The Effect of Ethchlorvynol on Pulmonary Ultrastructure in Dogs Lyle C. Dearden, PhD, Frederick L. Glauser, MD, and Deborah Smeltzer, BS
The ultrastructure of alveolar septae in dogs is investigated at times ranging from 30 seconds to 60 minutes after intravenous injection of ethchlorvynol (Placidyl). Pulmonary edematous fluid first appears in alveolar spaces 5 minutes after injection and becomes progressively more prominent with increasing time. Alveolar septae are initially somewhat fibrotic, and subsequently, most interstitial spaces become swollen and hydrated. Vesicles in endothelial cells increase with postinjectional time, and they seem to form channels or pores interconnecting capillaries and interstitial spaces. Similar vesicles in epithelial cells (Type 1) show an increase after 30 minutes, and they also seem to form channels or pores interconnecting interstitial spaces and the alveolus. Vesicles, whether in endothelial or epithelial cells, contain a flocculent filamentous material similar to plasma protein and the filamentous proteinaceous material in edematous fluid in alveolar spaces. Ethchlorvynol injection rapidly induces a nonhemodynamic form of pulmonary edema. Since cell junctions of both endothelial and epithelial cells remained intact, it is proposed that transalveolar transport of edematous proteinaceous fluid is mediated by means of endothelial and epithelial vesicles. (Am J Pathol 87:525-536, 1977)
PULMONARY EDENMA can be arbitrarily classified into two types -hemodynamic (characterized by elevated pulmonary capillary hydrostatic pressures) and nonhemodynamic [characterized by normal pulmonary capillary hydrostatic pressures with an associated increase in the permeability of the alveolar capillary membrane (ACM)]. The latter type of pulmonary edema (clinically termed adult respiratory distress syndrome, ARDS) has become of much interest in the last decade with the appreciation of its ubiquitousness. Although a multitude of insults can lead to a "leaky" ACM, the immediate cause of this increased permeability has, so far, escaped detection.' One of the common entities associated with ARDS is the injection or ingestion of various drugs, the best known of which is heroin.2'3 A less well recognized cause of ARDS is the intravenous injection of ethchlorvvnol (Placidyl), a hypnotic, sedative drug designed for oral use. We,' as well as others,5 have reported pulmonary edema following the intravenous use of ethchlorvynol in young adults. Acute studies in animals produced the From the Department of Anatomv, Universitv of California. College of Medicine. Irvine. California, and the Department of Medicine, Veterans Administration Hospital, Long Beach. California. Accepted for publication January 10, 1977. Address reprint requests to Dr. Frederick L Glauser, Chief, Pulmonary Section B Veterans Administration Hospital, 5901 East Seventh Street, Long Beach, CA 90801 525
526
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following within 5 minutes of injection of 15 to 25 mg/kg ethchlorvynol: pulmonary edema, decreased arterial oxygen pressure, transient decreases in cardiac output, transient hypotension, no effect on pulmonary wedge pressure, and a relative bradycardia.' We interpret these findings as consistent with a nonhemodynamic form of pulmonary edema. Further unpublished studies from our laboratory 6 have shown that ethchlorvynol fluxes into and out of pulmonary tissue within 5 minutes of intravenous injection (following the laws of passive diffusion). Permeability studies, employing the in vivo saline-filled dog lung model,7 have shown that substances with molecular weights of up to 90,000 appear in "alveolar" liquid samples following injection into the venous system. Using the same model after ethchlorvynol injection,6 we have demonstrated an increase in permeability of the ACM to high-molecular-weight dextrans (500,000) but with a lag time of 30 minutes when compared with lower molecular weight (< 200,000) substances. From the above data it would appear that intravenous injection of ethchlorvynol leads to a rapid and marked functional decrease of ACM integrity. It, therefore, could be anticipated that marked structural alterations at the ACM level might parallel these physiologic findings. This study examines the pulmonary ultrastructure following the intravenous injection of ethchlorvynol in dogs. Materials and Methods Twenty-two mongrel dogs were distributed according to body weight into control and experimental groups. All dogs were anesthetized with 30 mg /kg pentobarbital, intubated, and ventilated with 100% oxygen. They were then placed in the 450 upright position. The experimental group (11 dogs) received an intravenous injection of 15 to 25 mg/kg ethchlorvynol (Placidyl, Abbott Laboratories, North Chicago, Ill.). The control group received an injection of 1 cu cm sterile saline solution. Lung samples from both groups were obtained at 30, 60, and 90 seconds, and at 2, 3, 4, 5, 15, 30, 45, and 60 minutes after injection. Only one lung sample was obtained from each animal. Lung tissue was obtained as follows: the sternum was split and retracted widely and a left lower lobe specimen was obtained. Tissues were immediately placed in cold (4 C) 2% glutaraldehyde buffered in 0.1 M cacodylate buffer (pH 7.4) and diced into small fragments (approximately 1-mm cubes). They were then transferred to fresh fixative for 2 hours and were postfixed for 1 hour in cacodylate-buffered 1% osmium tetroxide (pH 7.4). Dehydration occurred in a graded series of acetone, and the specimens were then embedded in Luft's Epon 812. Thick sections (0.5 to 1.0 ,) were cut and stained with 0.5% aqueous toluidine blue. These sections were used for routine examination and for selecting areas for thin sectioning. Thin sections with gray to gold interference colors were cut, mounted on uncoated or on Formvar-coated copper grids, and observed and photographed in a Philips EM 300 electron microscope after staining with uranyl acetate and lead citrate.
