Experimental post-traumatic lung insufficiency in dogs: ultrastructure of lung lesions

Acta path. microbiol. immunol. scand. Sect. A, 90: 113-123, 1982.

EXPERIMENTAL POST-TRAUMATIC LUNG INSUFFICIENCY IN DOGS Ultrastructure of Lung Lesions B. LIUMI, A. 0. AASEN2,O. D. SAUGSTAD2, I. GULDVOG2, K.NORDSTOGAI and E. AMUNDSEN2 Department of Pathology, The Veterinary College of Norway1 and Institute for Surgical Research, National Hospital, Oslo, Norway2

Lium, B., Aasen, A. O., Sewtad, 0. D., Guldvog, I., Nordstoga, K. & Amundsen, E. Experimental post-traumatic lung insufficiency in dogs. Ultrastructure of lung lesions. Acta path. microbiol. immunol. scand. Sect. A, 90: 113-123, 1982. The ultrastructure of developing lung lesions in two groups of dogs exposed to a combination of haemorrhagic hypotension and liver trauma was studied with particular anention to changes at the alveolar level and lung micro-vessels. Lung samples were obtained every four hours and at collapse in one group and 12 hrs after initiation of the trauma in the other. An interstitial oedema was recognized in biopsies obtained 4 hrs after initiation of the trauma, and before marked lesions were observed at the ultrastructural level in endothelial cells. Endothelial damage was, however, evident in biopsies obtained at 8 hrs and at collapse. Aggregates of degranulated and degenerated leucocytes and platelets were occasionally found to obstruct respiratory capillaries together with erythrocytes, some of which seemed to be haemolysing. A considerable amount of protein-rich oedema, cellular debris and fibrhoid material was found in alveolar lumina at collapse. The present experiments indicate that increased vascular permeability in lung micro-vessels IS of importance for the development of the characteristic lesions seen in shock lungs. Possible pathogenetic mechanisms, initiating the lung lesions, are discussed with special emphasis on the significance of kinin activation and the presence of polymorphonuclear leucocytes and microthrombi. Key words: Post-traumatic shock lung; ultrastructure; dog.

B. Lium, Department of Pathology, The Veterinary College of Norway, P. 0. Box 8146, Dep., Oslo 1, Norway.

Received 2S.vii.81

Accepted 9.x.81

An important, but poorly understood phenomenon associated with many different etiologic factors, is the progressive development of respiratory insufficiency, often refered to as adult respiratory distress syndrome (ARDS) (2). Knowledge of the morbid anatomy of this syndrome has been obtained mainly from autopsy studies of fatal human cases (2). Ultrastructure of pulmonary damage in human shock lungs has been described

during the past few years (27, 30, 37). In a very recent report Schnells et al. (32) described the progressive development of ultrastructural lung changes in biopsies from various time intervals after trauma in human patients. Several experimental models have been used to induce progressive respiratory insufficiency in animals (4, 6, 1 2, 26, 35). None of these reports describe the progressive ultrastructural changes in lung biopsies from various stages of the shock lung development or

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attempt to correlate these with changes in biochemical and physical parameters at the same time interValS. In an attempt to identify pathogenetic mechanisms participating in initiation of post traumatic lung insufkiency, we designed an experimental model in which shock was induced by a standardi-

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zed combination of hypotension and liver trauma in anaesthetizeddogs. The presentpaper describes the progressive development of ultrastructural changes at the alveolar level in these dogs. Changes in central haemodynamics, blood gases and several other blood parameters have been reported previously ( I , 1 3, 29).

MATERIALS AND METHODS Lung insufficiency was induced in anaesthetized dogs as described in an earlier paper (19). In group I (n=4) haemorrhagic hypotension to 50 mm Hg for 2 hours was combined with occlusion of hepatic artery, portal vein and common bile duct for the first 20 minutes of the hypotensive period. In group I1 experiments (n = 7) the procedure also included a left side thoracotomy. Samples for electron microscopic studies were fixed in 3 % glutaraldehyde and processed to semi-thin sections as described previously (19). Ultra-thin sections from representative areas in blocks from all dogs and all time intervals were cut with diamond knives, mounted unsupported on copper grids, stained with uranyl acetate and lead citrate and examined in a Siemens Elmiscope IA at 60 kw.

