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Scanning electron microscopy of canine lung transplants
Scanning electron microscopy of canine lung transplants Yoshio Kondo, M.D., M. Don Turner, Ph.D., Virginia G. Lockard, M.S., Charles Hallford, B.S., and James D. Hardy, M.D., Jackson, Miss.
HistopatholOgic aspects of pulmonary allograft rejection have been documented by many investigators using light microscopy (LM)"' II, '" and transmission electron microscopy (TEM),"'" 11, I, but as yet there has been no report of the application of scanning electron microscopy (SEM) in this field. Although the nature of lung rejection appears to be identical to the classical rejection of other organ allografts, the unique anatomy of the lung, which communicates directly with the atmosphere by the alveolar membrane system, modifies the morphologic expression of the rejection reaction to a large extent. This is especially true for bilateral lung transplantation, in which the respiratory function of the recipient depends entirely upon the transplanted lungs. Alveolar edema, ischemic injury of the bronchial lining, and infection of the bronchial system are all common after this procedure whether immunosuppressive agents are employed or not." The resulting accumulations of fibroproteinaceous exudate, mucus plugs, and cell debris in the bronchial tree impair gas exchange preventing consistent survival of the recipients. The scanning electron microscope is a From the Departments of Surgery and Pathology, The University of Mississippi Medical Center, Jackson, Miss, 39216. Supported by U. S. Public Health Service Grant No. HL11730. Received for publication Feb. 26, 1973.
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valuable instrument for studying lung morphology because relatively large samples of tissues can be examined at both low and high magnification with great depth of focus. The open structure of the lung also makes it particularly suitable for study by SEM. These studies were designed to elucidate the stereoscopic findings in the lung rejection process by means of SEM and to correlate these findings with those observed by LM and TEM. Materials and methods
Forty-six blocks of lung tissue procured from 12 dogs in five different categories were studied. Normal lung. Biopsy specimens obtained from lungs of 3 normal dogs served as controls. Unmodified unilateral allotransplantation. Three dogs had unilateral lung allotransplantation without immunosuppressive treatment. Serial open biopsies were taken from the graft immediately after operation and I, 3, 4, 7, and 11 days postoperatively. Modified bilateral allotransplantation. One dog received bilateral lung allotransplants which were successfully maintained with azathioprine and prednisolone for over 2 months. Open lung biopsy was carried out 26 days postoperatively. Simultaneous allotransplantation with contralateral reimplantation. Four dogs had unilateral lung allotransplantation and con-
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tralateral lung reimplantation. No immunosuppressive drugs were given. Three of these dogs died on the fourth postoperative day, and autopsies were performed shortly after death. The fourth dog, moribund on the fourth day, was put to death after open lung biopsy. Bilateral reimplantation. An open lung biopsy was obtained from 1 healthy dog subjected to bilateral reimplantation of the lung 2 years earlier. The technical details of bilateral lung transplantation have been described previously.? The operation was performed as a one-stage procedure of successive right and left lung transplantation, the right side first. Penicillin, streptomycin, and Chloromycetin were given for 2 weeks postoperatively. For SEM, small blocks of the tissue taken by open biopsy were immediately put in cacodylate-buffered glutaraldehyde and kept in a refrigerator. When an entire lobe was taken at autopsy, the lung was inflated with glutaraldehyde through the bronchus before blocks were cut out. After 24 to 72 hours, the glutaraldehyde-fixed tissue was sectioned in rectangles approximately 5 by 5 by 3 mm., wrapped in one layer of a thin plastic film (Saran), and quick-frozen in a bath of acetone and Dry Ice. The tissue was then freeze-dried, and a new, even surface was exposed by cutting with a razor blade. The surface was then coated with a carbon-goldpalladium layer approximately 100 A thick by vapor deposition and examined with an AMR Model 900 high-resolution scanning electron microscope. Magnification for direct SEM ranged from 50,000 to 100,000 x. For TEM, adjacent portions of glutaraldehyde-fixed tissue were cut into 1 mm. cubes which were further fixed for 2 hours at 0° C. in 1 per cent osmium tetroxide (pH 7.4) containing 0.25M sucrose. The blocks of tissue were rapidly dehydrated in a graded series of ethyl alcohol and embedded in Epon 812. Ultrathin sections were mounted 'on uncoated copper grids and stained with uranyl acetate and lead citrate prior to examination in a Philips EM-300 electron microscope.
