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Into deepest lung with drug and camera
Neuroscience & BiobehavioralReviews, Vol. 12, pp. 315-320. © Pergamon Press pie, 1988. Printed in the U.S.A.
0149-7634/88 $3.00 + .00
Into Deepest Lung With Drug and Camera T. K. A K E R S A N D D. J. C R I T T E N D E N 1 D e p a r t m e n t o f Physiology, U N D S M , Grand Forks, N D 58202
AKERS, T. K. AND D. J. CRITTENDEN. Into deepest lung with drug and camera. NEUROSCI BIOBEHAV REV 12(3/4) 315-320, 1988.--Alveoli of guinea pigs subjected to high partial pressures of oxygen (pO2 500 mmHg) for 6 days were studied with the scanning electron microscope. After six days the alveoli of untreated guinea pigs were engorged with an increased volume of alveolar type II cells and macrophages resulting in death of approximately half of the animals. Guinea pigs that had been pretreated with reserpine and phenoxybenzamine catecholamine inhibitors did not show the damaging ultrastructural changes seen in the untreated controls, although phenoxybenzamine was less effective in preventing the changes than reserpine. The results support previous experiments implicating the sympathetic nervous system in the production of pulmonary pathology due to oxygen toxicity. Guinea pigs Electron microscopy Phenoxybenzamine
Alveolar ultrastructure
Oxygen toxicity
Reserpine
or 0.6 mg phenoxybenzamine in saline, and sealing the other end of the tubing (7). A trochar was used to implant the capsules under the skin between the shoulder blades. Six of the guinea pigs from each treatment group were pressurized to 1.1 atmospheres absolute (ATA) with a pO2 of 500 mmHG, six were pressurized to 20 ATA with a pO~ of 500 mmHg, and six more were maintained in air at I ATA. Helium was the diluent gas in the pressurized groups. Six guinea pigs (two reserpine-treated, two phenoxybenzaminetreated, and two saline-treated) from each environment were removed after 2, 4 and 6 days of exposure. The animals in the 20 ATA environment were decompressed following a 4-hour continuous decompression schedule (2). All animals were anesthetized with sodium pentobarbital (35 mg/kg) and prepared for electron microscopy. The lungs were perfused through the trachea at a hydrostatic pressure of 50 cm H20 with glutaraldehyde-paraformaldehyde buffered at a pH of 7.3 with 0.2 N sodium cacodylate (12). After the lungs were fully perfused the thoracic cavity was massaged gently to insure insuffiation of the fixative. The lungs were allowed to fix in situ for 2 hours at which time the thoracic cavity was opened and the lungs were surgically removed. They were immersed in a chilled beaker of fixative for an additional 2 hours. Two tissue cubes were cut from the apex and base of the right lobe of each lung, rinsed in 0.2 N cacodylate buffer for 20 min (20), postfixed in 4% cacodylate buffered 0504 for 1 hr (18), and rinsed again in 0.144 N cacodylate buffer. The tissue was dehydrated in a graded series of acetone solutions, critical point dried in a Sorvall Critical Point System using liquid COs, mounted on specimen stubs with plastic adhesive tape, and coated with carbon and gold/palladium (Technics Hummer II) for SEM viewing on Cambridge Stereoscan. The second set was dried in serial alcohols, then propylene oxide, and finally Epon 812 (Tousimis Research Corporation). Tissue blocks were sectioned on Porter-Blum MT-I Ultramicrotome with a glass knife. Silver sections were mounted on 200 mesh copper grids, stained with uranyl
A previous study in this laboratory using scanning electron microscopy (SEM) showed deterioration of alveolar surface structure in guinea pigs exposed to high levels of oxygen in the range of 400 to 600 mmHg (19). Other studies using pharmacologic techniques have strongly implicated the sympathetic nervous system in producing the symptoms of pulmonary oxygen toxicity (21), and pharmacological sympathectomy using reserpine to deplete catecholamines has been shown to reduce lung damage significantly in rats exposed to oxygen at high pressures (9). Since the protection known to be afforded by sympatholytic drugs had not been studied at the level of alveolar uitrastructure, experiments were performed to see if the protection afforded by reserpine and phenoxybenzamine against oxygen toxicity might be related to changes in alveolar surface topography. SEM was used to permit easier visualization of surface structure as well as to facilitate examination of larger areas of tissue. METHOD Guinea pigs weighing 400 to 700 grams were obtained from Rockland Farms (PA). The animals were maintained in pathogen-free animal quarters in the High Pressure Life Laboratory at the University of North Dakota, Grand Forks. Beginning four days before placement in experimental chambers, 18 guinea pigs were given intramuscular injections of 0.25 mg per day of reserpine (Serpasil; CIBA), 18 more were given 0.30 mg per day of phenoxybenzamine (Dibenzyline; Smith Kline and French Laboratories), and a third group of 18 guinea pigs was given saline injections to act as control. The day before placement in experimental chambers Silastic (Dow Coming) capsules containing 0.5 mg reserpine or 0.6 mg phenoxybenzamine were implanted in guinea pigs receiving reserpine and phenoxybenzamine, respectively. The Silastic capsules were prepared by sealing one end of a 1.5 ml length of Silastic tubing, partially Idling the tubing with a saline solution or 0.5 mg reserpine in saline
1Present address: Cardiopulmonary Sciences, College of Health, University of Central Florida, Orlando, FL 32816.
