Vet. Pathol. 26:479-487 (1989)

Histopathologic Findings and Energy Dispersive X-ray Spectroscopic Analysis of Experimentally Induced Foreign-body Pneumonias in Rats R. F. SLOCOMBE, M. G. EVANS,AND F. J. DERKSEN Departments of Pathology and Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI Abstract. To document the diagnostic features of foreign-body pneumonias, four commonly used orally administered medicaments were instilled into the lungs of Sprague-Dawley rats. Rats in each group received a single 0.4 ml dose of either barium sulfate suspension (BaSO,), mineral oil, Pepto-bisrnol", or Kaopectate" inoculated into a lung via a mainstem bronchus. The other lung served as a non-inoculated control. Rats were euthanatized on post-inoculation day 2 or 7 in order to document fully-developed, acute pulmonary lesions and developing, chronic pulmonary lesions, respectively. Light microscopic features of BaS0,-inoculated lungs were distinctive from changes in mineral oil-inoculated lungs at both post-inoculation days. On post-inoculation day 2, rats inoculated with BaSO, had pneumonia characterized by large numbers of alveolar macrophages containing green-to-brown granular material. There was minimal interstitial involvement. On post-inoculation day 2, mineral oil caused pneumonia characterized by giant cells and alveolar macrophages that had cytoplasms distended with variably-sized clear vacuoles. Lungs inoculated with BaSO, or mineral oil had changed little on post-inoculation day 7 compared to the light microscopic features observed on day 2. On post-inoculation day 2, rats inoculated with either Pepto-bismol" or Kaopectate had broncho-interstitial pneumonia with areas of necrosis and hemorrhage. On post-inoculation day 7, lungs inoculated with Pepto-bismol@or Kaopectatem had extensive fibrosis within alveolar lumens. Energy dispersive spectroscopy performed on sections of lung from rats given BaSO,, Pepto-bismol@,and KaopectateB yielded a unique elemental spectrum for each compound in situ on post-inoculation days 2 and 7. We conclude that pulmonary responses differ among these compounds and that energy dispersive spectroscopy is a useful diagnostic adjunct for the definitive identification of elements comprising BaSO,, Pepto-bismol@,and KaopectateB in situ in affected lungs. @

Oral medicaments may accidentally be deposited into qualitative and semi-quantitative determinations can the lungs and cause pneumonia with few specific di- be made for detectable elements.I3 Elements below agnostic features. Such commonly used compounds in atomic number 11 cannot generally be detected unless veterinary medicine include barium sulfate (BaSO,), more specialized equipment is used. Another disadmineral oil, Pepto-bismol@,and KaopectateB . Light vantage is that samples containing column-contamimicroscopic changes in pulmonary tissue affected by nating materials, such as oils or waxes, should not be these materials can be dramatic and may include gran- inserted into the scanning electron microscopy colulomatous, exudative, necrotizing, or fibrotic reac- umn. Energy dispersive spectroscopy is used increasingly tions. There may be histologic evidence of foreign material. The value of light microscopy in the reliable as a method for foreign substance detection in biomedetiological identification of foreign substances is, how- ical sciences. It has been used to identify foreign maever, often limited. Energy dispersive spectroscopy in terials in the airways of human patient^',^,^,^,'^,^^ and to Other conjunction with scanning electron microscopy is a characterize various lesions in method that can provide definitive identification of methods for identifying foreign materials in histopaththe elemental composition of pulmonary foreign ma- ologic specimens have been described." Most of these terial in situ,4-6J6including extracellular and intracel- involve tissue destruction (such as incineration) or lack the elemental specificity of energy dispersive spectroslular constituents. Advantages of the energy dispersive spectroscopy COPY. The purpose of this study was to determine the histechnique include the following: 1) the ability to find several elements simultaneously, 2) preservation of tis- tologic features of pneumonia associated with the pulsue architecture, and 3) direct visualization of the in monary inoculation of each of these compounds and situ location and distribution of foreign material. Both to determine the usefulness of energy dispersive spec479

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Fig. 1. Alveolar macrophages containing BaSO,. Minimal alveolar interstitial reaction, post-inoculation day 2. Bar

75 pm.