Corresponding parts of tissue blocks were fixed in formalin, and paraffin sections were stained with hematoxylin and eosin for LM. Results Normal lung. Because of the enormous depth of focus in SEM, multifaceted structures of the alveolar duct and the alveolar sac were clearly demonstrated (Fig. 1, A). The alveoli were small chambers of fairly equal size communicating with each other through fenestrae and pores (Kohn's alveolar pore, Fig. 1, B). The surface of the thin alveolar wall was lined by an almost smooth membrane with scattered elevations presumably representing type II alveolar epithelial cells, alveolar macrophages, and other migrating cells in and on the wall (Fig. 1, C). Although fine irregular wrinkles were observed in some areas at high magnification, neither the junctions of type I or type II alveolar epithelial cells nor the characteristic surface of the type II cells covered with microvilli were discernible. The surface of the small bronchioles appeared extremely pebbly and rugged at lower magnification. On higher magnification of the area extending from the terminal bronchiole to the respiratory bronchiole, there were protrusions of the Clara cells; these were 2 to 4ft in diameter and had many pits and sulci on their surfaces. Ciliated cells with relatively few strands of cilia were arranged irregularly between the protrusions (Fig. 1, D). The surface of the more proximal bronchiole exhibited abundant cilia. Nonciliated goblet cells appeared sporadically (Fig. 1, E, and F). Unmodified unilateral allotransplantation. Fig. 2 shows SEM of the serial open lung biopsies taken 24, 72, 96 hours and 11 days postoperatively. Thickening of the alveolar wall was apparent on SEM 24 hours after transplantation, but the surface texture appeared almost normal (Fig. 2, A). At 72 hours, the surface became rough, with numerous cells on the wall. These cells differed in size and shape but maintained the configuration characteristic of active cells from various origins. The alveolar spaces
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Fig.!. Scanning electron microscop y of normal canine lung . A . Alveolar sac. B. Kohn's pore . C. T ypical alveolar surface. D. Bronchiolar wall with Clara cells and some ciliated epithelial cells. E. Bronchiolar wall with ciliated epithelial cells. F . Higher magnification of E. (Original magnifications: A >:500; B >: 10,000; C x500; D x2,OOO; E x l ,OOO; F ':5,000.)
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Fig. 2. Scanning electron microscopy of an unmodified lung allotransplant. A, After 24 hours. B, After 72 hours. C, After 96 hours. D, After 11 days. (Original magnification x500. )
appeared to be restricted (Fig. 2, B) . By LM , a moderate amount of perivascular cell infiltration with pleomorphic cell proliferation in the alveolar wall was observed, along with interstitial and intra-alveolar edema and hemorrhage. These changes were mor e extensive at 96 hours (Fig. 2, C) and 7 da ys. Fig. 3 shows a typical TEM finding at 96 hours. The specimen taken on the eleventh da y revealed ad vanced hemorrhagic necrosis by LM , and by SEM it appeared as an amorphous mass with only remnants of deformed alveolar spaces (Fig. 2, D) .
SEM finding s in the bronchioles wer e noteworthy. E ven at this advanced stage, the Clara cells seemed to be well preserved , yet the ciliated cells had lost their characteristic app earance (F ig. 4, A). In the proximal bronchioles, destruction of the wall became more severe . Fig. 4, B shows a devastated area with piles of the desquamated epithelium . Modified bilateral allotransplantation. On the da y of biop sy (twenty-sixth da y ) , the animal showed no dyspnea at rest and only mild distress on moderate exercise. Th e ar-
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Fig. 3. Transmission electron microscopy of an unmodified unilateral lung allotransplant after 96 hours. Endothelial destruction (en ). Degenerating type I epithelial cell (ep-l) exhibiting extrcme vacuolization (arrows ) around the nucleus and in the membrane cytoplasm. Degenerating type II epithelial cell (ep-2) showing severe vacuolization but fairly well-preserved lamellar bodies . Alveolar lumen showing extensive edema and hemorrhage (at ). (Original magnification xS,200. )
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Fig. 4. Scann ing electron microscopy of bronchioles from an unmodified unilateral lung allotransplant. A, Distal bronchiolar wall with well-preserved Clara cells, B, Proximal bronchiolar wall showing a deteriorated ciliated epithelium. (Orig inal magnification x5,000.)
Fig. 5. Scanning electron microscopy of modified bilateral lung allotransplant, A, Well-preserved alveolar structure. B, Alveol ar sac. (Original magnifications : A >< 100; B ><500. )
terial blood Po, was 61 mm. Hg, Pco, was 30 mm. Hg, and pH was 7.38. SEM disclosed that the honeycomb structure of the alveoli was well preserved . However, the surface of some areas of the alveolar wall revealed an increase in cellularity and accumulation of debris, probably the remnants of an earlier rejection crisis (Fig. 5, A and B ).
TEM of the specimen showed localized edema of the endothelial cells; increase in collagen and elastic fibers in the subendothelial space; proliferation of type II alveolar epithelial cells, with marked vacuolization and decreased electron densit y of osmiophilic lamellar bodies; and deposition of fibrin and debris in the alveolar space (Fig. 6) .
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Fig. 6. Transmission electron microscopy of a modified bilateral lung allotransplant. Type II epithelial cell (ep-Z] showing severe vacuolization and diminished electron density of lamellar bodies. Degenerated endothelial cell (en). Fibrin deposit in the alveolar lumen (fi). (Original magnification >:7,000.)