Results
Following the injection of ethchlorvynol, the diameter of most alveolar septae increased progressively through 30 minutes and retained this in-
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creased diameter through 60 minutes (Figure IA and B, Table 1). Some septae in the experimental group were markedlv thickened by fibrous connective tissue (Figure 1B). Alveolar capillaries increased in prominence up to 15 minutes after injection of the drug and exhibited this enhanced vascularity throughout the ensuing 45 minutes (Figure LA and B, Table 1). Ervthrocvtes in alveoli were more prominent than in controls 30 through 60 minutes after injection. Comparison of controls and of experimental groups at the electron microscopic level (Figure 2A and B) revealed alterations in terms of the degree of fibrillaritv and elasticitv in interstitial spaces, involvement of endothelial and epithelial (Type 1) cells, numbers of vesicles and vacuoles in both of these tvpes of cells, slight modifications in greater alveolar cells (Type 2), enhanced macrophage activity, and leakage of fluid into alveolar spaces.
Within a minutes after injection of ethchlorovvnol there was evidence of widening of the interstitial spaces in most alveolar septae concurrent, Table 1-Pulmonary Alterations Following Ethchlorvynol Injection (5 through 60 minutes)
Experimental animals
Control animals
Diameter of alveolarsepta Vacularity of septa Fibrosis of septa
Endothelial vesicles Endothelial vacuoles Endothelial blebs Hydration of interstitial spaces
45
60
++±
++±
++±
++±
+±
++±
++
++± ++± ++± ++++
++++
+±
+±
+++
-
i
+
-
-
-
-
+
+±
+±
±
-
+
i
+
i
+
++±
+++
+++
+±
++
++
++
+±
+±
++±
++
++±
+
+
+
+
+
+
+
++
++
+++
++
++
++
++
++
+
*
++
++
++
+
+
+
+
+
+±
++
++
++
++
+
+±
+±
+
+±
+± ++± ++±
+++
++
-
-
-
-
-
++
+±+
++±
30
15
5
15
30
45
60
5
+
i
+
+±
+
+±
++
+
+±
+
+
+±
++
++±
++
++
++
+±
++
+±
++
i -
+++
+++
+++
++ +++ ++±
+++
++
+±
+±
+++
Epithelial vesicles
+±
Epithelial vacuoles Lamellar bodies Type II cells WBC + platelets Macrophage population Edema fluid in alveolus
++++ to
- =
the degree of preponderance.
i
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with some alteration or disruption of basement membranes including the presence of enhanced quantities of elastin and of thick (up to 400 A) collagen fibrils. These fibrils were not dispersed, but rather occurred in thick bundles observable both in cross and longitudinally oriented profiles (Table 1). The significant difference between these interstitial areas and those in the controls related primarily to the increased frequency of fibrillarity of the interstitial spaces in the experimental animals. This was possibly related to the frequent observation of fibrocytes in interstitial spaces which were more common than they were in controls. In uninvolved interstitial spaces in the controls and in the experimental groups the basement membrane remained relatively constant, averaging about 650 A when the endothelial and epithelial basement membranes were fused. Subsequently (15, 30, 45, and 60 minutes) there was a progressive increase of interstitial involvement, including apparent hydration in the experimental animals and a proportionate decrease in the occurrence of "thin segments" in the ACM. Fibrocytes continued to be prominent in these latter periods of time. As noted previously, when the basement membrane was intact it did not differ in diameter from that in corresponding controls. The first indications of involvement of endothelial cells occurred at 5 minutes and consisted of increasing numbers of plasmalemmal vesicles (Table 1). These vesicles, in control and in experimental animals, averaged about 600 A in diameter, and often contained a flocculent filamentous material similar to plasma (inset, Figure 3). Some endothelial cells were swollen and lucent. Intracellular vacuoles measuring up to 0.5 A in diameter and containing flocculent material were often observed in endothelial cells 15 minutes after injection (Figure 2B, Table 1). At this same time occasional blebs were observed protruding from these cells into their capillary lumen, and they also contained flocculent material similar to that in vacuoles, vesicles, and the lumen of capillaries. Blebbing of the endothelium was not a prominent feature at this or at subsequent times (Table 1). By 30 minutes, lucency of endothelial cells was fairly common and they contained numerous vesicles (Figure 3 and inset). It was sometimes possible to observe vesicles as apparent channels or pores interconnecting the capillary lumen with interstitial spaces (arrows, Figure 4). Maximum dimensions of the openings of these channels or pores varied between 200 and 350 A. Cell-to-cell junctions remained apparently intact and did not differ from those in controls. Epithelial (Type 1) cells showed no consistent alterations until 30 minutes after injection. At this time there was evidence of lucency, swelling, and vacuolization of some of these cells (Figure 3). Vesicles were
Vol. 87, No. 3 June 1977
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fairly prominent and contained the same kind of flocculent material observed in similar vesicles in endothelial cells, endothelial vacuoles and blebs, and epithelial vacuoles (inset, Figure 3). The membranes of vesicles sometimes appeared to fuse, forming an apparent channel through epithelial cells (Figure 5), while other vesicles seemed to fuse with and empty their content into vacuoles (inset, Figure 3) which subsequently fused with the plasmalemma and were then exocytosed. As in endothelial vesicles, the maximum dimensions of openings of epithelial channels or pores varied between 200 to 350 A. Somewhat similar observations were made in corresponding controls, but they were not common, and vesicles were also less commonly observed than they were in the experimental animals. Epithelial cell junctions in experimental and control animals remained intact. Greater alveolar cells (Type 2) showed only minor modifications from those in controls and these consisted of a reduction of microvilli (both numerically and in length) and a reduction of the osmiophilic electrondense bands typically found in these cells (Table 1). These alterations were somewhat transitory (through 15 minutes), and subsequently, these cells were comparable with similar cells in controls. Cellular debris (arrows, Figure 2B) was first observed in alveoli of the experimental animals 15 minutes after injection of Placidyl and increased in amount with