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FIG. I. This scanning electron micrograph shows an alveolus from a control guinea pig breathing normobaric room air. Note the red blood cells in the pulmonary microvasculature easily visible through a thin air-blood barrier. A few Kohn's pores are apparent. Granular material suggestive of surfactant material is noticeable in a very few areas primarily in corners of the alveolus. In general, the alveolar surface is smooth and clean, presenting minimal diffusion distance for respiratory gases.
acetate, counterstained with lead citrate and viewed on an EM 2000 Transmission Electron Microscope (Philips). RESULTS
Guinea Pigs Breathing Air The alveoli of all untreated, reserpine-treated, and phenoxybenzamine-treated guinea pigs breathing air appeared normal under S E M (Fig. 1). The outstanding feature of the normal alveolus was the thinness of the air-blood barrier. Erythrocytes were easily visualized in the microvasculature under the thin alveolar lining. Alveolar spaces were free of debris. K o h n ' s pores, although few in numbers, were patent, permitting pressure equalization and c o m m u n i c a t i o n between adjacent alveoli.
Guinea Pigs Exposed to 1.1 ATA, 500 mmHg pO.,_ The alveoli of guinea pigs exposed for 2 days did not show appreciable changes from alveoli of air-breathing guinea pigs in untreated, reserpine-treated, or p h e n o x y b e n z a m i n e treated groups. After 4 days exposure, alveoli of untreated guinea pigs showed thickening of alveolar septa, obliteration of K o h n ' s pores, a marked increase in alveolar type II cells, and infiltration of macrophages (Fig. 2, Plate lib). In contrast, alveoli of reserpine- and p h e n o x y b e n z a m i n e - t r e a t e d guinea pigs remained normal in appearance after 4-day exposures. After 6 days the proliferative and invasive changes intensified in untreated guinea pigs, resulting in alveolar engorgement due to wall thickening, type II cell proliferation, and macrophage infiltration. Globular secretory mate-
FACING PAGE FIG. 2. This composite scanning electron micrograph shows the progression of ultrastructural damage to the air-blood barrier of alveolus from exposure to high partial pressures of oxygen (500 mmHg). In Plate lla (upper left) is an essentially normal alveolus from a guinea pig exposed 2 days to 500 mmHg pO~. In Plate lib some thickening of the air-blood barrier after 4 days is evidenced by difficulty in visualizing RBC's through the alveolar wall, and congestion of the air space is increasing due to macrophage infiltration. Plate llc (upper right) shows massive obliteration of the gas exchange structure by continued thickening of the air-blood barrier (evidenced by complete inability to visualize RBC's through the alveolar wall) and extensive infiltration of macrophages into the alveolar space. These alveoli are from guinea pigs exposed to 500 mmHg pOz tbr six days. Plate lid (lower right) shows three alveolar type 11 cells. It is easily seen that due to their cuboidal nature, they protrude into the air space, thickening the air-blood barrier. The granular material covering the surfaces of the cells may represent pulmonary surfactant, the surface tension-lowering phospholipoprotein complex necessary for normal lung function.
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FIG. 3. This composite transmission electron micrograph s h o w s essential features o f the normal alveolus in cross section. Plate Ilia (upper left) s h o w s an alveolar type 11 cell typically located in a corner o f an alveolus. The type 11 cell is easily identified by the presence of several lamellar bodies in the cytoplasm and microvilli at the free edge of the cell. Red blood cells are easily seen in cross section in the microvessels to the right of the airspace. In Plate lllb (lower left), the right air-blood barrier of Plate ilia comprising the area within the lines has been enlarged to show the close apposition of capillary endothelium and thin alveolar epithelium, separated only by the fused b a s e m e n t m e m b r a n e s of the two layers with no interstitial tissue. Plate lllc (upper right) s h o w s a free edge of an alveolar type 11 cell extruding intracellular contents, presumably pulmonary surfactant, via exocytosis into the alveolar airspace. Plates llla, b, and c represent lung tissue from normobaric air breathing guinea pigs. Plate llld (lower right), on the other hand, s h o w s the air blood barrier of a guinea pig exposed to 500 m m H g pO._, for six days. In contrast to the congested surface features s h o w n by the scanning micrographs in Plate lic, the cross-sectional ultrastructure shown in this transmission electron micrograph reveals no damage to the endo- or epithelial cells or the fused basement m e m b r a n e s or the two. This evidence suggests that the increased thickness of the air-blood b a m e r seen in the scanning micrographs has come about as a result ot the prohteration of the thicker, cuboidal type 11 cells combined with massive influx of macrophages, not as a result of destruction of the integrity of the type I epithelium or separation of the fused b a s e m e n t m e m b r a n e s .