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Fig. 7. Scanning electron microscopy of simultaneous lung allotransplant (A, C. and D) with contralateral reimplant (B). A, Fibrin and cell debris on the alveolar surface. B, Serous exudate on the alveolar wall. C, Thickened alveolar septa. The asterisk indicates a small pulmonary artery. D . Terminal bronchiole. (Original magnifications: A and B / 500; C " 200: D v: 1,000.)
Simultaneous allotransplantation with contralateral reimplantation. At autopsy, both the reimplant and the allotransplant showed severe edema and congestion. No difference was discernible macroscopically, and the gross weights of the allotransplant and the reimplant had increased to the same degree. SEM of the allotransplant revealed an exudate containing abundant cells; fibrin and debris covered the entire surface of the freeze-dried specimen, so that alveolar architecture was barely visible (Fig. 7, A). In contrast, no cellular or fibrin deposits were observed in the reimplant. Some thickening
of the cut edges of interalveolar septa was evident , and the surface had a waxy appearance, indicating a film of serous exudate (Fig. 7, B). Perivascular cell infiltration and interstitial proliferation in the allograft resulted in severe thickening of these areas (Fig. 7, C). A portion of the terminal bronchiole of the same specimen is shown in Fig. 7, D. The surfaces of the ciliated epithelium and the Clara cells were heavily veiled by exudate and cellular deposits throughout the bronchial tree. In the reimplant, the surface of the bronchial epithelium was better preserved.
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Fig. 8. Scanning electron microscopy of a bilateral reimplant after 2 years. (Original magnification x500.)
Bilateral reimplantation. Despite daily activity and normal appearance for 2 years, this dog had a subnormal POe and a normal Pco., indicating mild diffusion impairment. SEM demonstrated an increased cellularity and slight thickening of the alveolar wall (Fig. 8). These findings were also confirmed byLM. Discussion
In recent years, SEM has become a valuable instrument for morphologic study of the normal and diseased lung,"- 12, 13, 14 Cellular distribution and topography of the surface structure are best obtained by SEM. However, there has been no report of the application of SEM to the study of lung transplants. Wheeler and co-workers" did describe interesting findings in SEM of the human heart valve allograft. For successful SEM of biological material, the methods of tissue preparation are crucial.' After many trials and errors using freeze-dried lung tissue, we found the present
method to be the most suitable. We did not employ the critical-point drying technique, a widely used procedure recommended by Anderson,' which might be worthwhile in further studies. Inflation of the lung through the bronchus with glutaraldehyde fixed the tissue faster and demonstrated the honeycomb structure of the organ better. To reveal the detailed texture of the mucosal surface, some investigators have used bronchial lavage with saline. However, we did not adopt this technique, since accumulation of exudate, mucus material, and cells represents an important pathophysiologic process in pulmonary allograft rejection. Furthermore, the severe destruction of the bronchial mucosa often prevented simple practice of this procedure. Probably for this reason, the ciliated epithelium of our specimens frequently appeared to be covered with a thin film. Also, we could not identify the microvilli on the surface of type II alveolar epithelial cells or in the junctions between alveolar epithelial cells.
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In many respects, SEM confirmed the earlier findings obtained by LM and TEM in alveoli of lung transplants. Accumulation of exudate and cells, thickening of the alveolar wall, and a decrease in the air-space of the alveoli were observed to parallel functional impairment. These changes were so striking that one could easily imagine severe restriction of oxygen exchange without any other factors. In long-term survivors of bilateral reimplantation and modified bilateral allotransplantation, there was a marked increase in cellularity on the alveolar surface. Many of these cells were identified as type II alveolar epithelial cells. Strong reactivity or regenerative activity of type II cells has been reported in different situations.t- 8, 1~, 16 Nagaishi'" described adenomatous hyperplasia of the alveolar lining elicited by hyperplasia of type II cells following cobalt-60 irradiation of the human lung, The Clara cell, which is localized in the terminal bronchiole, remained relatively intact in the severely rejected lung. The transplantation procedure itself is stressful and may cause degeneration of type I cells and proliferation of type II cells in the alveoli. Ciliated epithelial cells are damaged more severely than are Clara cells in the bronchiole. The closer relationship of Clara cells to type II alveolar epithelial cells has been recognized for many years!"; therefore, the similar response of these cells in the rejection process is quite understandable. In addition, immunologic processes certainly enhance and modify these changes. Distinctions between simultaneous allotransplantation and contralateral reimplantation were revealed much better by SEM than by LM or TEM. Surfaces of the alveoli and bronchioles were heavily covered by cells, fibrin, and debris in the allograft, whereas no such deposits were observed in the reimplant. Thus, despite both grafts exhibiting almost the same grade of edema macroscopically, the different nature of the alveolar exudate sharply reflected the effect of the local immune reaction. Pathogenesis of severe pulmonary edema in the contralateral reimplant represents a new, attractive