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Fig. 2. Thickened alveolar walls and confinement of BaSO, within macrophages, post-inoculation day 7. Bar = 120 pm.

derian gland and submaxillary salivary gland were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 pm, stained with hematoxylin and eosin, and examined by light microscopy. To examine for histologic evidence of foreign material removal by lymphatic drainage, sections of mediastinal lymph node were similarly fixed, Materials and Methods embedded, sectioned, and stained. Paraffin-embedded secThirty-four, clinically healthy, female Sprague-Dawley rats tions of lungs were cut at 3 Km, stained with hematoxylin (Charles River Corp., Portage, MI) weighing between 180 and eosin, and evaluated by light microscopy. and 200 grams were divided into four groups of eight rats For energy dispersive spectroscopic analysis, formalin-fixed each, with two rats in a fifth group inoculated with saline sections of the affected regions of the inoculated lung and serving as inoculated controls. Each rat was anesthetized with randomly chosen regions from the control lung from all groups a mixture of 10 mg/kg of xylazine (Rompun@,Haver-Lock- (except the group given mineral oil) were cut to a thickness hart, Shawnee, KS) and 90 mg/kg of ketamine hydrochloride of 5 mm, dehydrated sequentially in graded ethanols, and (Ketaseta , Bristol Laboratories, Syracuse, NY) that was in- critical point dried (Ivan Sorvall, Inc., Newtown, CT). To jected intra-mu~cularly.~ The rats then received 0.4 ml of make the specimens conductive, each sample was carboneither barium sulfate suspension (BaSO,, Micropaque Pow- coated by vapor deposition (Ladd Research Industries, Burder@Picker Corp., Cleveland, OH), mineral oil (White Oil@, lington, VT) and attached (Television Tube Coat@,GC ElecUSP 31, Amoco Oil Co., Southfield, MI), Pepto-bismolm tronics, Rockford, IL) to carbon planchets (Ernest F. Fullam (Norwich-Eaton Pharmaceuticals, Inc., Nonvich, NY),or Inc., Latham, NY). Carbon planchets were then mounted Kaopectatea (The Upjohn Co., Kalamazoo, MI) instilled onto aluminum stubs (Electron Microscopy Sciences, Fort through a polyethylene catheter (Becton, Dickinson & Co., Washington, PA), and specimens were analyzed at a magParsippany, NJ) wedged into the left sublobar bronchus via nification of 2,000 x , an accelerating voltage of 15 kiloeleca tracheotomy. The right lung served as a non-inoculated tron volts, a 60 second collection time, and a beam current control. to yield a dead time of 10-15% with a JEOL JSM-35-C Four rats from each group were euthanatized by ether an- scanning electron microscope (Japanese Electron Optics Co., esthesia (Mallinckrodt, Inc., Paris, KY) and decapitation on Tokyo, Japan) with energy dispersive spectroscopy capability post-inoculation day 2, and four other rats were killed sim- (Tracor Northern, Middleton, WI). Spectra from six ranilarly on day 7. A necropsy was performed, gross lesions were domly chosen areas from each non-inoculated control lung recorded, and sections of lung were submitted for detection sample were compared with six spectra generated from loof bacterial pathogens (including anaerobes), Mycoplasma, cations chosen at random within macroscopically affected Sendai virus, and sialodacryoadenitis virus (SDAV). Other areas from each inoculated lung sample. A mean peak height sections of lung were fixed in 10% neutral buffered formalin was calculated as previously described. l 3 Significant differvia positive pressure infusion at 25 mm Hg for 24 hours. To ences between peak heights of elements characteristic for the examine for histologic evidence of SDAV, sections of Har- inoculated compounds in treatment groups and peak heights troscopy in identifying characteristicelements of BaSO,, P e p t o - b i s m o l @, and Kaopectatem in p u l m o n a r y parenchyma.

Foreign-body Pneumonias in Rats

of non-inoculated controls were compared by a Student's t-test (P < 0.05). Suspensions of BaSO,, Pepto-bismol m, and Kaopectate" were similarly placed on carbon planchets, mounted on aluminum stubs, air-dried, carbon-coated, and analyzed with energy dispersive spectroscopy to determine the elemental composition of these compounds in the absence of pulmonary tissue. This was done to ascertain which elements were present in BaSO,, Pepto-bismol@,and Kaopectate .