increasing time (Table 1). Concurrent with the first appearance of debris, leukocytes and platelets in capillaries were more commonly observed than previously (Table 1). Phagocytic cells (Figure 2B) were much more common in interstitial spaces and alveoli of the experimental groups than they were in controls (Table 1). These cells appeared to be phagocytosing cellular debris in alveolar spaces. Edematous fluid containing flocculent filamentous proteinaceous material similar to plasma protein was first observed in scanty amounts 5 minutes after injection of ethchlorvynol (Table 1). Subsequently, filamentous material increased in alveolar spaces (Figure 3). This material often appeared to be in close association with vesicles opening on the surface of epithelial cells (arrows, Figure 6) and with exocytosing vacuoles. The amount of this material in alevoli was maximal at 60 minutes after ethchlorvynol injection. This indicated a time-related increase in permeability of the ACM. Leakage of edematous fluid through the ACM was not observed at any time in the controls. Discussion Intravenous injection of ethchlorvynol induces several alveolar responses referable to pulmonary edema which are demonstrable ultrastructur-
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ally. Pulmonary edema is characterized ultrastructurally as consisting of all or most of the following: a) swelling and hydration of interstitial spaces of alveolar septa,8`'0 b) blebbing of capillary endothelium, "" c) swelling and vesiculation of endothelial and epithelial (Type 1) cells,"'2 and d) the accumulation of excessive amounts of proteinaceous fluid in alveolar spaces. Treatment with ethchlorvynol induces all of these aspects of pulmonary edema either directly or indirectly. Moreover, it seems to induce some aspects typical of inflammation, and these consist of the following: 1) enhancement of collagen and elastic tissue deposition in insterstitial spaces, 2) an influx of leukocytes and platelets in alveolar capillaries, 3) increasing quantities of cellular debris in alveolar spaces, and 4) a marked increase in the number of macrophages and in their phagocytic activity, particularly in alevolar spaces. These latter observations might account, at least in part, for the increased vascularity observed after treatment. Most of these phenomena are time related and become progressively more prominent with increasing time postinjection. These observations are particularly interesting since ethchlorovynol is known to flux into and out of pulmonary tissue within 5 minutes after injection. Physiologic studies indicate that endothelium is normally permeable to small water-soluble and lipid-soluble solutes as well as to larger molecules such as albumin. These substances are thought to move across the endothelium, enter interstitial spaces of the ACM, and pass along bronchioles to enter lung lymphatics."3 We have observed endothelial channels opening between the capillary lumen and interstitial spaces and these may be the morphologic pathway for passage of these solutes. Indeed, tracer techniques employing glycogens and dextrans 14 or small heme-peptides 1 have provided good evidence that capillary membrane transport may be mediated by means of plasmalemmal vesicles forming transport channels. The enhanced widening and hydration of interstitial spaces in the ethchlorvynol-treated dogs suggests that the rate of transport of substances into these spaces is indeed more rapid than it is in corresponding controls. Vesicles in endothelial cells of ethchlorovynol-treated dogs are more numerous than they are in controls, and if transendothelial transport is by means of vesicles as we suspect, the increase in numbers of endothelial vesicles would correlate well with the interstitial edema observed in these animals. The increase in permeability of the endothelium does not seem to relate to an increase in diameter of endothelial cell junctions because these junctions are comparable in diameter with those in controls and the interstitial spaces in controls are considerably less edematous. The earliest appearance of filamentous flocculent edematous fluid in alveolar spaces occurs 5 minutes after injection of ethchlorvynol and this
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correlates well with previous physiologic findings in this model.4 At this time, the increased permeability of the ACM does not seem to relate to increased diameters of cell junctions in endothelial or epithelial cells since these diameters are comparable with those in controls and no leakage of fluid into alveolar spaces is observed in control animals. Subsequently, even with increasing quantities of edematous fluid entering alveolar spaces, the endothelial and epithelial cell junctions seem to remain intact. In this regard, it has been shown that neither horseradish peroxidase nor hemoglobin escape the vascular bed through interendothelial junctions under near normal perfusion pressures (15 to 20 mm of mercur).'3 Moreover, leakage of these substances through both endothelial and epithelial cell junctions occurs only when pressures greater than 50 mm of mercury are used.13 In physiologic studies of similar ethchlorvvnol-treated dogs,' we have observed a decrease in pulmonary arterial pressure and a systemic hypotension when compared with controls, and therefore, a leakage through epithelial cell junctions would not be expected. However, vesiculation and vacuolization of both endothelial and epithelial cells increases with increasing postinjectional time, and it seems reasonable that these structures might be involved in transalveolar transport. Indeed, epithelial channels or pores have been observed interconnecting interstitial and alveolar spaces, and vacuoles seem to empty their filamentous content into alveoli. It would seem, therefore, that vesicular transport through the ACM, probably at an increased rate, might be one explanation for the enhanced permeability of this membrane after treatment with ethchlorvynol. However, these studies do not exclude the possibility of transport via intercellular junctions as previously suggested,'3 '6',7 and it is possible that substances with low molecular weights and dimensions of less than 10 A might penetrate the ACM using intercellular spaces (about 10 A in diameter for epithelial cell junctions) to gain access to alveoli. Regardless of the mechanism, the present study reveals a time-related increase in permeability of the ACM following ethchlorvynol injection. In this regard, flocculent proteinaceous material becomes prominent in alveolar spaces 30 minutes after injection and this correlates with the increase in vesicles and vacuoles in Type 1 cells and with previous observations regarding a time delay of epithelial involvement in pulmonary edema.6'13 This timing and involvement of epithelial cells also corresponds with the postinjectional time for passage of high-molecularweight (250,000 to 500,000) dextrans through the ACM in this model.6 The effect of ethchlorvynol on alveolar capillary permeability has been suggested to be either direct, or indirect.' In view of the rapid flux of this drug into and out of alveolar capillaries (5 minutes) and the increased
