 0, \ ticles as a function of radiation dose to lung. There I is reasonabl good correlation between survival time O0 a 20 and dose. L.J
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vascular disease still showed early changes. For this reason, one animal may demonstrate the histologic features of several different degrees and forms of pulmonary vascular injury. Hence, in this discussion and in the figure legends, the degree of damage relates only to the particular vessel shown and not necessarily to the animal as a whole. Where possible, we have indicated the time sequence for the development of various forms of vascular lesions. Edema and Vascular Infiltration
A commonly encountered early vascular event in the lung injured by radiation was the presence of edema within vessel walls accompanied by leukocytic infiltration into the edematous media and adventitia as well as perivascular leukocytic accumulation. Dilation of perivascular lymphatic chanhiels and occasional periarterial lymphangiectasia were also noted. In Qontrast to neutrophils, which were the major infiltrating cells in lesions characterized by fibrinoid necrosis with arteritis, the leukocytes most commonly encountered in these early edematous vessel lesions were generally lymphocytes, with only scattered neutrophil polymorphonuclear le'ukocytes present (Figure 1). Such changes were encountered most commonly within and about small thin-walled pulmonary arterioles but were also seen in pulmonary veins (Figure 2). While such changes were often more commonly seen in the acute phases of lung injury (to 60 days after exposure), they were also encountered in the lungs of animals dying at later times after exposure, at a time when pulmonary fibrosis and occlusive vascular lesions were well developed. Vasculitis and Fibnnoid Vascular Necrosis
Lesions characteristic of fibrinoid vascular necrosis were seen in many of the dogs. These lesions were typically seen at two different time frames in the course of events after exposure and involved both the pulmonary and systemic (bronchial) arteries. At early times after exposure (less than 1 month), fibrinoid necrosis primarily involved the bronchial arteries (Figure 3); similar changes were less commonly encountered in the muscular pulmonary arteries and arterioles. Conversely, the second time-frame for the appearance of fibrinoid necrosis was late in the progression of the disease (over 150 days after exposure), when the lesions almost exclusively involved muscular pulmonary arteries and arterioles, with sparing of the bronchial arteries (Figure 4). Although some variation was seen in this two-phase appearance of fibrinoid vascular necrosis in the two circulations, the pattern generally held true. These lesions characteristically occurred in medium-sized and small
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muscular arterioles and included necrosis and swelling of the media, which often presented a glossy or smudgy appearance, with loss of nuclei, and usually took the staining reactions for fibrin. In some vessels, the medial necrosis was accompanied by an intense, reactive acute inflammatory response around and within the necrotic vessel wall with prominent accumulation of neutrophil leukocytes. There was often loss or disruption of the elastic laminae in affected vessels, and the lesions in general were either segmental or uniform in their involvement of the vessel wall. Hemorrhage into the media was also encountered. Occasionally vessels exhibited changes suggestive of a healing phase of previous vascular necrosis. Such arteries were characterized by the appearance of granulation tissue within the media or by intensely staining hemosiderin or hematoidin deposits within the vessel walls, reflecting prior hemorrhage. Sometimes small capillaries ramified through such granulating lesions, making distinction from healing thrombosis with recanalization difficult. Chronic vasculitis with intense medial lymphocytic infiltrations and luminal reduction was also sometimes seen, and suggested a healing phase of previous acute arteritis (Figure 5). Fibromuscular hypertrophy of the vascular media was a characteristic feature of the middle and later stages of the development of chronic radiation pneumonitis. It was often seen in conjunction with intimal proliferation, although it sometimes appeared to have preceded changes in the intima. Fibromuscular hypertrophy of the media was sometimes accompanied by medial elastosis with thickening of the elastic laminae and a marked increase in the amount of elastic fibers within the media. Fibrosis of the media between the elastic fibers and muscular bundles and fibrous replacement of the muscle bundles was also sometimes encountered. In general, medial changes were less commonly encountered than were intimal changes, and when medial hypertrophy was present it was often accompanied by intimal proliferation which in combination sometimes served to virtually occlude the vascular lumen (Figure 6). hvn Pioaon an Thk_rrg
Lesions affecting the intima of vessels were commonly encountered. It was often difficult to place such lesions in a time frame, as proliferative intimal lesions (early changes) were often seen in the same lungs which exhibited occlusive and relatively acellular (chronic) intimal sclerotic changes. Thus, it seemed that the process of intimal thickening was a continuing one, with variations in severity, extension, and histologic