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On post-inoculation day 2, the barium sulfate suspension (BaSO,) remained relatively localized to the inoculation site so that a clearly defined inoculated region of the lung was evident. No inoculum was found in the non-inoculated lung. Macroscopically, multiple foci of affected parenchyma had a chalky white appearance. Microscopically, the inoculum was found in alveolar sites adjacent to the bronchioles, with the bronchioles themselves remaining free of foreign material. Affected regions of pulmonary parenchyma were partially atelectatic. Alveolar macrophages and fewer numbers of neutrophils had phagocytized all BaSO, (Fig. l), except in focal areas where large amounts of inoculum filled alveolar lumens. The histologic appearance of BaSO, in lungs on postinoculation day 2 consisted of finely granular, yellowbrown to olive-green material that lacked birefringence when viewed with polarized light. On post-inoculation day 7, large amounts of BaSO, were entirely confined within macrophages and were associated with lymphomonocytic inflammation (Fig. 2), patchy alveolar epithelial hyperplasia, mild interstitial fibroplasia and microatelectasis. Pulmonary vessels adjacent to these lesions had prominent lymphocytic perivascular cuffing. The energy dispersive spectroscopic analysis of BaS0,-inoculated lungs was similar on post-inoculation days 2 and 7, with significant peaks of sulfur (2.3 12 KeV) and barium (4.470,4.844,5.174, and 5.535 KeV, Fig. 3). Non-inoculated lungs lacked peaks for these elements and had background levels of other endogenous elements. Mineral oil group

On post-inoculation day 2, lungs instilled with mineral oil had a gelatinous and semitranslucent appearance macroscopically. Microscopically, mineral oil-induced lesions were characterized by mild interstitial pneumonia consisting of lympho-monocytic infiltrates within alveolar septa, mild alveolar epithelial hyperplasia, and scattered accumulations of macrophages within alveolar lumens. Many macrophages had distinctive intracytoplasmic vacuoles (Fig. 4). A few giant

ENERGY (Ke V)

VFS = 2048

Fig. 3. Energy dispersive spectroscopy spectra, lung inoculated with BaSO, (upper line) compared with non-inoculated lung (lower line). Characteristic peak of sulfur (S) and multiple peaks of barium (Ba). VFS = vertical full scale.

cells were seen as well. Perivascularly, there was mild edema and mild inflammatory cell infiltrates consisting of lymphocytes, neutrophils, eosinophils, and macrophages. The most intensely inflamed regions had severe atelectasis and neutrophilic infiltrates in addition to lympho-monocytic cells. On post-inoculation day 7, the intensity of interstitial inflammation had lessened, but alveolar lumens still contained foamy macrophages (Fig. 5). Lymphocytic perivascular cuffing was prominent, and, within these infiltrates, there were scattered eosinophils and neutrophils. Medial smooth muscle hypertrophy of larger blood vessels was also noted. No birefringence of the foreign material was noted upon examination with polarized light microscopy. Energy dispersive spectroscopic analysis was not done on mineral oil-inoculated lungs because mineral oil is volatilized by the electron beam, resulting in contamination of the column housing of the scanning electron microscope. Pepto-bismols group

Pulmonary instillation of Pepto-bismol8 caused severe broncho-interstitial pneumonia that was grossly evident as either necrohemorrhagic areas or pink areas due to the color of the medicament. On post-inoculation day 2, histologic lesions were variable but consisted principally of necrohemorrhagic pneumonia with edema, fibrin, and mixed inflammatory cell infiltrates (Fig. 6). These lesions sometimes extended into bronchioles, and alveolar lumens were distended with Pepto-bismol@. Other areas had inflammation characterized by large numbers of macrophages and fewer numbers of neutrophils. In less severely affected areas, alveolar septa were thickened because of mild lymphomonocytic infiltrates and epithelial cell hyperplasia.

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Fig. 4. Alveolar macrophages with intracytoplasmic vacuoles, mineral oil, post-inoculation day 2. Lympho-monocytic cells in alveoli and mixed inflammatory cells perivascularly. Bar = 75 pm. Fig. 5. Interstitial pneumonia, mineral oil, post-inoculation day 7. Foamy macrophages within alveoli. Prominent lymphocytic perivascular cuffing. Bar = 190 pm. Fig. 6. Clumps of amphophilic amorphous material and crystals present within terminal bronchiole and its terminal branches, Pepto-bismol@,post-inoculation day 2. Fibrin and mixed inflammatory cells. Bar = 190 pm. Fig. 7. Radially arranged black spicules and ovoid crystals (arrows), Pepto-bismol@,post-inoculation day 2. Bar = 18 pm.