project for the study of lung transplantation, which will be described in a separate paper. to Summary
Stereoscopic micro-findings in various phases of lung transplantation have been studied with a scanning electron microscope, and the findings were correlated with those obtained by LM and TEM. Forty-six blocks of lung tissue were procured from dogs with normal lungs, unmodified unilateral allotransplants, modified bilateral allotransplants, simultaneous allotransplants with contralateral reimplants, and long-functioning bilateral reimplants. The great depth of focus with the scanning electron microscope dramatized the rapid and gross damage to alveolar walls during acute rejection. Accumulation of exudate and cells, thickening of the alveolar wall, and a decrease in the air-space of the alveoli were observed to parallel functional impairment. These changes were so striking that one could easily imagine severe restriction of gas exchange. In long-term survivors of bilateral reimplantation and modified bilateral allotransplantation, there was a marked increase in type II alveolar epithelial cells and slight thickening of the alveolar wall, explaining the persistent nature of functional impairment. Although simultaneous allotransplantation with contralateral reimplantation caused almost the same grade of edema macroscopically, the different characters of alveolar exudate were clearly demonstrated by SEM. We are grateful for the assistance furnished us by Dr. Katherine Mather and for the use of the scanning electron microscope at the U. S. Army Engineers Waterways Experiment Station, Vicksburg, Mississippi. REFERENCES Anderson, T. F.: Techniques for the Preservation of Three Dimensional Structure in Preparing Specimens for the Electron Microscope, Trans. N. Y. Acad. Sci. (Ser. II) 13: 130, 1951. 2 Barnes, B. A., Flax, M. R., Burke, J. F., and Barr, G.: Experimental Pulmonary Homografts
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4 5
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in the Dog. I. Morphological Studies, Transplantation 1: 351, 1963. Becker, N. H., Sinha, S. B. P., Hagstrom, J. W. C., Bliirncke, S., and Veith, F. J.: Fine Structure Alterations in Canine Lung Transplants, J. THORAe. CARDIOVASC. SURG. 63: 81, 1972. Boyde, A., and Wood, c.: Preparation of Animal Tissue for Surface-Scanning Electron Microscopy, J. Microbiol. 90: 221, 1969. Daly, B. D. T., and McNary, W. F.: Type II (Granular Alveolar) Pneumocyte in Ultrastructural Analysis of Stressed Human Lung, Surg. Forum 22: 38, 1971. Greenwood, M. F., and Holland, P.: The Mammalian Respiratory Tract Surface: A Scanning Electron Microscopic Study, Lab. Invest. 27: 296, 1972. Gondos, B., White, P., and Benfield, J. R.: Ultrastructural Alterations in Canine Lung Allografts, Am. J. Pathol. 64: 373, 1971. Kapanci, Y., Weibel, E. R., Kaplan, H. P., and Robinson, F. R.: Pathogenesis and Reversibility of the Pulmonary Lesions of Oxygen Toxicity in Monkeys. II. Ultrastructural and Morphometric Studies, Lab. Invest. 20: 10 I, 1969. Kondo, Y., Isin, E., Cockrell, J. V., and Hardy, J. D.: One-Stage Bilateral Allotransplantation of Canine Lungs: Further Studies. Early Rejection and Prolonged Survival With Immunosuppression, J. THORAe. CARDIOVASC. SURG. 64: 897, 1972. Kondo, Y., Cockrell, J. V., Turner, M. D., and Hardy, J. D.: Mechanism of Postallotransplantation Pulmonary Edema: Examination With Simultaneous Reimplant With Contralateral Allograft. In preparation.
11 Molokhia, F. A. S., Ponn, R. B., Asimacopolos, P. J., and Norman, J. C.: Microscopic and Ultrastructural Changes in Unmodified Canine Lung Allografts, Arch. Surg. 103: 490, 1971. 12 Nagaishi, C.: Functional Anatomy and Histology of the Lung, Baltimore, 1972, University Park Press, pp. 29-65. 13 Nowell, J. A., and Tyler, W. S.: Scanning Electron Microscopy of the Surface Morphology of Mammalian Lungs, Am. Rev. Resp. Dis. 103: 313, 1971. 14 Tyler, W. S., de Lorimier, A. A., Manus, A. G., and Nowell, J. A.: Surface Morphology of Hypoplastic and Normal Lungs From Newborn Lambs, ill Jahari, 0., and Corvin, I., editors: Scanning Electron Microscopy/1971, Proceedings of the Fourth Annual Scanning Electron Microscopy Symposium, Chicago, 1971, lIT Research Institute, pp. 305-312. 15 Veith, F. 1., and Hagstrom, J. W.: Alveolar Manifestations of Rejection: An Important Cause of the Poor Results With Human Lung Transplantation, Ann. Surg, 175: 336, 1972. 16 Wang, N. S., Huang, S. N., Sheldon, H., and Thurlbeck, W. M.: Ultrastructural Changes of Clara cells and Type II Alveolar Cells in Adrenalin-Induced Pulmonary Edema in Mice, Am. J. Pat hoI. 62: 237, 1971. 17 Warren, B. A., and de Bono, A. H. B.: The Ultrastructure of Early Rejection Phenomena in Lung Homografts in Dogs, Br. J. Exp. Pathol. 50: 593, 1969. 18 Wheeler, E. E., Gavin, J. B., and Herdson, P. B.: A Scanning Electron Microscopy Study of Human Heart Valve Allografts. Pathology 4: 185, 1972.