L U N G A L V E O L I A N D OXYGEN TOXICITY riai was abundant (Fig. 2, Plate IIc). Reserpine-treated guinea pigs still showed normal alveoli after 6 days exposure. However, any protection due to phenoxybenzamine was ineffective after 6 days, as alveoli from phenoxybenzaminetreated guinea pigs were engorged with macrophages, type II cells and fibrin strands.
Guinea Pigs Exposed to 1.1 ATA, 500 mmHg p02 Transmission Electron Micrographs (TEM) Alveoli of guinea pigs exposed in the normal, untreated state for six days appear as in Fig. 3, Plate IIIa. In the upper left one can see both alveolar type I cells and endothelial cells surrounding blood cells and the alveolar type II cells in the corner of the alveolus, containing surfactant vacuoles with lamellar bodies present. The average number of lamellar bodies found in normal alveoli usually was six. In alveoli which are exposed to oxygen this number increased in the cross section to approximately thirteen. Figure 3, Plate IIIb, lower left, shows quite clearly the relationship between the air space and the red blood cell with the alveolar type I cell, the basement membrane and the endothelial cell with a few vesicles present in normal. In alveoli exposed to 500 mmHg pO2 after 4 and 6 days not only the lamellar bodies increase but the number of microvilli on the surface of the alveolar type II cells increased. In Fig. 3, Plate IIc, upper right, one can see the great increase in microvilli across the surface of the surfactant cell and a lameUar body extruding plates, presumably of pulmonary surfactant, out into the air space via exocytosis. One other change was noted and this was a very great increase in the number of vesicles in both the endothelial cells and in the alveolar type I cells when cells have been exposed to oxygen for 4 to 6 days (see Fig. 3. Plate llId, lower right). DISCUSSION Our results showed that the sympatholytic drugs suppressed or delayed the "alveolar congestion and death in pulmonary oxygen toxicity. The primary ultrastructural change occurring in unprotected guinea pigs due to oxygen toxicity was an increase in number and volume of alveolar type II cells, occurring relatively early. Macrophages increased in number following the increased alveolar type II cell activity, presumably to ingest excess alveolar contents (8). In animals unprotected by the sympatholytic drugs the type II proliferation and subsequent macrophage invasion contributed to disrupt diffusion of respiratory gases across the respiratory membranes. Ultimately, hypoxemia led to acidosis, terminating in death of the animal. Type II cell proliferation preceded any noticeable behavioral symptoms of oxygen toxicity, and SEM failed to reveal any prior ultrastructural damage or desquamation of alveolar type I cells. An earlier team of investigators in our laboratory also applied SEM to pulmonary oxygen toxicity, and while most of their results confirmed previous results obtained by the numerous TEM studies in the literature, they were first to report that microvilli of the alveolar type II cells were elongated in oxygen toxicity. This occurred long before other overt ultrastructural or symptomatic changes were apparent (19). Recently another group of investigators found a 50 percent increase in volume of alveolar type II cells without any prior manifestation of alveolar lesions in rats following administration of metabolite VIII of Bromexine over periods of 3 to 6 days (5). Although the increased volume appeared to be due to type II cell hypertrophy, hyperplasia could not be ruled out.