Pepto-bismolm had a characteristic histologic appearance in these sections. Birefringent structures of ovoid shape, 1.5 x 3 pm, were scattered throughout clumps of amorphous, slightly eosinophilic to basophilic material (Fig. 7). Also observed were black spicules radially arranged into oval aggregates, 5-15 pm in diameter (Fig. 7). On post-inoculation day 7, lesions were characterized by persistence of Pepto-bismol@ within alveolar lumens, extensive lymphocytic and granulomatous interstitial pneumonia with aggressive

fibrosis within alveolar lumens (Fig. 8), and widespread formation of foreign-body giant cells. Black, spiculate aggregates remained within the foreign material on post-inoculation day 7. There were mixed inflammatory cells in perivascular cuffs and scattered areas of medial muscular hypertrophy of vessel walls. The energy dispersive spectroscopic analysis of Pepto-bismol@-inoculated lungs was similar on postinoculation days 2 and 7 and revealed significant peaks of aluminum (1.475 KeV), silica (1.746 KeV), and bis-

Foreign-body Pneumonias in Rats

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Fig. 9. Energy dispersive spectroscopic spectra, lung inoculated with Pepto-bismolm (upper line) compared with non-inoculated lung (lower line). Characteristic peaks of aluminum (Al), silica (Si), and bismuth (Bi). Other peaks of sodium (Na), magnesium (Mg), phosphorus (P), potassium (K), and calcium (Ca). VFS = vertical full scale. Fig. 8. Severe diffuse interstitial pneumonia, Pepto-bismol@,post-inoculation day 7. Alveolar walls thickened with lymphocytes, macrophages, and fibroblasts. Extensive fibrosis in alveolar lumens. Foreign material in bronchioles and proteinaceous exudates in alveolar lumens. Bar = 190 pm.

of vascular smooth muscle. Randomly scattered macrophages were distended with pigmented and refractile granular material (Fig. 12). The energy dispersive spectroscopic analysis of Kaopectate -inoculated lungs was similar on post-inoculation days 2 and 7. Significant peaks of aluminum @

muth (2.459 KeV) from regions with lesions (Fig. 9), but there were negligible amounts of these elements from non-inoculated lungs. Peaks representing sodium and phosphorus, as well as minor peaks of magnesium, potassium, and calcium, were also detectable. Kaopectateo group

Gross lesions were apparent as segments with pronounced pallor compared to adjacent normal lung. Histologically, on post-inoculation day 2, Kaopectate caused pneumonia characterized by large numbers of alveolar macrophages. Kaopectate had a characteristic histologic appearance, with anisotropic birefringent crystals of highly variable size and shape scattered within a light pink-to-gray amorphous background material (Fig. 10). Also present were yellow, brown, and black non-crystalline granules, 1-2 pm in diameter. On post-inoculation day 2, some KaopectateB had been phagocytized by macrophages. Less commonly, other areas had multiple foci of intense suppuration without an alveolar interstitial reaction. On post-inoculation day 7, Kaopectate had resolved to principally a granulomatous pneumonia with fibrosis within alveolar lumens and little extracellular KaFig. 10. Pneumonia with macrophages in alveolar luopectate@ (Fig. 11). In other areas, alveolar septa1 mens, Kaopectatea, post-inoculation day 2. Foreign material thickening was minimal except in a few locations in appears as amphophilic, amorphous clumps in which black, which alveolar septa were infiltrated with mononuclear 1-2 pm diameter, non-crystalline granules are embedded. cells. There was patchy medial muscular hypertrophy Bar = 42 pm. @

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Slocombe, Evans, and Derksen

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Fig. 11. Granulomatous pulmonary nodules, Kaopectate 0 , post-inoculation day 7. Dense accumulation of pigmented granules within macrophages. Extensive fibrosis. Bar = 190 pm. Fig. 12. Refractile foreign material in alveolar macrophages, Kaopectatem, post-inoculation day 7. Bar = 30 pm.