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permeability of the ACM 30 minutes after injection, it would seem that either the effects of the drug once initiated are long lasting or the enhanced permeability is an indirect result. The present study does not prove either of these possibilities, but the wealth of the information obtained is supportive of the hypothesis that ethchlorvynol induces pulmonary edema secondarily or indirectly to its primary effect on the ACM. If this is true, the primary effect of the drug on the ACM has not been determined by these studies, but it has been established that pulmonary edema is rapidly induced. References 1. Hayes JA, Shiga A: Ultrastructural changes in pulmonary oedema produced experimentally,with ammonium sulfate. J Pathol. 100:281-286, 1970 2. Steinberg, AD, Karliner, JS: The clinical spectrum of heroin pulmonary edema. Arch Inter Med 122:122-127, 1968 3. Frand IU, Shim CS, Williams MH: Heroin-induced pulmonary edema: Sequential studies of pulmonary function. Ann Intern Med 77:29-35, 1972 4. Glauser FL, Smith WR, Caldwell, A, Hoshiko M, Dolan GS, Baer H, Olsher N: Ethchlorvynol (Placidyl')-induced pulmonary edema. Ann Intern Med 84:46-48, 1976 5. Algeri EJ, Katsas, GG, Luongo MA: Determination of ethchlorvynol in biologic mediums and report of two fatal cases. Am J Clin Pathol 38:125-130, 1962 6. Dearden LC, Glauser, FL, Smeltzer D: Unpublished data 7. Theodore J, Robin ED, Gaudio R, Acevedo J: Transalveolar transport of large polar solutes (sucrose, inulin, and dextran). Am J Physiol 229:989-996, 1975 8. Meyrick B, Miller J, Reid L: Pulmonary oedema induced by ANTU, or by high or low oxygen concentrations in rat: An electron microscopic study. Br J Exp Pathol 53:347-358, 1972 9. Cottrell TS, Levine OR, Senior RM, Wiener J, Spiro D, Fishman AP: Electron microscopic alterations at the the alveolar level in pulmonary edema. Circ Res 21:783-797, 1967 10. Teplitz C: The ultrastructural basis for pulmonary pathophysiology following trauma. J Trauma 8:700-714, 1968 11. Heath D, Moosavi H, Smith P: Ultrastructure of high altitude pulmonary edema. Thorax 28:694-700, 1973 12. Staub NC, Nagano H, Pearce ML: Pulmonary edema in dogs, especially the sequence of fluid accumulation in lungs. J Appl Phvsiol 22:227-240, 1966 13. Szidon JP, Pietra GG, Fishman AP: The alveolar-capillary membrane and pulmonary edema. N Engl J Med 286:1200-1204, 1972 14. Simionescu N, Palade GE: Dextrans and glycogens as particulate tracers for studying capillary permeability. J Cell Biol 50:616-624, 1971 15. Simionescu N, Simionescu M, Palade GE: Permeability of muscle capillaries to small heme-peptides. J Cell Biol 64:586-607, 1975 16. Karnovsky MJ: The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J Cell Biol 35:213-236, 1967
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17. Schneeberger-Keeley EE, Karnovsky MJ: The ultrastructural basis of alveolarcapillary membrane permeability to peroxidase used as a tracer. J Cell Biol 37:781-793, 1968
Acknow dgment The authors are indebted to Teresa Espinosa for her expert technical assistance in all aspects of this electron microscopic study.
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Legend for Figures Figure IA-Pulmonary alveoli of a control. B-Pulmonary alveoli 30 minutes after ethchlorvynol injection. Note the widening of alveolar septae and the increased vascularity in B. Some septae (B) are composed of dense connective tissue. (Semithin plastic sections stained with
toluidine blue, X 800)
Figure 2A-Part of an alveolar septum in a control. B-Alveolar septae 15 minutes after ethchlorvynol injection. Comparison of these figures reveals increased fibrillarity and elasticity of interstitial spaces, lucency, swelling, and vacuolization of endothelial cells, cellular debris and a macrophage in the alveolus of the ethchlorvynol treated specimens and absence of most of these alterations in the control. "Thin sections" of the ACM are less commonly observed in B than in A. (Uranyl acetate and lead citrate, x 19,800
Figure 3-Part of an alveolar septum 30 minutes after injection of ethchlorvynol. Observe extensive amounts of flocculent filamentous edematous fluid in the alveolus. The epithelium is swollen, lucent and vacuolated and contains numerous vesicles. The content of vacuoles corresponds with that in the alveolus and is similar to that in capillaries. The endothelium is also lucent and has numerous vesicles; an intact endothelial cell junction is present in the capillary at the left. The interstitial space appears hydrated. Inset-ACM, 30 minutes after injection. Observe the numerous vesicles in the endothelium. They contain material similar to that in the capillary (bottom). Epithelial vesicles also contain similar filamentous material. One epithelial vesicle has fused (arrow) with a vacuole while other vesicles appear to open into the alveolus. (Uranyl acetate and lead citrate; x 27,500; inset, x 38,000) Figure 4-Part of a "thin segment" of the ACM showing vesicles (arrows) interconnecting the capillary lumen (left) and interstitial area (right). Sixty minutes after ethchlorvynol injection. (Uranyl acetate and lead citrate, x 108,000) Figure 5-Part of a Type 1 cell showing an epithelial channel (arrows) interconnecting the interstitial area and the alveolus (left). Other vesicles are in close association to each other. In an intact thin segment of the ACM (lower picture), a vesicle (arrows) is interconnecting the alveolus with the interstitium. Forty-five minutes after ethchlorvynol injection. (Uranyl acetate and lead citrate, x 132,000) Figure 6-Enlargement of a portion of Figure 3 to show the intimate association of filamentous material in the alveolus with openings of vesicles at the cell surface (arrows) (Uranyl acetate and lead citrate, x 38,750).