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appearance being continijally present. It was necessary, therefore, to try to reconstruct the sequence of appearance of various intimal changes as they related to individual vessels rather than to the lung as a whole or to the time after radiation injury at which those lungs were examined. For purposes of this report, we have considered that the cellular intimal proliferative changes (especially endothelial proliferation) represented early events, whereas changes in elastic tissue, collagen, reticulin, and acellular fibrosis of the intima were late-occurring changes within any one individual vessel. Intimal lesions encountered in the dogs in this study were often focal and were best appreciated when a vessel was sectioned in a nearly longitudinal plane. Proliferative intimal lesions were encountered as early as 24 days after exposure and as late as 220 days after exposure. They generally consisted of either a loose proliferation of endothelial elements or of accumulations of several cell layers between the internal elastic membrane and the endothelium. In more advanced lesions, the marked cellular proliferation was gradually replaced by a preponderance of collagen and reticulin fibers with marked luminal reduction (Figure 7). Admixed elastic fibers were sometimes encountered, and there often was an outer circular fibrous layer with elastosis and an inner, more cellular layer, suggesting different phases in the process of intimal fibrosis. Severe intimal proliferative changes were sometimes seen in larger, elastic arteries as well as in the smaller, muscular walled vessels (Figure 8). Chronic Obliterative Vascular Lesions
Lesions which partially or totally occluded the vascular lumina were seen with increasing frequency related to the length of time after exposure. Thus, lungs from dogs surviving to 150 days after exposure almost always exhibited some chronic obliterative vascular lesions, and the longest surviving dogs in this experiment had the most severe lesions. In general, it was difficult to reconstruct the histologic events that led to fibrous obliteration of the vasculature. The lesions were often more pronounced in medium-sized and smaller vessels than in larger muscular arteries or elastic arteries and were generally widespread within all lobes of the lung. Bronchial arteries were not spared by the obliterative process although the changes were often more pronounced in branch pulmonary arterioles in the lung parenchyma itself. Within some larger vessels, the luminal obliteration was more cellular and proliferative, with fibroplasia and peripheral recanalization particularly prominent (Figure 9). Around smaller muscular arterioles in the lung parenchyma where obliterative changes were most pronounced, a dense collar of collagenous tissue often
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surrounded the adventitial aspects of the obliterated vessels (Figure 10). When sectioned longitudinally, such scarred and totally obliterated vessel tracts were difficult to define as vascular structures. Obliterative changes were also seen in the smallest identifiable pulmonary arterioles, and capillary level obliterative changes were often seen in association with the process of interstitial fibrosis. NVonpuKAry Cardiovasr Parameters
Right ventricular hvpertrophy, often with marked dilatation, was seen in many of the dogs at necropsy. Since we did not directly quantitate pulmonarv hypertension in this group of dogs by measuring pulmonary arterv mean pressure and pulmonary vascular resistance, comparisons of cardiac weights were made between the exposed group of dogs and a similar number of age-matched and sex-matched control beagle dogs which had been sacrificed for other studies. The mean age of the control group was not different from that of the dogs with pneumonitis when tested statisticallv (P = 1.65). None of the control dogs exhibited pulmonarv or cardiac lesions. Comparisons of heart weight/body weight ratios were not useful, as many of the dogs with pneumonitis exhibited significant weight loss, which unfairly biased the data. We therefore compared only total heart weight between the exposed and control groups as an index of the cardiac manifestations of pulmonary vascular disease. As shown in Table 1, a highly significant difference (P < 0.001) was found between the means of the heart weights even though there was considerable overlap in the ranges for the two groups. This difference in part reflects a substantial increase in heart weight for dogs dying of pneumonitis at longer times after exposure when occlusive pulmonary vascular lesions were well developed, rather than a uniform or generalized change throughout the exposed group. Table 1-Heart Weight in Dogs Wih Radiation Pneumonitis Compared to Sex and Age Matched Controls Pneumonitis Controls No.ofdogs 34 29 Male 17 13 Female 17 16 Mean age ± SE (Days)* 448 27 509 10 Heart weight (g) Mean ± 1 SDt 82.9 ± 18.6 61.2 ± 15.5 SEM 3.2 2.6 Range 43.3-121.5 45.3-99.3 Mean age + SE in days; the means are not statistically different (P = 1.65). t Mean heart weight ± 1 SD; the means are statistically different (P < 0.001) as dtermined by a two-tailed t test.