from which an alpha hemolytic Streptococcus was isolated from the affected lungs. This isolate was not judged to be a pulmonary pathogen. No anaerobes were isolated. Attempts to isolate Mycoplasma were negative as were fluorescent antibody tests and virus isolation procedures for sialodacryoadenitis virus and Sendai Other findings virus. Saline-inoculated lungs were not different from nonAttempts to isolate bacterial pathogens from lungs yielded no bacterial growth from all rats except for two inoculated lungs of other groups, either grossly, histologically, or by energy dispersive spectroscopic analysis. One rat did have areas of intrapulmonary hemorrhage associated with aspiration of blood at the time of decapitation. 'Oo0 Mediastinal lymph nodes were mildly hyperplastic with sinus histiocytosis, hemorrhage, and hemosiderosis in all treatment groups on post-inoculation days 2 and 7. Foreign material consistent with the microscopic appearance of BaSO, was present in the mediastinal lymph nodes of BaS0,-inoculated rats, but foreign material was not found in mediastinal lymph nodes from other treatment groups. There were mild degenerative and cystic changes in salivary glands and Harderian glands in all post-in. . oculation day 2 rats. Organs other than lungs collected VFS= 1024 from rats on post-inoculation day 7 appeared histoFig. 13. Energy dispersive spectroscopy spectra from lung logically normal. inoculated with Kaopectatem (upper line) compared with nonSuspensions of BaSO,, Pepto-bismol@and Kaopecinoculated lung (lower line). Characteristic peaks of alumitate@ were analyzed in the absence of pulmonary num (Al) and silica (Si). Other peaks of sodium (Na), phosphorus (P), sulfur (S), calcium (Ca), and copper (Cu). VFS = tissue, and resulting spectra (data not shown) were vertical full scale. compared with BaS0,-, Pepto-bismol@, and Kaopec-

(1.485 KeV) and silica (1.750 KeV) were present (Fig. 13). Non-inoculated lungs lacked peaks for these elements. Minor peaks representing the elements sodium, phosphorus, sulfur, calcium, and copper were also present.

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Foreign-body Pneumonias in Rats

tate -inoculated lungs, respectively. We found no evidence that the presence of formalin-fixed pulmonary tissue interfered with the ability of energy dispersive spectroscopy to detect those elements unique to each instilled compound. Characteristic elements of the compounds found in the absence of tissue were fully detectable in inoculated lungs in situ on post-inoculation days 2 and 7. Other elements were present, however, on spectra from inoculated lungs that were absent on spectra from pure suspensions of foreign materials, suggestingthat energy dispersive spectroscopy is of sufficient sensitivity to detect elements endogenous to pulmonary parenchyma or to detect increases in elements associated with increased numbers of inflammatory cells. @

Discussion Definitive identification of foreign material in the lungs can be difficult when only light microscopy is used. Histopathologic changes associated with the pulmonary instillation of the compounds used in this study generally became less distinctive at post-inoculation day 7 when compared to day 2. Tissue reaction to barium sulfate (BaSO,) has been previously studied in the lungloand other tissues.12The inflammatory reaction in lungs in an earlier studylo was similar to our observations and included, principally, an infiltrate of macrophages that was not accompanied by extensive fibrosis, edema, or mixed inflammatory cell infiltrates in 7-day-old lesions. Pulmonary instillation of BaSO, resulted in no observed birefringence when examined with polarized light microscopy. We have previously observed that some commercial preparations of BaSO, are indeed birefringent in sections of pulmonary tissue. The BaSO, used in this study is described by the manufacturer as “micropulverized,” resulting in a very small particle size of the BaSO, crystals. Perhaps the small particle size of the BaSO, was responsible for its lack of birefringence in this study. To our knowledge, there have not been descriptions of pulmonary tissue reactions induced specifically by Pepto-bismolm or Kaopectatem although the pulmonary responses to kaolin clays have been previously described.l 4 When deposited into murine lungs, Peptobismolm and Kaopectatem caused severe fibrosis within alveolar lumens when compared to the other compounds used in this study. The pulmonary lesions induced by these compounds differed in one aspect: the presence of black radially arranged spicules found in Pepto-bismolm -induced pulmonary disease were absent in Kaopectate -induced disease. The significance of these structures is unknown. We were unable to detect them with scanning electron microscopy, therefore, their specific elemental composition remains un@