RESULTS Controls. Ultrastructural findings in alveolar septa from control dogs (Fig. 1) were in accordance with descriptions of the normal ultrastructure of the distal lungs ( 1 8). A moderately increased number of leucocytes, however, were observed and a few free lamellar structures were sporadically seen in arterioles. Both leucocytes and platelets showed normal ultrastructures and no tendency to aggregate. Group I. Electron microscopic examinations of lung samples from these animals revealed perivascular oedema with marked widening of the perivascular connective tissue (Fig. 2). The thick segments of alveolar septa were markedly oedematow, with separation of the basement membranes. Respiratory capillaries were dilated and partly plugged with aggregates of polymorphonuclear leucocytes (Fig. 5). Some leumytes showed loss of specific granules and a marked increase in glycogen-

like deposits within the cytoplasm. Other leucocytes were degenerated or necrotic. Small aggregates of degranulated platelets were also found in respiratory capillaries together with a lamellar material and round to oval bodies representing degenerated cells of uncertain origin (Figs. 3 & 4). Some endothelial cells had swollen mitochondria and cytoplasmic vacuoles. The number of pinocytotic vesicles was not significantly changed compared with the controls. The endothelial cells maintained their structural integrity, and no evidence of endothelial disruption was seen. Epithelial cells were essentially normal except for some cytoplasmic vacuoles. Occasionally macrophages, but no oedema fluid, were seen in alveolar lumina. Group II. The ultrastructure of biopsies obtained at 0 hour (Fig. 6) was similar to that described for control dogs, except that intracapillary leucocytes were only sporadically observed. The first signs of endothelial damage were seen at 4 hours and consisted of cytoplasmic vacuoles and moderate swollen mitochondria in a few scattered cells. Platelets and polymorphonuclear leucucytes were observed more frequently than in the 0 hour biopsies, and they were occasionally seen as aggregates. Incipient degranulation and increase in intracytoplasmic glycogen deposits were occasionally recognized in leucocytes. Intracapillary, membrane-bound vesicles were frequently seen at this time (Fig. 7); they had varying forms and sizes, and a finely granulated electron-lucent material was seen in some of them. Interstitial oedema was observed around blood vessels and to a minor extent in the interstitial tissue of the thick parts of alveolar septa. Vacuoles and myelin figures were occasionally found in oedematous areas.

All the illustrations, Figs. 2-1 7 are electron micrographs of sections of lung tissue from dogs with experimental lung lesions whereas Fig. I shows similar tissue from a normal control dog. Fig. 1. The thin (asterisk)and thick (double asterisk)portion of an alveolar septum is shown. Alv: alveolar lumen; AI:

type I alveolar epithelial cell; Cap: capillary lumen; Coll: collagen fibrils; E: capillary endothelium. x 18900. Fig. 2. Perivascular oedema with marked widening of the interstitial space (In) and separation of collagen fibrils. Vesicles are present in the interstitium (arrows).Alv: alveolar lumen; E: endothelial cell; Ery: erythrocyte. Group 1. x 6750. Fig. 3 . A respiratory capillary obstructed by degranulated and partly disintegrated platelets (PL) is shown. E: capillary endothelium. Group I. x 12500. Fig. 4 . A cluster of vesicles (V) of unknown origin is present in the lumen of a respiratory capillary. E: capillary endothelium. Group 1. x 16250. Fig. 5 . A distended respiratory capillary is seen to be obstructed by aggregates of partly degranulated leucocytes (L) and degenerated leucocytes (DL). The expanded interstitium (In), containing dispersed collagen fibrils, is an indication of interstitial oedema. Group 1. x 9900.