319 Although type II cell hyperplasia most commonly indicates previous alveolar tissue damage, these two studies in conjunction with the present one suggest that other events may infuence the metabolism and activity of type II ceils in the absence of tissue damage. For example, in the developing rat lung attenuation of type II cells to form type I cells appears to be under the influence of the developing vasculature. The bulging action of new capillaries pushing into the alveoli may transform type II cells into the attenuated type I forms (1, 15). Similar structural changes occurring before detectable tissue damage may have contributed to the alveolar type II cell proliferation seen in the present study. In this regard Kapanci and associates (11) have demonstrated contractile interstitial cells, located in the alveolar walls between alveolar spaces and around pre- or postcapillary vessels, that react to hypoxia, epinephrine and possibly other vasoactive amines with contraction resulting in microvasculature infolding and bulging of the alveolar surfaces into the alveolar space. In view of reported increased adrenal gland activity following intermittent exposure to toxic levels of oxygen (3), presumably resulting in increased levels of circulating catecholamines, this bulging action may have occurred in the present study, and may have provided a stimulus for type II cell proliferation. Furthermore, the, protection afforded by reserpine might be explained on the basis of a depleted pool of catecholamines resulting in reduced interstitial cell contraction, thereby reducing structural stimuli for type II cell activity. The greater efficacy of reserpine compared to phenoxybenzamine with respect to suppressing type II cell proliferation is consistent with reports implicating betaadrenergic receptors but not alpha-adrenergic receptors in increasing alveolar surfactant content in rabbits following increased ventilation (17) and in rats following isoproterenol administration (16). Since the alveolar type II cell is responsible for surfactant synthesis and secretion (13), betaadrenergic receptors may mediate some aspects of type II cell activity. Dobbs and Mason (6) have shown that isolated alveolar type II cells release disaturated phosphatidylcholine in response to direct beta-adrenergic stimulation. Mechanisms for accomplishing this are not clear but an ultrastructural base for neural influence on surfactant secretion (and possibly alveolar type II cell function) has been obtained in two separate laboratories. Meyrick and Reid (14) reported electron microscopic observations of nerves in the alveolar walls of rats lungs. Hung and associates (10) found nerve endings containing large dense-cored vesicles (suggestive of a motor function) located in the interstitium of the alveolar walls near alveolar type II cells. They speculated that these nerve endings were related to and influenced the activities of the type II cells. An earlier study using flourescence techniques demonstrated a large number of adrenergic nerve terminals in lung parenchyma, suggesting that the nerve endings identified by Hung and associates may in fact subserve adrenergic pathways (4). The results of the present study are consistent with two interpretations of stimuli for alveolar type II cell activity, one structural and the other neural, neither of which require prior damage to the type I epithelium to exert their influence. The possibility exists that both kinds of stimuli may interact with each other and with other mechanisms (6) under stressful conditions, including oxygen toxicity, to modulate proliferative as well as secretory aspects of alveolar type II cell activity. The ability of the sympatholytic drug reserpine and to a lesser extent phenoxybenzamine to suppress the ultrastruc-
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tural r e s p o n s e to o x y g e n suggests that the p a r t i c i p a t i o n o f the s y m p a t h e t i c n e r v o u s s y s t e m , particularly b e t a - a d r e n e r -
gic p a t h w a y s , is n e c e s s a r y for full e x p r e s s i o n o f o x y g e n toxic effects.
REFERENCES I. Adamson, I. Y. R., Bowden, D. Derivations of type I epithelium from type 2 cells in the developing rat lung. Lab. Invest. 32:736--745; 1975. 2. Bares, W. A. Optimum driving profiles. Bulletin No. 74307, Instrument Society of America. Pittsburgh, PA; 1974:29-30. 3. Bean, J. W. Adrenal alteration induced by oxygen at high pressure. Fed. Proc. 10:11; 1951. 4. Bean, J. W. ; Nakamoto, T. Direct effect of sympathetics on the lung in centrogenic pulmonary pathology due to O2 at high pressure and other stress. In: Trapp, W. G.; Bannister, E. W.; Davison, J. A.; Trapp, D. A., eds. 5th international hyperbaric conference proceedings. Burnaby, Canada: Simon Fraser University Press; 1974:37-44. 5. Cerutti, P.; Kapanci, Y. Effects of metabolite VIII of Bromexine (Na 872) on type I1 epithelium of the lung. Respiration 37:241-251 ; 1979. 6. Dobbs, L. G.; Mason, R. J. Pulmonary alveolar type II cells isolated from rats: release of phosphatidylcholine in response to /3-adrenergic stimulation. J. Clin. Invest. 63:378-387; 1979. 7. Folkman, J.; Long, D. M. The use of silicone rubber as a carrier for prolonged drug therapy. J. Surg. Res. 4:139-142; 1964. 8. Goldenberg, V. E.; Buckingham, S.; Sommers, S. C. Pilocarpine stimulation of granular pneumocyte secretion. Lab. Invest. 20:147-158; 1969. 9. Hammond, R. E.: Akers, T. K. The effects of catecholamine depletion and potentiation on hyperbaric oxygen toxicity in the rat. Physiologist 15:159; 1972. 10. Hung, K. S.; Hertweck, M. S.; Hardy, J. D.; Loosli, C. G. Electron microscopic observations of nerve endings in the alveolar walls of mouse lungs. Am. Rev. Respir. Dis. 108: 328-333; 1973.