485

known. While these structures may be a unique feature of Pepto-bismol -induced pulmonary disease, the possibility that they represent artifacts created during routine tissue processing of lungs instilled with this medicament cannot be ruled out. These structures were not observed in lungs inoculated with any other compounds used in this study. Pulmonary histopathologic lesions induced by BaSO,, mineral oil, Pepto-bismolm , and Kaopectatem were not resolved by post-inoculation day 7, possibly because of the toxicity of these compounds to phagocytes or because of limited ability of these cells to remove these substances from alveolar lumens. Our observations suggest that instillation of adequate amounts of mineral oil, Pepto-bismolm, or Kaopectatem may lead to severe pulmonary disease with parenchymal destruction, with Pepto-bismolm and Kaopectate causing marked fibrosis within alveolar lumens by post-inoculation day 7. Additionally, mineral oil, Pepto-bismol@,and Kaopectatem induced medial muscular hypertrophy of vessel walls. This vascular response may have resulted from severe pulmonary disease leading to hypoxia, but other factors may be involved as well. Although lungs instilled with each compound used in this study still had characteristic histologic features on post-inoculation day 7, these distinctive features were less apparent than on day 2; thus, as chronic pulmonary lesions develop, energy dispersive spectroscopy may be increasingly useful for definitive determination of the elemental etiology of foreign-body p n e u m o n i a ~ . ~ , ~ X-rays are created when an electron undergoes a decrease in energy that occurs during an electron’s movement to a lower energy level (or “shell”), usually from the displacement of another electron by the beam of the scanning electron microscope. Each individual element in a sample has a potential spectrum of detectable X-ray energies. This is seen in the energy dispersive spectroscopy spectrum of lungs instilled with BaSO,, in which multiple peaks of barium were observed. There is a risk of erroneous elemental identification if many elements are present in a sample and multiple X-ray energies are found by energy dispersive spectroscopy. In the energy dispersive spectroscopy system we used, however, computer-based searches are generated to best match X-ray energies with appropriate elements, thereby minimizing the possibility of inaccurate determination of elemental composition. The energy dispersive spectroscopic analysis of lungs instilled with Pepto-bismolm had significant peaks of elements characteristic of that compound (aluminum, silica, and bismuth). Peaks representing sodium and phosphorus were also present. Sodium and phosphorus were not present in spectra from specimens of Pepto-bismolm analyzed in the absence of pulmonary @

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tissue. Sodium and phosphorus probably represent elements endogenous to pulmonary tissues or to inflammatory cells within pulmonary parenchyma. Conversely, minor peaks of magnesium, potassium, and calcium were detectable in specimens of Pepto-bismol@analyzed in the absence of pulmonary tissue. These peaks most likely represent minor basic elements of Pepto-bismola. The energy dispersive spectroscopic analysis of Kaopectate -inoculated lungs had peaks that were characteristic of aluminum and silica, which are the elements present in [email protected] representing sodium, phosphorus, sulfur, and calcium were also detected and were interpreted as elements not characteristic of Kaopectate@. Rather, these elements were endogenous to pulmonary parenchyma or to increased numbers of inflammatory cells because these elements were not present on spectra from specimens of JSaopectate analyzed in the absence of pulmonary tissue. A copper peak, not present in specimens of Kaopectate@analyzed in the absence of pulmonary tissue, was of unknown significance. The histologic similarities between Pepto-bismol@and Kaopectate -inoculated lungs correlated with similar elemental determinations of aluminum and silica. Finding aluminum and silica in the energy dispersive spectroscopy spectra of these compounds is most likely associated with the presence of kaolin, a clay-based material composed largely of aluminum silicate, that is commonly used in commercial antidiarrheal preparations, The presence of a bismuth peak (present as the ingredient bismuth subsalicylate in Pepto-bismolm) in the energy dispersive spectroscopic spectrum of Pepto-bismol@-inoculated lungs distinguishes Pepto-bismol@-induced airway disease from that caused by instillation of Kaopectatea. Because of the similarities in the pneumonia caused by Peptobismol@and KaopectateB, we speculate that aluminum silicate, common to both compounds, may have been responsible for the similar pulmonary inflammatory reaction caused by these medicaments. Mediastinal lymph node enlargementwas associated with pulmonary instillation of BaSO, and suggests that inoculum removal from the lungs via movement of inflammatory cells occurs following BaSO, inoculation. With other compounds, mediastinal lymph node enlargement was present but was not accompanied by histologically detectable accumulations of mineral oil, Pepto-bismola , or Kaopectate within these lymph nodes. Fixatives such as glutaraldehyde are suitable for tissues to be analyzed by energy dispersive spectroscopy. Post-fixatives, such as osmium tetroxide, can be used, but osmium will remain in the specimen and appear on the energy dispersive spectroscopy spectrum. For@