HistopatholOgic aspects of pulmonary allograft rejection have been documented by many investigators using light microscopy (LM)"' II, '" and transmission electron microscopy (TEM),"'" 11, I, but as yet there has been no report of the application of scanning electron microscopy (SEM) in this field. Although the nature of lung rejection appears to be identical to the classical rejection of other organ allografts, the unique anatomy of the lung, which communicates directly with the atmosphere by the alveolar membrane system, modifies the morphologic expression of the rejection reaction to a large extent. This is especially true for bilateral lung transplantation, in which the respiratory function of the recipient depends entirely upon the transplanted lungs. Alveolar edema, ischemic injury of the bronchial lining, and infection of the bronchial system are all common after this procedure whether immunosuppressive agents are employed or not." The resulting accumulations of fibroproteinaceous exudate, mucus plugs, and cell debris in the bronchial tree impair gas exchange preventing consistent survival of the recipients. The scanning electron microscope is a From the Departments of Surgery and Pathology, The University of Mississippi Medical Center, Jackson, Miss, 39216. Supported by U. S. Public Health Service Grant No. HL11730. Received for publication Feb. 26, 1973.
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valuable instrument for studying lung morphology because relatively large samples of tissues can be examined at both low and high magnification with great depth of focus. The open structure of the lung also makes it particularly suitable for study by SEM. These studies were designed to elucidate the stereoscopic findings in the lung rejection process by means of SEM and to correlate these findings with those observed by LM and TEM. Materials and methods
Forty-six blocks of lung tissue procured from 12 dogs in five different categories were studied. Normal lung. Biopsy specimens obtained from lungs of 3 normal dogs served as controls. Unmodified unilateral allotransplantation. Three dogs had unilateral lung allotransplantation without immunosuppressive treatment. Serial open biopsies were taken from the graft immediately after operation and I, 3, 4, 7, and 11 days postoperatively. Modified bilateral allotransplantation. One dog received bilateral lung allotransplants which were successfully maintained with azathioprine and prednisolone for over 2 months. Open lung biopsy was carried out 26 days postoperatively. Simultaneous allotransplantation with contralateral reimplantation. Four dogs had unilateral lung allotransplantation and con-
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tralateral lung reimplantation. No immunosuppressive drugs were given. Three of these dogs died on the fourth postoperative day, and autopsies were performed shortly after death. The fourth dog, moribund on the fourth day, was put to death after open lung biopsy. Bilateral reimplantation. An open lung biopsy was obtained from 1 healthy dog subjected to bilateral reimplantation of the lung 2 years earlier. The technical details of bilateral lung transplantation have been described previously.? The operation was performed as a one-stage procedure of successive right and left lung transplantation, the right side first. Penicillin, streptomycin, and Chloromycetin were given for 2 weeks postoperatively. For SEM, small blocks of the tissue taken by open biopsy were immediately put in cacodylate-buffered glutaraldehyde and kept in a refrigerator. When an entire lobe was taken at autopsy, the lung was inflated with glutaraldehyde through the bronchus before blocks were cut out. After 24 to 72 hours, the glutaraldehyde-fixed tissue was sectioned in rectangles approximately 5 by 5 by 3 mm., wrapped in one layer of a thin plastic film (Saran), and quick-frozen in a bath of acetone and Dry Ice. The tissue was then freeze-dried, and a new, even surface was exposed by cutting with a razor blade. The surface was then coated with a carbon-goldpalladium layer approximately 100 A thick by vapor deposition and examined with an AMR Model 900 high-resolution scanning electron microscope. Magnification for direct SEM ranged from 50,000 to 100,000 x. For TEM, adjacent portions of glutaraldehyde-fixed tissue were cut into 1 mm. cubes which were further fixed for 2 hours at 0° C. in 1 per cent osmium tetroxide (pH 7.4) containing 0.25M sucrose. The blocks of tissue were rapidly dehydrated in a graded series of ethyl alcohol and embedded in Epon 812. Ultrathin sections were mounted 'on uncoated copper grids and stained with uranyl acetate and lead citrate prior to examination in a Philips EM-300 electron microscope.