11. Kapanci, Y.; Mo Costabella, P.; Gabbiani, G. Location and function of contractile interstitial cells of the lungs. In: Bouht~ys, A., eds. Amsterdam: Elsevier/North-Holland Biomedical Press; 1976:69-82. 12. Karnovsky, M. J. A formaldehyde-glutaraldehyde fLxative of high osmolality for use in electron microscopy. J. Cell. Biol. 27:137A; 1965. 13. Mason, R. J.; Williams, M. C. Type II alveolar cell: defender of the alveolus. Am. Rev. Respir. Dis. 115:81-91; 1977. 14. Meyrick, B.; Reid, L. Nerves in rat intra-acinar alveoli: an electron microscopic study. Respir. Physiol. 11:367-377; 1971. 15. Noack, W.; Schwarz, W. Electron-microscopic studies on the development of the lung of rats (16 days a.p. - 10 days p.p.). Z. Anat. Entwickl. Gesch. 134:343-360; 1971. 16. Olsen, D. G. Neurohumoral-hormonal secretory stimulation of pulmonary surfactant in the rat. Physiologist 15:230; 1972. 17. Oyarzun, M. J.; Clements, J. A. Control of lung surfactant by ventilation and /3-adrenergic mediator in rabbits. Fed. Proc. 37:719; 1978. 18. Palade, G. A study of fixation for electron microscopy. J. Exp. Med. 95:285-297; 1952. 19. Ross, B. K.; Akers, T. K. Scanning electron microscopy of normoxic and hyperoxic hyperbaric exposed lungs. Undersea Biomed. Res. 3:283-299; 1976. 20. Sabatini, D. C.; Bensch, K.; Barrnett, R. J. Cytochemistry and electron microscopy: the preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell. Biol. 17:19-58; 1963. 21. Smith, C. W.; Bean, J. W. Adrenal factors in toxic action of O~ at atmospheric pressure. Fed. Proc. 14:140; 1955.
0149-7634/88 $3.00 + .00
Into Deepest Lung With Drug and Camera T. K. A K E R S A N D D. J. C R I T T E N D E N 1 D e p a r t m e n t o f Physiology, U N D S M , Grand Forks, N D 58202
AKERS, T. K. AND D. J. CRITTENDEN. Into deepest lung with drug and camera. NEUROSCI BIOBEHAV REV 12(3/4) 315-320, 1988.--Alveoli of guinea pigs subjected to high partial pressures of oxygen (pO2 500 mmHg) for 6 days were studied with the scanning electron microscope. After six days the alveoli of untreated guinea pigs were engorged with an increased volume of alveolar type II cells and macrophages resulting in death of approximately half of the animals. Guinea pigs that had been pretreated with reserpine and phenoxybenzamine catecholamine inhibitors did not show the damaging ultrastructural changes seen in the untreated controls, although phenoxybenzamine was less effective in preventing the changes than reserpine. The results support previous experiments implicating the sympathetic nervous system in the production of pulmonary pathology due to oxygen toxicity. Guinea pigs Electron microscopy Phenoxybenzamine
Alveolar ultrastructure
Oxygen toxicity
Reserpine
or 0.6 mg phenoxybenzamine in saline, and sealing the other end of the tubing (7). A trochar was used to implant the capsules under the skin between the shoulder blades. Six of the guinea pigs from each treatment group were pressurized to 1.1 atmospheres absolute (ATA) with a pO2 of 500 mmHG, six were pressurized to 20 ATA with a pO~ of 500 mmHg, and six more were maintained in air at I ATA. Helium was the diluent gas in the pressurized groups. Six guinea pigs (two reserpine-treated, two phenoxybenzaminetreated, and two saline-treated) from each environment were removed after 2, 4 and 6 days of exposure. The animals in the 20 ATA environment were decompressed following a 4-hour continuous decompression schedule (2). All animals were anesthetized with sodium pentobarbital (35 mg/kg) and prepared for electron microscopy. The lungs were perfused through the trachea at a hydrostatic pressure of 50 cm H20 with glutaraldehyde-paraformaldehyde buffered at a pH of 7.3 with 0.2 N sodium cacodylate (12). After the lungs were fully perfused the thoracic cavity was massaged gently to insure insuffiation of the fixative. The lungs were allowed to fix in situ for 2 hours at which time the thoracic cavity was opened and the lungs were surgically removed. They were immersed in a chilled beaker of fixative for an additional 2 hours. Two tissue cubes were cut from the apex and base of the right lobe of each lung, rinsed in 0.2 N cacodylate buffer for 20 min (20), postfixed in 4% cacodylate buffered 0504 for 1 hr (18), and rinsed again in 0.144 N cacodylate buffer. The tissue was dehydrated in a graded series of acetone solutions, critical point dried in a Sorvall Critical Point System using liquid COs, mounted on specimen stubs with plastic adhesive tape, and coated with carbon and gold/palladium (Technics Hummer II) for SEM viewing on Cambridge Stereoscan. The second set was dried in serial alcohols, then propylene oxide, and finally Epon 812 (Tousimis Research Corporation). Tissue blocks were sectioned on Porter-Blum MT-I Ultramicrotome with a glass knife. Silver sections were mounted on 200 mesh copper grids, stained with uranyl
A previous study in this laboratory using scanning electron microscopy (SEM) showed deterioration of alveolar surface structure in guinea pigs exposed to high levels of oxygen in the range of 400 to 600 mmHg (19). Other studies using pharmacologic techniques have strongly implicated the sympathetic nervous system in producing the symptoms of pulmonary oxygen toxicity (21), and pharmacological sympathectomy using reserpine to deplete catecholamines has been shown to reduce lung damage significantly in rats exposed to oxygen at high pressures (9). Since the protection known to be afforded by sympatholytic drugs had not been studied at the level of alveolar uitrastructure, experiments were performed to see if the protection afforded by reserpine and phenoxybenzamine against oxygen toxicity might be related to changes in alveolar surface topography. SEM was used to permit easier visualization of surface structure as well as to facilitate examination of larger areas of tissue. METHOD Guinea pigs weighing 400 to 700 grams were obtained from Rockland Farms (PA). The animals were maintained in pathogen-free animal quarters in the High Pressure Life Laboratory at the University of North Dakota, Grand Forks. Beginning four days before placement in experimental chambers, 18 guinea pigs were given intramuscular injections of 0.25 mg per day of reserpine (Serpasil; CIBA), 18 more were given 0.30 mg per day of phenoxybenzamine (Dibenzyline; Smith Kline and French Laboratories), and a third group of 18 guinea pigs was given saline injections to act as control. The day before placement in experimental chambers Silastic (Dow Coming) capsules containing 0.5 mg reserpine or 0.6 mg phenoxybenzamine were implanted in guinea pigs receiving reserpine and phenoxybenzamine, respectively. The Silastic capsules were prepared by sealing one end of a 1.5 ml length of Silastic tubing, partially Idling the tubing with a saline solution or 0.5 mg reserpine in saline
1Present address: Cardiopulmonary Sciences, College of Health, University of Central Florida, Orlando, FL 32816.