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Discussion
The pulmonary vascular lesions described here were prominent histopathologic features of radiation pneumonitis as produced experimentally in the dog by inhalation exposure to aluminosilicate particles containing the radionuclide "Y. Although the vascular component of this disease was prominent, it was not unique, as similar vascular lesions have been reported in dogs that inhaled 144Ce in aluminosilicate particles 22 as well as in animals and man that received external radiation.2'3 The pathogenesis of the pulmonary vascular lesions seen in radiation pneumonitis is poorly understood. We believe that there are four major factors involved in the development of these lesions: direct radiation injury, local chronic hypoxia, interstitial fibrosis, and the development of pulmonary hypertension.
Direct radiation-induced injury to the lung and its vasculature certainly is a primary cause of damage. Direct vascular injury is marked in radiation pneumonitis and probably initiates an inflammation and repair sequence that leads to more chronic changes as sequelae of the tissue repair process. Some of the early necrotizing changes in bronchial arteries, for example, may represent direct radiation-induced changes. The progressive development of changes in the pulmonary vasculature as detailed here, however, suggests that processes other than those associated with the initial injury are necessary to the development and pathogenesis of the more chronic lesions. This is particularly evident when one considers that the lesions persist and progress at much later times after exposure than the 9 days required to deliver 90% of the infinite dose from inhaled 90Y particles, and also because lesions evidencing various developmental stages were often found in the same lung. Thus, while direct radiation-induced injury may initiate the inflammatory process, the persistance and progression of the vascular lesions suggest that continuing radiation is not necessary for the maintenance and amplification of the injury process. In addition to the repair processes activated after initial vascular injury, changes secondary to chronic hypoxia, interstitial fibrosis, and the development of pulmonary Iypertension as sequelae to the initial insult seem implicated in the pathogenesis of the progressive vascular lesions. Chronic hypoxia can lead to pulmonary vascular disease 32,33 and may well be a contributing cause in the pathogenesis of the vascular changes seen in these dogs with radiation pneumonitis. The pathophysiologic features in dogs have been previously detailed.2'24 Pulmonary vascular changes have also been observed in a variety of other situations in which chronic hypoxia is present, including progressive muscular dystrophy with shallow breathing,34 damage to the respiratory center,35 simple obesity,36
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and various other disease states.32 The hypoxia in such situations is due to inadequate intake of ambient air of normal oxygen content and leads to carbon dioxide retention. Similar changes have been observed in patients with kyphoscoliosis who develop "heart failure of the hunchback.'"37 Chronic hypoxia can also result during normal breathing or even hvperventilation of air with a low oxygen content, such as encountered at high altitudes. Such a situation can lead to right ventricular hypertrophy, increased pulmonary vascular resistance and pulmonarv hvpertension, and marked medial hypertrophy of small muscular pulmonarv arteries.3' Similar observations have been made in animals living at high altitude41 or under conditions simulating high altitude.42 Chronic hvpoxia can also lead to pulmonary arterial hypertension in experimental animals," but the mechanism of its development is uncertain. It mav be brought about and maintained by constriction of the pulmonarv vascular tree. Acetylcholine administration can lower the elevated arterial pressure," although the autonomic nervous svstem plavs a questionable role, since pulmonary hypertension is uninfluenced by sympathectomy or atropine. Hypoxia itself may act directly on the muscular pulmonarv arteries causing them to constrict and