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Fig. 6 . An alveolar septum from a 0 hr biopsy in group 11, shows normal structures.AI: type I alvedlar epithelial cell; Cap: capillary lumen; Coll: collagen fibrils; E: capillary endothelium. x 15000. Fig. 7. A cluster of vesicles (V) of unknown origin is seen in the lumen of a respiratory capillary (Cap). From lung biopsy of a group I1 animal 4 hrs after the initiation of the trauma. Alv: alveolar lumen; AI: type I alveolar epithelial cell; E: capillary endothelium. x 26250. Fig. 8. A marked endothelial swelling with cytoplasmic oedema and loss of pinocytotic vesicles (El)is illustrated. Note the interendothelial junction (arrows). Lung biopsy 8 hrs after initiation of trauma. Alv: alveolar lumen; Cap. capillary lumen. E2:normal endothelial cell. x 14000. Fig. 9. An alveolar septum from an 8 hrs biopsy of a group I1 animal, showing interstitial oedema with widening of the interstitium (In) and separation of the collagen fibrils. Vesicles (V), a platelet (PL)and a leucocyte (L) are seen in the capillary lumen. E: capillary endothelium. x 8750.

At 8 hours there was a more definite evidence of damage of endothelial cells, although most cells still showed normal structures. As shown in Fig. 8, the extent of endothelial damage was very variable even within the same section. Cytoplasmic vacuoles and 116

myelin figures were more frequent than in biopsies obtained at 4 hours. Degenerated cells had reduced numbers of pinocytotic vesicles. Intracapillary membrane-bound blebs occurred, however, at a higher frequency, and interstitial oedema seemed to

Figs. 10-12 show lung samples from dogs of group I1 at collapse. Fig. 10.Perivascular oedema with marked widening of the interstitial space (In) and separation of collagen fibrils are present. Cellular fragments (arrows) a leucocyte (LI)and a mast cell (MI are seen in the interstitium. Alv: alveolar lumen; E: endothelial cell; Fib.: fibrocyte. L: leucocyte. x 5800. Fig. f I . A swollen endothelial cell (E) with loss of pinocytotic vesicles is seen to be partly detached from the basement membrane (arrows) in the thin part of an alveolar septum. AI: type I alveolar epithelial cell; P: proteinous material in alveolar lumen; L: leucocyte. x 24000. Fig. f 2. A marked oedematous swelling of endothelial cells (El and type I alveolar epithelial cells is present (AD. The capillary lumen (Cap) is partly occluded by swollen endothelial cells which show loss of pinocytotic vesicles. Both endothelial and epithelial cells contain intracytoplasmic vacuoles which perhaps represent degenerated mitochondria. Alv: alveolar lumen. x 17500. 117

Fig. 13. An alveolar septum from a dog of group I1 at collapse. Note the disruption of the respiratory capillaries with extravasation of partly degranulated leucocytes (L).An erythrocyte (Ery), cellular debris and necrotic material of unknown origin (Ne) is also present. Alv: alveolar lumen. x 10200.

Fig. 14. A distended respiratory capillary obstructed by a conglomerate of degenerated leucocytes (DL) and haemolysed erythrocytes (HE)is shown. The alveolar lumina M v ) contain oedema fluid. E: capillary endothelium; M: mast cell. Tissue from a dog of group I1 at collapse. x 1 1700.

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Figs. 15-1 7 show lung samples from dogs of group I1 at collapse. Fig. 15. Fibrinoid material (F)is seen between remnants of a capillary endothelial cell (E)and a type I alveolar epithelial cell (AI). Alv: alveolar lumen, L: leucocyte in capillary lumen. x 13000. Fig. 16. A type I1 alveolar epithelial cell with electron-lucent apical cytoplasmic swelling (asterisk) is Seen to project into the alveolar lumen (Alv). x 8 100. Fig. 17. Some plasma-like oedema fluid is present in alveolar lumen which also contains clusters of fibrinoid material

(F),an erythrocyte (Ery I) and some cellular debris (arrows). An erythrocyte (Ery3 and some degenerated cytoplasmic material (D) are seen in the lumen of a respiratory capillary. The thin portion of an alveolar septum is indicated by arrow-heads. Dispersed collagen fibrils are observed in the interstitium (In). E: capillary endothelium. x 9750.