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malin was chosen for this study because we wanted to simulate those conditions under which tissues would be submitted to our laboratory for routine diagnostic procedures. Because characteristic elements of BaSO,, Pepto-bismol@,and Kaopectate were detectable when compounds were analyzed as pure suspensions as well as when analyzed in the presence of pulmonary tissues, we conclude that fixation with formalin does not interfere with the X-ray detection of the characteristic elements of these compounds in lungs. The compounds used in this study induced pulmonary disease with differing histopathologic features that became less distinctive by post-inoculation day 7 depending on the compound instilled. We conclude that energy dispersive spectroscopy is a useful diagnostic adjunct for detection and confirmatory identification of the specific elemental composition of certain foreign materials in lungs. Acknowledgements This research was supported by the Animal Health Diagnostic Laboratory, the Center for Environmental Toxicology, the Center for Electron Optics, and the Pulmonary Laboratory, Michigan State University. The authors thank Mrs. Cheryl Assaff and Drs. Stan Flegler, Robert Leader, and Allan Trapp for assistance.

References 1 Abraham JL: Recent advances in pneumoconiosis: the pathologist's role in etiologic diagnosis. In: The Lung: Structure, Function, and Disease, ed. Thurlbeck WM and Abell MR, pp. 96-1 37. Williams and Wilkins, Baltimore, MD, 1978 2 Bunton TE, Baksi SM, George SG, Frazier JM: Abnormal hepatic copper storage in a teleost fish (Morone americana). Vet Path01245 15-524, 1987 3 de Oliveira AC, Rosenbruch M, Schulz L-Cl: Intimal asteroid bodies in horses: light and electron microscopic observations. Vet Pathol 22:226-23 1, 1985 4 Evans MG, Slocombe RF, Schwartz LD: Pulmonary silicosis in captive ring-necked pheasants: definitive diagnosis by electron probe X-ray microanalysis. Vet Patho1 25239-241, 1988 5 Funahashi A, Pintar K, Siegesmund KA: Identification of foreign material in lung using energy dispersive x-ray analyses. Arch Environ Health 30:285-289, 1975 6 Funahashi A, Siegesmund K, Dragen RF, Pintar K: Energy dispersive X-ray analysis in the study of pneumoconiosis. Br J Ind Med 34:95-101, 1977 7 Green CJ, Knight J, Precious S, Simpkin S: Ketamine alone and combined with diazepam or xylazine in laboratory animals: a 10-year experience. Lab Anim 15: 163-170, 1981 8 Hargest TE, Gay CV, Leach RM: Avian tibia1 dyschondroplasia. 111. Electron probe analysis. Am J Pathol 119: 199-209, 1985 9 Herbert A, Sterling G, Abraham J, Corrin B: Desqua-

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10 11 12 13

mative interstitial pneumonia in an aluminum welder. Hum Pathol 13:694-699, 1982 Huston J, Wallach DP, Cunningham GJ: Pulmonary reaction to barium sulfate in rats. Arch Pathol 54:430438, 1952 Johnson FB: Crystals in pathologic specimens. Pathol Annu 7:321-344, 1972 Kay S: Tissue reaction to barium sulfate contrast medium. Arch Pathol 57:279-284, 1954 Postek MT, Howard KS, Johnson AH, McMicheal KL: X-ray analysis. In: Scanning Electron Microscopy. A Student’s Handbook, pp. 69-1 13. Ladd Research Industries, Inc, Burlington, VT, 1980

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14 Stookey JL, Moe JB: The respiratory system. In: Pathology of Laboratory Animals, ed. Benirschke K, Garner FM, and Jones TC, 1st ed., vol. 1, pp. 96-97. Springer-Verlag, New York, NY, 1979 15 Vallyathan NV, Green FHY, Craighead JE: Recent advances in the study of mineral pneumoconiosis. In: Pathology Annual, Part 2, ed. Sommer SC and Rosen PP, pp. 77-104. Appleton-Century-Crofts, Nonvalk, CT, 1980 16 Yao Y, Wang N, Michel RP, Poulsen RS: Mineral dusts in lungs with scar or scar cancer. Cancer 54: 18 14-1 823, 1984

Request reprints from Dr. M. G. Evans, Harvard Medical School, New England Regional Primate Research Center, One Pine Hill Drive, Southborough, MA 0 1772 (USA).