Corresponding parts of tissue blocks were fixed in formalin, and paraffin sections were stained with hematoxylin and eosin for LM. Results Normal lung. Because of the enormous depth of focus in SEM, multifaceted structures of the alveolar duct and the alveolar sac were clearly demonstrated (Fig. 1, A). The alveoli were small chambers of fairly equal size communicating with each other through fenestrae and pores (Kohn's alveolar pore, Fig. 1, B). The surface of the thin alveolar wall was lined by an almost smooth membrane with scattered elevations presumably representing type II alveolar epithelial cells, alveolar macrophages, and other migrating cells in and on the wall (Fig. 1, C). Although fine irregular wrinkles were observed in some areas at high magnification, neither the junctions of type I or type II alveolar epithelial cells nor the characteristic surface of the type II cells covered with microvilli were discernible. The surface of the small bronchioles appeared extremely pebbly and rugged at lower magnification. On higher magnification of the area extending from the terminal bronchiole to the respiratory bronchiole, there were protrusions of the Clara cells; these were 2 to 4ft in diameter and had many pits and sulci on their surfaces. Ciliated cells with relatively few strands of cilia were arranged irregularly between the protrusions (Fig. 1, D). The surface of the more proximal bronchiole exhibited abundant cilia. Nonciliated goblet cells appeared sporadically (Fig. 1, E, and F). Unmodified unilateral allotransplantation. Fig. 2 shows SEM of the serial open lung biopsies taken 24, 72, 96 hours and 11 days postoperatively. Thickening of the alveolar wall was apparent on SEM 24 hours after transplantation, but the surface texture appeared almost normal (Fig. 2, A). At 72 hours, the surface became rough, with numerous cells on the wall. These cells differed in size and shape but maintained the configuration characteristic of active cells from various origins. The alveolar spaces
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Fig.!. Scanning electron microscop y of normal canine lung . A . Alveolar sac. B. Kohn's pore . C. T ypical alveolar surface. D. Bronchiolar wall with Clara cells and some ciliated epithelial cells. E. Bronchiolar wall with ciliated epithelial cells. F . Higher magnification of E. (Original magnifications: A >:500; B >: 10,000; C x500; D x2,OOO; E x l ,OOO; F ':5,000.)
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Fig. 2. Scanning electron microscopy of an unmodified lung allotransplant. A, After 24 hours. B, After 72 hours. C, After 96 hours. D, After 11 days. (Original magnification x500. )
appeared to be restricted (Fig. 2, B) . By LM , a moderate amount of perivascular cell infiltration with pleomorphic cell proliferation in the alveolar wall was observed, along with interstitial and intra-alveolar edema and hemorrhage. These changes were mor e extensive at 96 hours (Fig. 2, C) and 7 da ys. Fig. 3 shows a typical TEM finding at 96 hours. The specimen taken on the eleventh da y revealed ad vanced hemorrhagic necrosis by LM , and by SEM it appeared as an amorphous mass with only remnants of deformed alveolar spaces (Fig. 2, D) .
SEM finding s in the bronchioles wer e noteworthy. E ven at this advanced stage, the Clara cells seemed to be well preserved , yet the ciliated cells had lost their characteristic app earance (F ig. 4, A). In the proximal bronchioles, destruction of the wall became more severe . Fig. 4, B shows a devastated area with piles of the desquamated epithelium . Modified bilateral allotransplantation. On the da y of biop sy (twenty-sixth da y ) , the animal showed no dyspnea at rest and only mild distress on moderate exercise. Th e ar-
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Fig. 3. Transmission electron microscopy of an unmodified unilateral lung allotransplant after 96 hours. Endothelial destruction (en ). Degenerating type I epithelial cell (ep-l) exhibiting extrcme vacuolization (arrows ) around the nucleus and in the membrane cytoplasm. Degenerating type II epithelial cell (ep-2) showing severe vacuolization but fairly well-preserved lamellar bodies . Alveolar lumen showing extensive edema and hemorrhage (at ). (Original magnification xS,200. )
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Fig. 4. Scann ing electron microscopy of bronchioles from an unmodified unilateral lung allotransplant. A, Distal bronchiolar wall with well-preserved Clara cells, B, Proximal bronchiolar wall showing a deteriorated ciliated epithelium. (Orig inal magnification x5,000.)
Fig. 5. Scanning electron microscopy of modified bilateral lung allotransplant, A, Well-preserved alveolar structure. B, Alveol ar sac. (Original magnifications : A >< 100; B ><500. )
terial blood Po, was 61 mm. Hg, Pco, was 30 mm. Hg, and pH was 7.38. SEM disclosed that the honeycomb structure of the alveoli was well preserved . However, the surface of some areas of the alveolar wall revealed an increase in cellularity and accumulation of debris, probably the remnants of an earlier rejection crisis (Fig. 5, A and B ).
TEM of the specimen showed localized edema of the endothelial cells; increase in collagen and elastic fibers in the subendothelial space; proliferation of type II alveolar epithelial cells, with marked vacuolization and decreased electron densit y of osmiophilic lamellar bodies; and deposition of fibrin and debris in the alveolar space (Fig. 6) .
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Fig. 6. Transmission electron microscopy of a modified bilateral lung allotransplant. Type II epithelial cell (ep-Z] showing severe vacuolization and diminished electron density of lamellar bodies. Degenerated endothelial cell (en). Fibrin deposit in the alveolar lumen (fi). (Original magnification >:7,000.)
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Fig. 7. Scanning electron microscopy of simultaneous lung allotransplant (A, C. and D) with contralateral reimplant (B). A, Fibrin and cell debris on the alveolar surface. B, Serous exudate on the alveolar wall. C, Thickened alveolar septa. The asterisk indicates a small pulmonary artery. D . Terminal bronchiole. (Original magnifications: A and B / 500; C " 200: D v: 1,000.)