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FIG. I. This scanning electron micrograph shows an alveolus from a control guinea pig breathing normobaric room air. Note the red blood cells in the pulmonary microvasculature easily visible through a thin air-blood barrier. A few Kohn's pores are apparent. Granular material suggestive of surfactant material is noticeable in a very few areas primarily in corners of the alveolus. In general, the alveolar surface is smooth and clean, presenting minimal diffusion distance for respiratory gases.
acetate, counterstained with lead citrate and viewed on an EM 2000 Transmission Electron Microscope (Philips). RESULTS
Guinea Pigs Breathing Air The alveoli of all untreated, reserpine-treated, and phenoxybenzamine-treated guinea pigs breathing air appeared normal under S E M (Fig. 1). The outstanding feature of the normal alveolus was the thinness of the air-blood barrier. Erythrocytes were easily visualized in the microvasculature under the thin alveolar lining. Alveolar spaces were free of debris. K o h n ' s pores, although few in numbers, were patent, permitting pressure equalization and c o m m u n i c a t i o n between adjacent alveoli.
Guinea Pigs Exposed to 1.1 ATA, 500 mmHg pO.,_ The alveoli of guinea pigs exposed for 2 days did not show appreciable changes from alveoli of air-breathing guinea pigs in untreated, reserpine-treated, or p h e n o x y b e n z a m i n e treated groups. After 4 days exposure, alveoli of untreated guinea pigs showed thickening of alveolar septa, obliteration of K o h n ' s pores, a marked increase in alveolar type II cells, and infiltration of macrophages (Fig. 2, Plate lib). In contrast, alveoli of reserpine- and p h e n o x y b e n z a m i n e - t r e a t e d guinea pigs remained normal in appearance after 4-day exposures. After 6 days the proliferative and invasive changes intensified in untreated guinea pigs, resulting in alveolar engorgement due to wall thickening, type II cell proliferation, and macrophage infiltration. Globular secretory mate-
FACING PAGE FIG. 2. This composite scanning electron micrograph shows the progression of ultrastructural damage to the air-blood barrier of alveolus from exposure to high partial pressures of oxygen (500 mmHg). In Plate lla (upper left) is an essentially normal alveolus from a guinea pig exposed 2 days to 500 mmHg pO~. In Plate lib some thickening of the air-blood barrier after 4 days is evidenced by difficulty in visualizing RBC's through the alveolar wall, and congestion of the air space is increasing due to macrophage infiltration. Plate llc (upper right) shows massive obliteration of the gas exchange structure by continued thickening of the air-blood barrier (evidenced by complete inability to visualize RBC's through the alveolar wall) and extensive infiltration of macrophages into the alveolar space. These alveoli are from guinea pigs exposed to 500 mmHg pOz tbr six days. Plate lid (lower right) shows three alveolar type 11 cells. It is easily seen that due to their cuboidal nature, they protrude into the air space, thickening the air-blood barrier. The granular material covering the surfaces of the cells may represent pulmonary surfactant, the surface tension-lowering phospholipoprotein complex necessary for normal lung function.