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The ultrastructure of alveolar septae in dogs is investigated at times ranging from 30 seconds to 60 minutes after intravenous injection of ethchlorvynol (Placidyl). Pulmonary edematous fluid first appears in alveolar spaces 5 minutes after injection and becomes progressively more prominent with increasing time. Alveolar septae are initially somewhat fibrotic, and subsequently, most interstitial spaces become swollen and hydrated. Vesicles in endothelial cells increase with postinjectional time, and they seem to form channels or pores interconnecting capillaries and interstitial spaces. Similar vesicles in epithelial cells (Type 1) show an increase after 30 minutes, and they also seem to form channels or pores interconnecting interstitial spaces and the alveolus. Vesicles, whether in endothelial or epithelial cells, contain a flocculent filamentous material similar to plasma protein and the filamentous proteinaceous material in edematous fluid in alveolar spaces. Ethchlorvynol injection rapidly induces a nonhemodynamic form of pulmonary edema. Since cell junctions of both endothelial and epithelial cells remained intact, it is proposed that transalveolar transport of edematous proteinaceous fluid is mediated by means of endothelial and epithelial vesicles. (Am J Pathol 87:525-536, 1977)
PULMONARY EDENMA can be arbitrarily classified into two types -hemodynamic (characterized by elevated pulmonary capillary hydrostatic pressures) and nonhemodynamic [characterized by normal pulmonary capillary hydrostatic pressures with an associated increase in the permeability of the alveolar capillary membrane (ACM)]. The latter type of pulmonary edema (clinically termed adult respiratory distress syndrome, ARDS) has become of much interest in the last decade with the appreciation of its ubiquitousness. Although a multitude of insults can lead to a "leaky" ACM, the immediate cause of this increased permeability has, so far, escaped detection.' One of the common entities associated with ARDS is the injection or ingestion of various drugs, the best known of which is heroin.2'3 A less well recognized cause of ARDS is the intravenous injection of ethchlorvvnol (Placidyl), a hypnotic, sedative drug designed for oral use. We,' as well as others,5 have reported pulmonary edema following the intravenous use of ethchlorvynol in young adults. Acute studies in animals produced the From the Department of Anatomv, Universitv of California. College of Medicine. Irvine. California, and the Department of Medicine, Veterans Administration Hospital, Long Beach. California. Accepted for publication January 10, 1977. Address reprint requests to Dr. Frederick L Glauser, Chief, Pulmonary Section B Veterans Administration Hospital, 5901 East Seventh Street, Long Beach, CA 90801 525
526
DEARDEN ET AL.
American Journal of Pathology
following within 5 minutes of injection of 15 to 25 mg/kg ethchlorvynol: pulmonary edema, decreased arterial oxygen pressure, transient decreases in cardiac output, transient hypotension, no effect on pulmonary wedge pressure, and a relative bradycardia.' We interpret these findings as consistent with a nonhemodynamic form of pulmonary edema. Further unpublished studies from our laboratory 6 have shown that ethchlorvynol fluxes into and out of pulmonary tissue within 5 minutes of intravenous injection (following the laws of passive diffusion). Permeability studies, employing the in vivo saline-filled dog lung model,7 have shown that substances with molecular weights of up to 90,000 appear in "alveolar" liquid samples following injection into the venous system. Using the same model after ethchlorvynol injection,6 we have demonstrated an increase in permeability of the ACM to high-molecular-weight dextrans (500,000) but with a lag time of 30 minutes when compared with lower molecular weight (< 200,000) substances. From the above data it would appear that intravenous injection of ethchlorvynol leads to a rapid and marked functional decrease of ACM integrity. It, therefore, could be anticipated that marked structural alterations at the ACM level might parallel these physiologic findings. This study examines the pulmonary ultrastructure following the intravenous injection of ethchlorvynol in dogs. Materials and Methods Twenty-two mongrel dogs were distributed according to body weight into control and experimental groups. All dogs were anesthetized with 30 mg /kg pentobarbital, intubated, and ventilated with 100% oxygen. They were then placed in the 450 upright position. The experimental group (11 dogs) received an intravenous injection of 15 to 25 mg/kg ethchlorvynol (Placidyl, Abbott Laboratories, North Chicago, Ill.). The control group received an injection of 1 cu cm sterile saline solution. Lung samples from both groups were obtained at 30, 60, and 90 seconds, and at 2, 3, 4, 5, 15, 30, 45, and 60 minutes after injection. Only one lung sample was obtained from each animal. Lung tissue was obtained as follows: the sternum was split and retracted widely and a left lower lobe specimen was obtained. Tissues were immediately placed in cold (4 C) 2% glutaraldehyde buffered in 0.1 M cacodylate buffer (pH 7.4) and diced into small fragments (approximately 1-mm cubes). They were then transferred to fresh fixative for 2 hours and were postfixed for 1 hour in cacodylate-buffered 1% osmium tetroxide (pH 7.4). Dehydration occurred in a graded series of acetone, and the specimens were then embedded in Luft's Epon 812. Thick sections (0.5 to 1.0 ,) were cut and stained with 0.5% aqueous toluidine blue. These sections were used for routine examination and for selecting areas for thin sectioning. Thin sections with gray to gold interference colors were cut, mounted on uncoated or on Formvar-coated copper grids, and observed and photographed in a Philips EM 300 electron microscope after staining with uranyl acetate and lead citrate.