Simultaneous allotransplantation with contralateral reimplantation. At autopsy, both the reimplant and the allotransplant showed severe edema and congestion. No difference was discernible macroscopically, and the gross weights of the allotransplant and the reimplant had increased to the same degree. SEM of the allotransplant revealed an exudate containing abundant cells; fibrin and debris covered the entire surface of the freeze-dried specimen, so that alveolar architecture was barely visible (Fig. 7, A). In contrast, no cellular or fibrin deposits were observed in the reimplant. Some thickening
of the cut edges of interalveolar septa was evident , and the surface had a waxy appearance, indicating a film of serous exudate (Fig. 7, B). Perivascular cell infiltration and interstitial proliferation in the allograft resulted in severe thickening of these areas (Fig. 7, C). A portion of the terminal bronchiole of the same specimen is shown in Fig. 7, D. The surfaces of the ciliated epithelium and the Clara cells were heavily veiled by exudate and cellular deposits throughout the bronchial tree. In the reimplant, the surface of the bronchial epithelium was better preserved.
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Fig. 8. Scanning electron microscopy of a bilateral reimplant after 2 years. (Original magnification x500.)
Bilateral reimplantation. Despite daily activity and normal appearance for 2 years, this dog had a subnormal POe and a normal Pco., indicating mild diffusion impairment. SEM demonstrated an increased cellularity and slight thickening of the alveolar wall (Fig. 8). These findings were also confirmed byLM. Discussion
In recent years, SEM has become a valuable instrument for morphologic study of the normal and diseased lung,"- 12, 13, 14 Cellular distribution and topography of the surface structure are best obtained by SEM. However, there has been no report of the application of SEM to the study of lung transplants. Wheeler and co-workers" did describe interesting findings in SEM of the human heart valve allograft. For successful SEM of biological material, the methods of tissue preparation are crucial.' After many trials and errors using freeze-dried lung tissue, we found the present
method to be the most suitable. We did not employ the critical-point drying technique, a widely used procedure recommended by Anderson,' which might be worthwhile in further studies. Inflation of the lung through the bronchus with glutaraldehyde fixed the tissue faster and demonstrated the honeycomb structure of the organ better. To reveal the detailed texture of the mucosal surface, some investigators have used bronchial lavage with saline. However, we did not adopt this technique, since accumulation of exudate, mucus material, and cells represents an important pathophysiologic process in pulmonary allograft rejection. Furthermore, the severe destruction of the bronchial mucosa often prevented simple practice of this procedure. Probably for this reason, the ciliated epithelium of our specimens frequently appeared to be covered with a thin film. Also, we could not identify the microvilli on the surface of type II alveolar epithelial cells or in the junctions between alveolar epithelial cells.
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Scanning electron microscopy
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In many respects, SEM confirmed the earlier findings obtained by LM and TEM in alveoli of lung transplants. Accumulation of exudate and cells, thickening of the alveolar wall, and a decrease in the air-space of the alveoli were observed to parallel functional impairment. These changes were so striking that one could easily imagine severe restriction of oxygen exchange without any other factors. In long-term survivors of bilateral reimplantation and modified bilateral allotransplantation, there was a marked increase in cellularity on the alveolar surface. Many of these cells were identified as type II alveolar epithelial cells. Strong reactivity or regenerative activity of type II cells has been reported in different situations.t- 8, 1~, 16 Nagaishi'" described adenomatous hyperplasia of the alveolar lining elicited by hyperplasia of type II cells following cobalt-60 irradiation of the human lung, The Clara cell, which is localized in the terminal bronchiole, remained relatively intact in the severely rejected lung. The transplantation procedure itself is stressful and may cause degeneration of type I cells and proliferation of type II cells in the alveoli. Ciliated epithelial cells are damaged more severely than are Clara cells in the bronchiole. The closer relationship of Clara cells to type II alveolar epithelial cells has been recognized for many years!"; therefore, the similar response of these cells in the rejection process is quite understandable. In addition, immunologic processes certainly enhance and modify these changes. Distinctions between simultaneous allotransplantation and contralateral reimplantation were revealed much better by SEM than by LM or TEM. Surfaces of the alveoli and bronchioles were heavily covered by cells, fibrin, and debris in the allograft, whereas no such deposits were observed in the reimplant. Thus, despite both grafts exhibiting almost the same grade of edema macroscopically, the different nature of the alveolar exudate sharply reflected the effect of the local immune reaction. Pathogenesis of severe pulmonary edema in the contralateral reimplant represents a new, attractive