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FIG. 3. This composite transmission electron micrograph s h o w s essential features o f the normal alveolus in cross section. Plate Ilia (upper left) s h o w s an alveolar type 11 cell typically located in a corner o f an alveolus. The type 11 cell is easily identified by the presence of several lamellar bodies in the cytoplasm and microvilli at the free edge of the cell. Red blood cells are easily seen in cross section in the microvessels to the right of the airspace. In Plate lllb (lower left), the right air-blood barrier of Plate ilia comprising the area within the lines has been enlarged to show the close apposition of capillary endothelium and thin alveolar epithelium, separated only by the fused b a s e m e n t m e m b r a n e s of the two layers with no interstitial tissue. Plate lllc (upper right) s h o w s a free edge of an alveolar type 11 cell extruding intracellular contents, presumably pulmonary surfactant, via exocytosis into the alveolar airspace. Plates llla, b, and c represent lung tissue from normobaric air breathing guinea pigs. Plate llld (lower right), on the other hand, s h o w s the air blood barrier of a guinea pig exposed to 500 m m H g pO._, for six days. In contrast to the congested surface features s h o w n by the scanning micrographs in Plate lic, the cross-sectional ultrastructure shown in this transmission electron micrograph reveals no damage to the endo- or epithelial cells or the fused basement m e m b r a n e s or the two. This evidence suggests that the increased thickness of the air-blood b a m e r seen in the scanning micrographs has come about as a result ot the prohteration of the thicker, cuboidal type 11 cells combined with massive influx of macrophages, not as a result of destruction of the integrity of the type I epithelium or separation of the fused b a s e m e n t m e m b r a n e s .
L U N G A L V E O L I A N D OXYGEN TOXICITY riai was abundant (Fig. 2, Plate IIc). Reserpine-treated guinea pigs still showed normal alveoli after 6 days exposure. However, any protection due to phenoxybenzamine was ineffective after 6 days, as alveoli from phenoxybenzaminetreated guinea pigs were engorged with macrophages, type II cells and fibrin strands.
Guinea Pigs Exposed to 1.1 ATA, 500 mmHg p02 Transmission Electron Micrographs (TEM) Alveoli of guinea pigs exposed in the normal, untreated state for six days appear as in Fig. 3, Plate IIIa. In the upper left one can see both alveolar type I cells and endothelial cells surrounding blood cells and the alveolar type II cells in the corner of the alveolus, containing surfactant vacuoles with lamellar bodies present. The average number of lamellar bodies found in normal alveoli usually was six. In alveoli which are exposed to oxygen this number increased in the cross section to approximately thirteen. Figure 3, Plate IIIb, lower left, shows quite clearly the relationship between the air space and the red blood cell with the alveolar type I cell, the basement membrane and the endothelial cell with a few vesicles present in normal. In alveoli exposed to 500 mmHg pO2 after 4 and 6 days not only the lamellar bodies increase but the number of microvilli on the surface of the alveolar type II cells increased. In Fig. 3, Plate IIc, upper right, one can see the great increase in microvilli across the surface of the surfactant cell and a lameUar body extruding plates, presumably of pulmonary surfactant, out into the air space via exocytosis. One other change was noted and this was a very great increase in the number of vesicles in both the endothelial cells and in the alveolar type I cells when cells have been exposed to oxygen for 4 to 6 days (see Fig. 3. Plate llId, lower right). DISCUSSION Our results showed that the sympatholytic drugs suppressed or delayed the "alveolar congestion and death in pulmonary oxygen toxicity. The primary ultrastructural change occurring in unprotected guinea pigs due to oxygen toxicity was an increase in number and volume of alveolar type II cells, occurring relatively early. Macrophages increased in number following the increased alveolar type II cell activity, presumably to ingest excess alveolar contents (8). In animals unprotected by the sympatholytic drugs the type II proliferation and subsequent macrophage invasion contributed to disrupt diffusion of respiratory gases across the respiratory membranes. Ultimately, hypoxemia led to acidosis, terminating in death of the animal. Type II cell proliferation preceded any noticeable behavioral symptoms of oxygen toxicity, and SEM failed to reveal any prior ultrastructural damage or desquamation of alveolar type I cells. An earlier team of investigators in our laboratory also applied SEM to pulmonary oxygen toxicity, and while most of their results confirmed previous results obtained by the numerous TEM studies in the literature, they were first to report that microvilli of the alveolar type II cells were elongated in oxygen toxicity. This occurred long before other overt ultrastructural or symptomatic changes were apparent (19). Recently another group of investigators found a 50 percent increase in volume of alveolar type II cells without any prior manifestation of alveolar lesions in rats following administration of metabolite VIII of Bromexine over periods of 3 to 6 days (5). Although the increased volume appeared to be due to type II cell hypertrophy, hyperplasia could not be ruled out.