Results
Following the injection of ethchlorvynol, the diameter of most alveolar septae increased progressively through 30 minutes and retained this in-
527
ETHCHLORVYNOL-INDUCED PULMONARY EDEMA
Vol. 87, No. 3 June 1977
creased diameter through 60 minutes (Figure IA and B, Table 1). Some septae in the experimental group were markedlv thickened by fibrous connective tissue (Figure 1B). Alveolar capillaries increased in prominence up to 15 minutes after injection of the drug and exhibited this enhanced vascularity throughout the ensuing 45 minutes (Figure LA and B, Table 1). Ervthrocvtes in alveoli were more prominent than in controls 30 through 60 minutes after injection. Comparison of controls and of experimental groups at the electron microscopic level (Figure 2A and B) revealed alterations in terms of the degree of fibrillaritv and elasticitv in interstitial spaces, involvement of endothelial and epithelial (Type 1) cells, numbers of vesicles and vacuoles in both of these tvpes of cells, slight modifications in greater alveolar cells (Type 2), enhanced macrophage activity, and leakage of fluid into alveolar spaces.
Within a minutes after injection of ethchlorovvnol there was evidence of widening of the interstitial spaces in most alveolar septae concurrent, Table 1-Pulmonary Alterations Following Ethchlorvynol Injection (5 through 60 minutes)
Experimental animals
Control animals
Diameter of alveolarsepta Vacularity of septa Fibrosis of septa
Endothelial vesicles Endothelial vacuoles Endothelial blebs Hydration of interstitial spaces
45
60
++±
++±
++±
++±
+±
++±
++
++± ++± ++± ++++
++++
+±
+±
+++
-
i
+
-
-
-
-
+
+±
+±
±
-
+
i
+
i
+
++±
+++
+++
+±
++
++
++
+±
+±
++±
++
++±
+
+
+
+
+
+
+
++
++
+++
++
++
++
++
++
+
*
++
++
++
+
+
+
+
+
+±
++
++
++
++
+
+±
+±
+
+±
+± ++± ++±
+++
++
-
-
-
-
-
++
+±+
++±
30
15
5
15
30
45
60
5
+
i
+
+±
+
+±
++
+
+±
+
+
+±
++
++±
++
++
++
+±
++
+±
++
i -
+++
+++
+++
++ +++ ++±
+++
++
+±
+±
+++
Epithelial vesicles
+±
Epithelial vacuoles Lamellar bodies Type II cells WBC + platelets Macrophage population Edema fluid in alveolus
++++ to
- =
the degree of preponderance.
i
528
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with some alteration or disruption of basement membranes including the presence of enhanced quantities of elastin and of thick (up to 400 A) collagen fibrils. These fibrils were not dispersed, but rather occurred in thick bundles observable both in cross and longitudinally oriented profiles (Table 1). The significant difference between these interstitial areas and those in the controls related primarily to the increased frequency of fibrillarity of the interstitial spaces in the experimental animals. This was possibly related to the frequent observation of fibrocytes in interstitial spaces which were more common than they were in controls. In uninvolved interstitial spaces in the controls and in the experimental groups the basement membrane remained relatively constant, averaging about 650 A when the endothelial and epithelial basement membranes were fused. Subsequently (15, 30, 45, and 60 minutes) there was a progressive increase of interstitial involvement, including apparent hydration in the experimental animals and a proportionate decrease in the occurrence of "thin segments" in the ACM. Fibrocytes continued to be prominent in these latter periods of time. As noted previously, when the basement membrane was intact it did not differ in diameter from that in corresponding controls. The first indications of involvement of endothelial cells occurred at 5 minutes and consisted of increasing numbers of plasmalemmal vesicles (Table 1). These vesicles, in control and in experimental animals, averaged about 600 A in diameter, and often contained a flocculent filamentous material similar to plasma (inset, Figure 3). Some endothelial cells were swollen and lucent. Intracellular vacuoles measuring up to 0.5 A in diameter and containing flocculent material were often observed in endothelial cells 15 minutes after injection (Figure 2B, Table 1). At this same time occasional blebs were observed protruding from these cells into their capillary lumen, and they also contained flocculent material similar to that in vacuoles, vesicles, and the lumen of capillaries. Blebbing of the endothelium was not a prominent feature at this or at subsequent times (Table 1). By 30 minutes, lucency of endothelial cells was fairly common and they contained numerous vesicles (Figure 3 and inset). It was sometimes possible to observe vesicles as apparent channels or pores interconnecting the capillary lumen with interstitial spaces (arrows, Figure 4). Maximum dimensions of the openings of these channels or pores varied between 200 and 350 A. Cell-to-cell junctions remained apparently intact and did not differ from those in controls. Epithelial (Type 1) cells showed no consistent alterations until 30 minutes after injection. At this time there was evidence of lucency, swelling, and vacuolization of some of these cells (Figure 3). Vesicles were
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fairly prominent and contained the same kind of flocculent material observed in similar vesicles in endothelial cells, endothelial vacuoles and blebs, and epithelial vacuoles (inset, Figure 3). The membranes of vesicles sometimes appeared to fuse, forming an apparent channel through epithelial cells (Figure 5), while other vesicles seemed to fuse with and empty their content into vacuoles (inset, Figure 3) which subsequently fused with the plasmalemma and were then exocytosed. As in endothelial vesicles, the maximum dimensions of openings of epithelial channels or pores varied between 200 to 350 A. Somewhat similar observations were made in corresponding controls, but they were not common, and vesicles were also less commonly observed than they were in the experimental animals. Epithelial cell junctions in experimental and control animals remained intact. Greater alveolar cells (Type 2) showed only minor modifications from those in controls and these consisted of a reduction of microvilli (both numerically and in length) and a reduction of the osmiophilic electrondense bands typically found in these cells (Table 1). These alterations were somewhat transitory (through 15 minutes), and subsequently, these cells were comparable with similar cells in controls. Cellular debris (arrows, Figure 2B) was first observed in alveoli of the experimental animals 15 minutes after injection of Placidyl and increased in amount with increasing time (Table 