hypertrophv.' Pulmonary fibrosis commonly develops in chronic radiation pneumonitis and may contribute to the pathogenesis of the vascular changes in the lung. Alveolar capillaries become compressed, distorted, and eventually obliterated by the excess collagenous tissue response. The greatly thickened and relatively avascular alveolar walls thus greatlv handicap the diffusion of oxygen from the alveolar spaces into the remaining pulmonarv capillaries. When interstitial fibrosis develops, as it does in radiation pneumonitis," the consequences to the pulmonarv vasculature can be great. The widespread obstruction and loss of the capillary bed of the lung at the alveolar level in interstitial fibrosis can lead to pulmonarv arterial hypertension, which in turn can produce hypertensive changes in the muscular pulmonary arteries and arterioles. In addition, severe injurv to the respiratory bronchioles in radiation pneumonitis 3 can lead to fibrous obliteration of these structures and when this occurs in the lung already compromised by interstitial fibrosis, a situation analogous in manv ways to "honeycomb lung" emerges. In addition to radiation, many other diseases can lead to interstitial fibrosis and honeycombing, with its attendant loss of capillaries, alveolar-capillary block, chronic hvpoxia due to reduced local ventilation, and the development of increased pulmonarv vascular resistance and pulmonar hypertension. Finally, the development of pulmonarv hvpertension itself can be a major contributor to the development of progressive vascular changes in the pulmonary vessels. A slight initial rise in pressure as a consequence of
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acute vascular damage, including edema of the vessel walls, can induce medial hypertrophy which can potentiate the further development of pulmonary vascular resistance. Medial hypertrophy as well as secondary intimal changes which narrow the arterial lumen can, therefore, cause further elevation of the pulmonary arterial pressure. A vicious circle can thus become established in which the initial vascular changes induce increased vascular resistance, which subsequently leads to pulmonary hypertension and its morphologic sequelae. A wide variety of pulmonary vascular lesions have been described in pulmonary hypertension, including medial hypertrophy, atherosclerosis, fibrosis of the intima, elastosis, plexiform lesions, dilated thin-walled arterial branches, fibrinoid necrosis of the media, and thrombosis with recanalization. While many of these changes were encountered in our dogs with radiation pneumonitis, we believe that the key lesion suggesting pulmonary hypertension was the development of fibrinoid vascular necrosis at times far removed from the period over which the radiation dose was delivered. The relatively high incidence of fibrinoid necrosis or necrotizing arteritis in idiopathic pulmonary hypertension tends to single this lesion out from other lesions seen in hypertensive pulmonary vascular disease.47 Thus, necrotizing arteritis when present is characteristic but not pathognomonic of pulmonary hypertension. Some authors 36 suggest that necrosis of the media suggests that the pulmonary hypertension was of rather sudden onset and was exaggerated to extreme heights. In our series of dogs, fibrinoid necrosis was seen relatively soon after exposure, largely in bronchial arteries, and as a late-occurring lesion in the pulmonary arterioles of dogs with welldeveloped radiation pneumonitis, pulmonar'y fibrosis, and obliterative vascular lesions. We believe that the former lesions may represent direct radiation effects while the latter, appearing well after the radiation dose was delivered, are manifestations of pulmonary hypertension. The cardiac hypertrophy seen in affected dogs tends to support this proposition. The pulmonary vascular lesions that developed in these dogs with radiation pneumonitis constitute an important part of the overall disease process. Pulmonary fibrosis also can occur in radiation pneumonitis; however, fibrous scar tissue in the lung eventually becomes neither ventilated nor perfused and hence can be considered physiologically resected. The development of pulmonary vascular lesions, on the other hand, can lead to self-perpetuating sequelae such as chronic hypoxia and hypertension, which increase rather than decrease in severity and importance as the disease progresses. References 1. Warren S: Effects of radiation on normal tissues. VI. Effects of radiation on the cardiovascular system. Arch Pathol 34:1070-1084, 1942