319 Although type II cell hyperplasia most commonly indicates previous alveolar tissue damage, these two studies in conjunction with the present one suggest that other events may infuence the metabolism and activity of type II ceils in the absence of tissue damage. For example, in the developing rat lung attenuation of type II cells to form type I cells appears to be under the influence of the developing vasculature. The bulging action of new capillaries pushing into the alveoli may transform type II cells into the attenuated type I forms (1, 15). Similar structural changes occurring before detectable tissue damage may have contributed to the alveolar type II cell proliferation seen in the present study. In this regard Kapanci and associates (11) have demonstrated contractile interstitial cells, located in the alveolar walls between alveolar spaces and around pre- or postcapillary vessels, that react to hypoxia, epinephrine and possibly other vasoactive amines with contraction resulting in microvasculature infolding and bulging of the alveolar surfaces into the alveolar space. In view of reported increased adrenal gland activity following intermittent exposure to toxic levels of oxygen (3), presumably resulting in increased levels of circulating catecholamines, this bulging action may have occurred in the present study, and may have provided a stimulus for type II cell proliferation. Furthermore, the, protection afforded by reserpine might be explained on the basis of a depleted pool of catecholamines resulting in reduced interstitial cell contraction, thereby reducing structural stimuli for type II cell activity. The greater efficacy of reserpine compared to phenoxybenzamine with respect to suppressing type II cell proliferation is consistent with reports implicating betaadrenergic receptors but not alpha-adrenergic receptors in increasing alveolar surfactant content in rabbits following increased ventilation (17) and in rats following isoproterenol administration (16). Since the alveolar type II cell is responsible for surfactant synthesis and secretion (13), betaadrenergic receptors may mediate some aspects of type II cell activity. Dobbs and Mason (6) have shown that isolated alveolar type II cells release disaturated phosphatidylcholine in response to direct beta-adrenergic stimulation. Mechanisms for accomplishing this are not clear but an ultrastructural base for neural influence on surfactant secretion (and possibly alveolar type II cell function) has been obtained in two separate laboratories. Meyrick and Reid (14) reported electron microscopic observations of nerves in the alveolar walls of rats lungs. Hung and associates (10) found nerve endings containing large dense-cored vesicles (suggestive of a motor function) located in the interstitium of the alveolar walls near alveolar type II cells. They speculated that these nerve endings were related to and influenced the activities of the type II cells. An earlier study using flourescence techniques demonstrated a large number of adrenergic nerve terminals in lung parenchyma, suggesting that the nerve endings identified by Hung and associates may in fact subserve adrenergic pathways (4). The results of the present study are consistent with two interpretations of stimuli for alveolar type II cell activity, one structural and the other neural, neither of which require prior damage to the type I epithelium to exert their influence. The possibility exists that both kinds of stimuli may interact with each other and with other mechanisms (6) under stressful conditions, including oxygen toxicity, to modulate proliferative as well as secretory aspects of alveolar type II cell activity. The ability of the sympatholytic drug reserpine and to a lesser extent phenoxybenzamine to suppress the ultrastruc-
320
AKERS AND CRITTENDEN
tural r e s p o n s e to o x y g e n suggests that the p a r t i c i p a t i o n o f the s y m p a t h e t i c n e r v o u s s y s t e m , particularly b e t a - a d r e n e r -
gic p a t h w a y s , is n e c e s s a r y for full e x p r e s s i o n o f o x y g e n toxic effects.
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11. Kapanci, Y.; Mo Costabella, P.; Gabbiani, G. Location and function of contractile interstitial cells of the lungs. In: Bouht~ys, A., eds. Amsterdam: Elsevier/North-Holland Biomedical Press; 1976:69-82. 12. Karnovsky, M. J. A formaldehyde-glutaraldehyde fLxative of high osmolality for use in electron microscopy. J. Cell. Biol. 27:137A; 1965. 13. Mason, R. J.; Williams, M. C. Type II alveolar cell: defender of the alveolus. Am. Rev. Respir. Dis. 115:81-91; 1977. 14. Meyrick, B.; Reid, L. Nerves in rat intra-acinar alveoli: an electron microscopic study. Respir. Physiol. 11:367-377; 1971. 15. Noack, W.; Schwarz, W. Electron-microscopic studies on the development of the lung of rats (16 days a.p. - 10 days p.p.). Z. Anat. Entwickl. Gesch. 134:343-360; 1971. 16. Olsen, D. G. Neurohumoral-hormonal secretory stimulation of pulmonary surfactant in the rat. Physiologist 15:230; 1972. 17. Oyarzun, M. J.; Clements, J. A. Control of lung surfactant by ventilation and /3-adrenergic mediator in rabbits. Fed. Proc. 37:719; 1978. 18. Palade, G. A study of fixation for electron microscopy. J. Exp. Med. 95:285-297; 1952. 19. Ross, B. K.; Akers, T. K. Scanning electron microscopy of normoxic and hyperoxic hyperbaric exposed lungs. Undersea Biomed. Res. 3:283-299; 1976. 20. Sabatini, D. C.; Bensch, K.; Barrnett, R. J. Cytochemistry and electron microscopy: the preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell. Biol. 17:19-58; 1963. 21. Smith, C. W.; Bean, J. W. Adrenal factors in toxic action of O~ at atmospheric pressure. Fed. Proc. 14:140; 1955.