project for the study of lung transplantation, which will be described in a separate paper. to Summary
Stereoscopic micro-findings in various phases of lung transplantation have been studied with a scanning electron microscope, and the findings were correlated with those obtained by LM and TEM. Forty-six blocks of lung tissue were procured from dogs with normal lungs, unmodified unilateral allotransplants, modified bilateral allotransplants, simultaneous allotransplants with contralateral reimplants, and long-functioning bilateral reimplants. The great depth of focus with the scanning electron microscope dramatized the rapid and gross damage to alveolar walls during acute rejection. Accumulation of exudate and cells, thickening of the alveolar wall, and a decrease in the air-space of the alveoli were observed to parallel functional impairment. These changes were so striking that one could easily imagine severe restriction of gas exchange. In long-term survivors of bilateral reimplantation and modified bilateral allotransplantation, there was a marked increase in type II alveolar epithelial cells and slight thickening of the alveolar wall, explaining the persistent nature of functional impairment. Although simultaneous allotransplantation with contralateral reimplantation caused almost the same grade of edema macroscopically, the different characters of alveolar exudate were clearly demonstrated by SEM. We are grateful for the assistance furnished us by Dr. Katherine Mather and for the use of the scanning electron microscope at the U. S. Army Engineers Waterways Experiment Station, Vicksburg, Mississippi. REFERENCES Anderson, T. F.: Techniques for the Preservation of Three Dimensional Structure in Preparing Specimens for the Electron Microscope, Trans. N. Y. Acad. Sci. (Ser. II) 13: 130, 1951. 2 Barnes, B. A., Flax, M. R., Burke, J. F., and Barr, G.: Experimental Pulmonary Homografts
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in the Dog. I. Morphological Studies, Transplantation 1: 351, 1963. Becker, N. H., Sinha, S. B. P., Hagstrom, J. W. C., Bliirncke, S., and Veith, F. J.: Fine Structure Alterations in Canine Lung Transplants, J. THORAe. CARDIOVASC. SURG. 63: 81, 1972. Boyde, A., and Wood, c.: Preparation of Animal Tissue for Surface-Scanning Electron Microscopy, J. Microbiol. 90: 221, 1969. Daly, B. D. T., and McNary, W. F.: Type II (Granular Alveolar) Pneumocyte in Ultrastructural Analysis of Stressed Human Lung, Surg. Forum 22: 38, 1971. Greenwood, M. F., and Holland, P.: The Mammalian Respiratory Tract Surface: A Scanning Electron Microscopic Study, Lab. Invest. 27: 296, 1972. Gondos, B., White, P., and Benfield, J. R.: Ultrastructural Alterations in Canine Lung Allografts, Am. J. Pathol. 64: 373, 1971. Kapanci, Y., Weibel, E. R., Kaplan, H. P., and Robinson, F. R.: Pathogenesis and Reversibility of the Pulmonary Lesions of Oxygen Toxicity in Monkeys. II. Ultrastructural and Morphometric Studies, Lab. Invest. 20: 10 I, 1969. Kondo, Y., Isin, E., Cockrell, J. V., and Hardy, J. D.: One-Stage Bilateral Allotransplantation of Canine Lungs: Further Studies. Early Rejection and Prolonged Survival With Immunosuppression, J. THORAe. CARDIOVASC. SURG. 64: 897, 1972. Kondo, Y., Cockrell, J. V., Turner, M. D., and Hardy, J. D.: Mechanism of Postallotransplantation Pulmonary Edema: Examination With Simultaneous Reimplant With Contralateral Allograft. In preparation.
11 Molokhia, F. A. S., Ponn, R. B., Asimacopolos, P. J., and Norman, J. C.: Microscopic and Ultrastructural Changes in Unmodified Canine Lung Allografts, Arch. Surg. 103: 490, 1971. 12 Nagaishi, C.: Functional Anatomy and Histology of the Lung, Baltimore, 1972, University Park Press, pp. 29-65. 13 Nowell, J. A., and Tyler, W. S.: Scanning Electron Microscopy of the Surface Morphology of Mammalian Lungs, Am. Rev. Resp. Dis. 103: 313, 1971. 14 Tyler, W. S., de Lorimier, A. A., Manus, A. G., and Nowell, J. A.: Surface Morphology of Hypoplastic and Normal Lungs From Newborn Lambs, ill Jahari, 0., and Corvin, I., editors: Scanning Electron Microscopy/1971, Proceedings of the Fourth Annual Scanning Electron Microscopy Symposium, Chicago, 1971, lIT Research Institute, pp. 305-312. 15 Veith, F. 1., and Hagstrom, J. W.: Alveolar Manifestations of Rejection: An Important Cause of the Poor Results With Human Lung Transplantation, Ann. Surg, 175: 336, 1972. 16 Wang, N. S., Huang, S. N., Sheldon, H., and Thurlbeck, W. M.: Ultrastructural Changes of Clara cells and Type II Alveolar Cells in Adrenalin-Induced Pulmonary Edema in Mice, Am. J. Pat hoI. 62: 237, 1971. 17 Warren, B. A., and de Bono, A. H. B.: The Ultrastructure of Early Rejection Phenomena in Lung Homografts in Dogs, Br. J. Exp. Pathol. 50: 593, 1969. 18 Wheeler, E. E., Gavin, J. B., and Herdson, P. B.: A Scanning Electron Microscopy Study of Human Heart Valve Allografts. Pathology 4: 185, 1972.