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2. Warren S. Gates 0: Radiation pneumonitis: Experimental and pathologic observations. Arch Pathol .30:440-460. 1940 3. Warren S, Spencer J: Radiation reaction in the lung. Am J Roentgenol 43:682-701, 1940
4. Jennings FL, Turner RA: Radiosensitivity of epithelium and endothelium in the lungs. Radiat Res 22:201. 1964 (Abstr) 3. Mladrazo A. Suzuki Y. Churg J: Radiation pneumonitis: Ultrastructural changes in the pulmonary alveoli following high doses of radiation. Arch Pathol 96:262-268, 1973 6. Phillips TL; An ultrastructural study of the development of radiation injury in the lung. Radiology 87:49-54, 1966 7. Reinhold HS, Buisman GH: Radiosensitivity of capillary endothelium. Br J Radiol 46:54-57, 1973 8. Benson EP: Radiation injury to large arteries. Radiology 106:195-197, 1973 9. Haywvard RH: Arteriosclerosis induced by radiation. Surg Clin North Am 52:359-366, 1972 10. Louis EL. NcLoughlin MJ. Wortzman G: Chronic damage to medium and large arteries following irradiation. J Can Assoc Radiol 25:94-104, 1974 11. Poon TP, Kanshepolsky J, Tehertkoff V: Rupture of the aorta due to radiation injury: Report of a case and electron microscopic study. JANIA 205:875-878, 1968 12. Stetson CG. Boland J: Experimental radiation pneumonitis: Radiographic and pathologic correlation. Cancer 20:2170-2183. 1967 1:3. Hopewvell JW: The late vascular effects of radiation. Br J Radiol 47:157-158. 1974 14. Jennings FL. Arden A: Development of experimental radiation pneumonitis. Arch Pathol 71:437-446. 1961 13. Phillips TL Benak S. Ross G: Ultrastructural and cellular effects of ionizing radiation. Front Radiat Ther Oncol 6:21-43. 1972 16. Kurohara S. Casarett GW: Effects of single thoracic x-ray exposure in rats. Radiat Res 52:263-290, 1972 17. Jennings FL. Arden A: Development of radiation pneumonitis: Time and dose factors. Arch Pathol 74:351-360 1962 1 8. Freid JR, Goldberg H: Post-irradiation changes in the lungs and thorax: The clinical. roentgenological, and pathological study with emphasis on late and terminal stages. Am J Roentgenol 43:877-895, 1940 19. Hampton JC. Rosario B: Permeability of arterial internal elastic laminae in irradiated mice. Exp Mol Pathol 17:307-316, 1972 20. Jacobsen %'C: The deleterious effects of deep roentgen irradiation on lung structure and function. Am J Roentgenol 44:2.35-249. 1940 21. Hobbs CH, Barnes JE. NMcClellan RO, Chiffelle TL Jones RK, Lundgren DL. \Mauderly JL. Pickrell JA. Rypka EW': Toxicity in the dog of inhaled 90Y in fused clay particles: Early biological effects. Radiat Res 49:4.30-460, 1972 22. \cClellan RO. Barnes JE. Boecker BB, Chiffelle TL, Hobbs CH, Jones RK. Mauderly JL. Pickrell JA. Redman HC: Toxicity of beta-emitting radionuclides inhaled in fused clay particles: An experimental approach. Morphology of Experimental Respiratory Carcinogenesis. Edited by P Nettesheim, MG Hanna Jr, JW Deatherage. AEC Symposium Series 21. Springfield. 'Va.. US Department of Commerce. 1970. pp 395-415 23. Slauson DO. Hahn FF. Benjamin SA, Chiffelle TL. Jones RK: Inflammatory se(iiences in acute pulmonary radiation injury. Am J Pathol 82:549-572. 1972 24. Mauderly JL. Pickrell JA. Hobbs CH, Benjamin SA. Hahn FF, Jones RK. Barnes JE: The effects of inhaled 9Y fused clay aerosol on pulmonary function and related parameters of the beagle dog. Radiat Res 56:8.3-96, 1973 25. Bames JE. McClellan RO. Hobbs CH. Kanapilly GM1: Toxicity in the dog of