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be more widespread (Fig. 9). A fmely granulated

intraalveolar oedema fluid, localized to the alveolar corners, was recognized in scattered alveoli. Collapse. Ultrastructural changes in animals that died before 8 hours were similar to those described for group I, and the following description refers to animalsthat lived for a b u t 12 hours after initiation of the trauma. The most constant and evenly distributed alterations were interstitial oedema seen as large electron-lucent spaces separating collagen fibers and other interstitial structures in perivascular and peribronchial connective tissues as well as in the thick collagen containing segments of the alveolar wall. Leucocytes and cytoplasmic fragments were identified in the oedematous spaces (Fig. 10). Endothelial cells in respiratory capillaries showed marked degenerative changes with swelling of mitochondria, cytoplasmic vacuoles and a reduced number of pinocytotic vesicles (Fig. 12). As shown in this figure, some capillaries were partly occluded by protruding oedematous endothelial cells. Myelin figure formation accompanied the cell degeneration. As in 8 hour biopsies, the extent of endothelial damage showed great variation; severely affected cells were sometimes seen adjacent to unchanged cells. In thin parts of the septa, endothelial cells were occasionally detached from the underlying basement membrane (Fig. 1 I). At this time, focal endothelial ruptures with extravasation of erythrocytes, leucocytes and cell fragments into the septa1 interstitium were seen (Fig. 13). A markedly increased number of polymorphonuclear leucocytes were found in respiratory capillaries. They tended to aggregate, and a great number of them showed pronounced degenerative changes with loss of specific granules. As described for group I, many leucocytes contained clusters of a granular material with an appearance consistent with glycogen. Some capillaries were distended and contained tightly-packed erythrocytes admixed with platelets or they were occluded by aggregates of degenerated leucocytes and erythrocytes, some of which were haemolyzed (Fig. 14). Lamellar structures and fragments of leucocytes were occasionally seen. Intracapillary fibrin deposits’ were never observed, whereas fibrinlike deposits were found in the pericapillary interstitial tissue of some specimens (Fig. 15). Type 1 pneumocytes sometimes showed extensive degenerative changes with irregular cytoplasmic vacoules and remnants of organelles (Fig. 12). Type I1 pneumocytes also revealed degenerative changes, and some of them released degenerated, electron-lucent cytoplasmic fragments into the alveolar lumina (Fig. 16). The pattern of epithelial damage was essentially focal. Alveolar lumina containing varying amounts of a 120

plasmalike material were frequently seen. In focal areas, alveolar lumina contained a markedly increased number of alveolar macrophages, polymorphonuclear leucocytes and red blood cells admixed with necrotic cells. Free cell organelles, haemolyzed erythrocytes, osmiophilic fibrillar material with an appearance compatible with that of fibrin and plasma-like &ma fluid were also present (Fig. 17). Alveolar macrophages were markedly swollen and vacuolated, and they showed active phagocytosis of necrotic cells and erythrocytes.

DISCUSSION Ultrastructural studies have demonstrated that interstitial oedema and trapping of leucocytes in lung microvessels were the prime morphologic changes in experimental pt-traumatic lung insufficiency in dogs. Biopsies from group I1 experiments clearly showed that lung &ma developed stepwise; the interstitial compartment seemed to act as a buffer zone, preventing early development of alveolar flooding. As also seen in other experimental models intitiating lung &ma (8, 10, 151, the intraseptal oedema fluid was primarily localized to the collagen containing thick segments of the alveolar septa. Blood gas measurements indicated that gas diffusion across the air-blood barrier was not severely compromized by oedema fluid in these regions (29). In both experimental groups, interstitial oedema was recognized before marked ultrastructural changes were observed in capillary endothelial cells. In isolated rabbit lungs Hovig et al. ( 1 4) demonstrated that evidently increased permeability lung oedema may occur without ultrastructura1 changes in microvascular endothelium. Endothelial injury was, however, recognized at 8 hours in group I1 experiments and was pronounced but focal at collapse. Both the light microscopic (1 9) and the ultrastructural changes indicated that increased permeability in lung micro-vessels was essential for the development of ))shocklunga in our experimental models. The present investigation, however, provides no evidence of whether the increased vascular permeability was restricted to bronchial or pulmonary vessels, to capillaries or arterioles/ venules. The pathogenetic mechanisms initiating increased vascular permeability and lung oedema in posttraumatic shock lungs are still poorly understood (1 6). In a previous paper we showed that hypotension and liver trauma in dogs significantly increased the activity of the kallikrein-kinin system (29). This suggested that activated kinins may have contributed to the increased vascular permeability and