1). Concurrent with the first appearance of debris, leukocytes and platelets in capillaries were more commonly observed than previously (Table 1). Phagocytic cells (Figure 2B) were much more common in interstitial spaces and alveoli of the experimental groups than they were in controls (Table 1). These cells appeared to be phagocytosing cellular debris in alveolar spaces. Edematous fluid containing flocculent filamentous proteinaceous material similar to plasma protein was first observed in scanty amounts 5 minutes after injection of ethchlorvynol (Table 1). Subsequently, filamentous material increased in alveolar spaces (Figure 3). This material often appeared to be in close association with vesicles opening on the surface of epithelial cells (arrows, Figure 6) and with exocytosing vacuoles. The amount of this material in alevoli was maximal at 60 minutes after ethchlorvynol injection. This indicated a time-related increase in permeability of the ACM. Leakage of edematous fluid through the ACM was not observed at any time in the controls. Discussion Intravenous injection of ethchlorvynol induces several alveolar responses referable to pulmonary edema which are demonstrable ultrastructur-
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ally. Pulmonary edema is characterized ultrastructurally as consisting of all or most of the following: a) swelling and hydration of interstitial spaces of alveolar septa,8`'0 b) blebbing of capillary endothelium, "" c) swelling and vesiculation of endothelial and epithelial (Type 1) cells,"'2 and d) the accumulation of excessive amounts of proteinaceous fluid in alveolar spaces. Treatment with ethchlorvynol induces all of these aspects of pulmonary edema either directly or indirectly. Moreover, it seems to induce some aspects typical of inflammation, and these consist of the following: 1) enhancement of collagen and elastic tissue deposition in insterstitial spaces, 2) an influx of leukocytes and platelets in alveolar capillaries, 3) increasing quantities of cellular debris in alveolar spaces, and 4) a marked increase in the number of macrophages and in their phagocytic activity, particularly in alevolar spaces. These latter observations might account, at least in part, for the increased vascularity observed after treatment. Most of these phenomena are time related and become progressively more prominent with increasing time postinjection. These observations are particularly interesting since ethchlorovynol is known to flux into and out of pulmonary tissue within 5 minutes after injection. Physiologic studies indicate that endothelium is normally permeable to small water-soluble and lipid-soluble solutes as well as to larger molecules such as albumin. These substances are thought to move across the endothelium, enter interstitial spaces of the ACM, and pass along bronchioles to enter lung lymphatics."3 We have observed endothelial channels opening between the capillary lumen and interstitial spaces and these may be the morphologic pathway for passage of these solutes. Indeed, tracer techniques employing glycogens and dextrans 14 or small heme-peptides 1 have provided good evidence that capillary membrane transport may be mediated by means of plasmalemmal vesicles forming transport channels. The enhanced widening and hydration of interstitial spaces in the ethchlorvynol-treated dogs suggests that the rate of transport of substances into these spaces is indeed more rapid than it is in corresponding controls. Vesicles in endothelial cells of ethchlorovynol-treated dogs are more numerous than they are in controls, and if transendothelial transport is by means of vesicles as we suspect, the increase in numbers of endothelial vesicles would correlate well with the interstitial edema observed in these animals. The increase in permeability of the endothelium does not seem to relate to an increase in diameter of endothelial cell junctions because these junctions are comparable in diameter with those in controls and the interstitial spaces in controls are considerably less edematous. The earliest appearance of filamentous flocculent edematous fluid in alveolar spaces occurs 5 minutes after injection of ethchlorvynol and this
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correlates well with previous physiologic findings in this model.4 At this time, the increased permeability of the ACM does not seem to relate to increased diameters of cell junctions in endothelial or epithelial cells since these diameters are comparable with those in controls and no leakage of fluid into alveolar spaces is observed in control animals. Subsequently, even with increasing quantities of edematous fluid entering alveolar spaces, the endothelial and epithelial cell junctions seem to remain intact. In this regard, it has been shown that neither horseradish peroxidase nor hemoglobin escape the vascular bed through interendothelial junctions under near normal perfusion pressures (15 to 20 mm of mercur).'3 Moreover, leakage of these substances through both endothelial and epithelial cell junctions occurs only when pressures greater than 50 mm of mercury are used.13 In physiologic studies of similar ethchlorvvnol-treated dogs,' we have observed a decrease in pulmonary arterial pressure and a systemic hypotension when compared with controls, and therefore, a leakage through epithelial cell junctions would not be expected. However, vesiculation and vacuolization of both endothelial and epithelial cells increases with increasing postinjectional time, and it seems reasonable that these structures might be involved in transalveolar transport. Indeed, epithelial channels or pores have been observed interconnecting interstitial and alveolar spaces, and vacuoles seem to empty their filamentous content into alveoli. It would seem, therefore, that vesicular transport through the ACM, probably at an increased rate, might be one explanation for the enhanced permeability of this membrane after treatment with ethchlorvynol. However, these studies do not exclude the possibility of transport via intercellular junctions as previously suggested,'3 '6',7 and it is possible that substances with low molecular weights and dimensions of less than 10 A might penetrate the ACM using intercellular spaces (about 10 A in diameter for epithelial cell junctions) to gain access to alveoli. Regardless of the mechanism, the present study reveals a time-related increase in permeability of the ACM following ethchlorvynol injection. In this regard, flocculent proteinaceous material becomes prominent in alveolar spaces 30 minutes after injection and this correlates with the increase in vesicles and vacuoles in Type 1 cells and with previous observations regarding a time delay of epithelial involvement in pulmonary edema.6'13 This timing and involvement of epithelial cells also corresponds with the postinjectional time for passage of high-molecularweight (250,000 to 500,000) dextrans through the ACM in this model.6 The effect of ethchlorvynol on alveolar capillary permeability has been suggested to be either direct, or indirect.' In view of the rapid flux of this drug into and out of alveolar capillaries (5 minutes) and the increased