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inhaled 90Y in fused clay particles: Distribution, retention, kinetics and dosimetry. Radiat Res 49:416-429, 1972 Boecker BB, Aguilar FL, Mercer TT: A canine inhalation exposure apparatus utilizing a whole-body plethysmograph. Health Phys 10:1077-1089, 1064 Cuddihy RG, Browstein DG, Raabe OG, Kanapilly GM: Respiratory tract deposition of inhaled polydisperse aerosols in beagle dogs. J Aerosol Sci 4:35-43, 1973 Raabe OG: Generation and characterization of aerosols. Inhalation Carcinogenesis. AEC Symposium Series 18. Edited by MG Hanna Jr, P Netesheim, JR Gilbert. Springfield, Va., US Department of Commerce, 1970, pp 123-172 Bielfelt SW, Wilson AJ, Redman HC, McCellan RO, Rosenblatt LS: A breeding program for the establishment and maintenance of a stable gene pool in a beagle dog colony to be utilized for long-term experiments. Am J Vet Res 30:2221-2229, 1969 Redman HC, Wilson AJ, Biefelt SW, McClellan RO: Beagle dog production experience at the Fission Product Inhalation Program (1961-1968). Lab Anim Care 20:61-68, 1970
31. Kanapilly GM, Newton GJ: A new method for the separation of multicurie (quantities of 90Y from 'SR. Int J Appl Radiat Isot 22:567-575, 1971 32. Hasleton PS, Heath D, Brewer DB: Hypertensive pulmonary vascular disease in states of chronic hypoxia. J Pathol Bacteriol 95:431-440, 1968 33. Heath D: Hvpoxic hypertensive pulmonary vascular disease. Prog Resp Res 5:13-16, 1970 34. De Fraiture DWH: Een zeldzame oorzaak van chronisch cor pulmonale. Ned T (Geneesk 101:399-401, 1957 35. Naeve RL: Alveolar hypoventilation and cor pulmonade secondary to damage to the respiratory center. Am J Cardiol 8:416-419, 1961 36. Wagenvoort CA, Heath D, Edwards JE: Pathology of the Pulmonary Vasculature. Springfield, Ill., Charles C Thomas, Publishers, 1964 37. Naeye RL: Kyphoscoliosis and cor pulmonale: A study of the pulmonary vascular hed. Am J Pathol 38:561-573, 1961 38. Arias-Stella J, Recavarren S: Right ventricular hypertrophy in native children living at high altitude. Am J Pathol 41:54-64, 1962 39. Naeye RL: Hypoxemia and pulmonary hypertension: A study of the pulmonary vasculatuire. Arch Pathol 71:447-452, 1961 40. Pefialoza D, Sime F, Banchero N, Gamboa R: Pulmonary hypertension in healthy man horn and living at high altitudes. Med Thorac 19:449-460, 1962 41. Will DH, Alexander AF, Reeves JT, Grover RF: High altitude-induced pulmonary hypertension in normal cattle. Cire Res 10:172-177, 1962 42. Valdivia E: Right ventricular hypertrophy in guinea pigs exposed to simulated high altituide. Circ Res 5:612-616, 1957 43. Dagher IK, Mishalanv HG, Simeone FA: Mechanisms of pulmonary hypertension in acuite hypoxia. J Thorac Cardiovase Surg 42:743-754, 1961 44. Harris P: Influence of acetylcholine on the pulmonary arterial pressure. Br Heart J 19:272-278. 1957
45. Pickrell JA, Harris DV, Mauderly JL, Hahn FF: Altered collagen metabolism in radiation-induced interstitial pulmonary fibrosis. Chest 69(Suppl):311-316, 1976 46. Heppleston AG: The pathology of honeycomb lung. Thorax 11:77-93, 1956 47. Heath D, Whitaker W, Brown JW: Idiopathic pulmonary hypertension. Br Heart J 19:8.3-92, 1958
Acknowledgments The authors greatly appreciate the efforts of the staff of the Inhalation Toxicology Research Institute, Lovelace Foundation for Medical Education and Research, in the conduct of these experiments. Without the input of manv individuals with diverse skills, this research could not have been performed. Dr. C. R. Crain and Laurel Southard were particularly helpful with the statistic analyses.
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Figure 1-Small pulmonary arteriole showing edema and mononuclear leukocyte infiltration of the vessel wall. There is irregular interstitial fibrosis in the surrounding lung parenchyma. Dog died 70 days after exposure, with cumulative dose to lung of 29,000 rads. (H&E, x 175) Figure 2-Medium-sized pulmonary vein with marked edema of vessel wall accompanied by a modest mononuclear leukocyte infiltrate. Dog died 64 days after exposure; the cumulative dose to lung was 30,000 rads. (H&E, X 280)
Figure 3-Fibrinoid necrosis in bronchial artery of dog dying 24 days after exposure with cumulative dose to lung of 41,000 rads. Note the smudgy appearance of the media, nuclear pyknosis, and perivascular leukocytic reaction. (H&E, x 200)
Figure 4-Fibrinoid necrosis and vasculitis in lung of dog dying at 214 days after exposure, with total cumulative dose to lung of 11,000 rads. Note the hemorrhage into the vessel wall, nuclear pyknosis, fibrinoid change, and cellular infiltration. (H&E, x 250)
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