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permeability of the ACM 30 minutes after injection, it would seem that either the effects of the drug once initiated are long lasting or the enhanced permeability is an indirect result. The present study does not prove either of these possibilities, but the wealth of the information obtained is supportive of the hypothesis that ethchlorvynol induces pulmonary edema secondarily or indirectly to its primary effect on the ACM. If this is true, the primary effect of the drug on the ACM has not been determined by these studies, but it has been established that pulmonary edema is rapidly induced. References 1. Hayes JA, Shiga A: Ultrastructural changes in pulmonary oedema produced experimentally,with ammonium sulfate. J Pathol. 100:281-286, 1970 2. Steinberg, AD, Karliner, JS: The clinical spectrum of heroin pulmonary edema. Arch Inter Med 122:122-127, 1968 3. Frand IU, Shim CS, Williams MH: Heroin-induced pulmonary edema: Sequential studies of pulmonary function. Ann Intern Med 77:29-35, 1972 4. Glauser FL, Smith WR, Caldwell, A, Hoshiko M, Dolan GS, Baer H, Olsher N: Ethchlorvynol (Placidyl')-induced pulmonary edema. Ann Intern Med 84:46-48, 1976 5. Algeri EJ, Katsas, GG, Luongo MA: Determination of ethchlorvynol in biologic mediums and report of two fatal cases. Am J Clin Pathol 38:125-130, 1962 6. Dearden LC, Glauser, FL, Smeltzer D: Unpublished data 7. Theodore J, Robin ED, Gaudio R, Acevedo J: Transalveolar transport of large polar solutes (sucrose, inulin, and dextran). Am J Physiol 229:989-996, 1975 8. Meyrick B, Miller J, Reid L: Pulmonary oedema induced by ANTU, or by high or low oxygen concentrations in rat: An electron microscopic study. Br J Exp Pathol 53:347-358, 1972 9. Cottrell TS, Levine OR, Senior RM, Wiener J, Spiro D, Fishman AP: Electron microscopic alterations at the the alveolar level in pulmonary edema. Circ Res 21:783-797, 1967 10. Teplitz C: The ultrastructural basis for pulmonary pathophysiology following trauma. J Trauma 8:700-714, 1968 11. Heath D, Moosavi H, Smith P: Ultrastructure of high altitude pulmonary edema. Thorax 28:694-700, 1973 12. Staub NC, Nagano H, Pearce ML: Pulmonary edema in dogs, especially the sequence of fluid accumulation in lungs. J Appl Phvsiol 22:227-240, 1966 13. Szidon JP, Pietra GG, Fishman AP: The alveolar-capillary membrane and pulmonary edema. N Engl J Med 286:1200-1204, 1972 14. Simionescu N, Palade GE: Dextrans and glycogens as particulate tracers for studying capillary permeability. J Cell Biol 50:616-624, 1971 15. Simionescu N, Simionescu M, Palade GE: Permeability of muscle capillaries to small heme-peptides. J Cell Biol 64:586-607, 1975 16. Karnovsky MJ: The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J Cell Biol 35:213-236, 1967
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17. Schneeberger-Keeley EE, Karnovsky MJ: The ultrastructural basis of alveolarcapillary membrane permeability to peroxidase used as a tracer. J Cell Biol 37:781-793, 1968
Acknow dgment The authors are indebted to Teresa Espinosa for her expert technical assistance in all aspects of this electron microscopic study.
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Legend for Figures Figure IA-Pulmonary alveoli of a control. B-Pulmonary alveoli 30 minutes after ethchlorvynol injection. Note the widening of alveolar septae and the increased vascularity in B. Some septae (B) are composed of dense connective tissue. (Semithin plastic sections stained with
toluidine blue, X 800)
Figure 2A-Part of an alveolar septum in a control. B-Alveolar septae 15 minutes after ethchlorvynol injection. Comparison of these figures reveals increased fibrillarity and elasticity of interstitial spaces, lucency, swelling, and vacuolization of endothelial cells, cellular debris and a macrophage in the alveolus of the ethchlorvynol treated specimens and absence of most of these alterations in the control. "Thin sections" of the ACM are less commonly observed in B than in A. (Uranyl acetate and lead citrate, x 19,800
Figure 3-Part of an alveolar septum 30 minutes after injection of ethchlorvynol. Observe extensive amounts of flocculent filamentous edematous fluid in the alveolus. The epithelium is swollen, lucent and vacuolated and contains numerous vesicles. The content of vacuoles corresponds with that in the alveolus and is similar to that in capillaries. The endothelium is also lucent and has numerous vesicles; an intact endothelial cell junction is present in the capillary at the left. The interstitial space appears hydrated. Inset-ACM, 30 minutes after injection. Observe the numerous vesicles in the endothelium. They contain material similar to that in the capillary (bottom). Epithelial vesicles also contain similar filamentous material. One epithelial vesicle has fused (arrow) with a vacuole while other vesicles appear to open into the alveolus. (Uranyl acetate and lead citrate; x 27,500; inset, x 38,000) Figure 4-Part of a "thin segment" of the ACM showing vesicles (arrows) interconnecting the capillary lumen (left) and interstitial area (right). Sixty minutes after ethchlorvynol injection. (Uranyl acetate and lead citrate, x 108,000) Figure 5-Part of a Type 1 cell showing an epithelial channel (arrows) interconnecting the interstitial area and the alveolus (left). Other vesicles are in close association to each other. In an intact thin segment of the ACM (lower picture), a vesicle (arrows) is interconnecting the alveolus with the interstitium. Forty-five minutes after ethchlorvynol injection. (Uranyl acetate and lead citrate, x 132,000) Figure 6-Enlargement of a portion of Figure 3 to show the intimate association of filamentous material in the alveolus with openings of vesicles at the cell surface (arrows) (Uranyl acetate and lead citrate, x 38,750).
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