thereby to the marked interstitial lung oedema in our experiments. The significance of vaSOactive agents for the development of increased endothelial permeability and oedema in lungs is, however, controversial (36). The marked endothelial damage seen terminally in our experiments may be of great importance since pulmonary endothelial cells, in addition to being components of the air-blood barrier, are the sites of specific enzymatic reactions for the metabolism of vasoactive substances, including kinins and prostaglandins (18). Teplirz (37) has thoroughly discussed the pathological basis for the development of acute respiratory insufficiency. He concluded that the severity and duration of pulmonary capillary endothelial injury and trans-capillary leakage are the major determinants of the clinical course and final outcome of adult respiratory insufficiency in man. During the experimental period there was a marked increase in leucocytes, principally polymorphonuclear leucocytes (PMN), trapped in pulmonary arterioles and capillaries. Aggregates of degenerated leucocytes and platelets devoid of their specific granules occluded lung micro-vessels.Vasoactive agents may thereby have been released from their lysosomes, together with proteolytic enzymes which may depolymerize the alveolar ground substance and activate the kallikrein-kinin and coagulation mechanisms proteolytically ( 3 , 4 , 5, 2 1, 22, 3 1, 38). The real importance of sequestered leucocytes for the development of characteristic morphological and clinical p t traumatic shock lung changes is disputable. Recent reports, however, indicate that PMN may be at least partly responsible for the increased vascular permeability Seen in shock lungs (1 7). Craddock et al. ( 9 ) have demonstrated that complement stimulated granulocytes may increase vascular permeability. In a morphometric analysis of human shock lungs Riede et al. (27) revealed a massive increase of leucocytes in alveolar capillaries and they suggested that this was essential for the development of characteristic shock lung changes in these patients. The microthrombi seen in our investigation were primarily localized to arterioles. They may be a result of release of procoagulant or vasoactive substances and cellular aggregates from the damaged liver, combined with reduced blood flow caused by the hypotension. Re-infusion of blood may be another source of thrombotic material. Electron microscopic studies revealed no fribin deposits in respiratory capillaries. Absence of intravascular fibrin has also been reported from other ultrastructural studies of canine shock lungs and in human shock lungs (6, 12, 25, 30). Mywoll and Brandberg (24) showed that increased capillary permeability

may be an important factor for the fall in fibrinogen level in endotoxin shock in dogs. In our group I1 experimentsfibrin was obviously lost to the alveolar lumina and pleural surface. .Disappearance of fibrinogen from the blood in our experiments (I 3) was therefore not necessarily caused only by DIC and trapping of fibrin in lungs or other tissues. Micro-embolization of the pulmonary vasculature and decreased rate of fibrin clearance from the lungs have been considered to be of pathogenetic significance for the development of shock lung (23, 28). Recent reports indicate that fibrin degradation products may increase the pulmonary microvascular permeability and thereby contribute to the development of interstitial lung oedema (1 1, 20). The observation of a fibrin-Like material in the interstitium between alveolar endothelial and epithelial cells in our dogs is similar to what Cosrabella et al. (7) described as characteristic for the delayed micro-embolism syndrome in man and experimental animals. This indicates that fibrin degradation products may have contributed to the increased vascular permeability and lung oedema in our models. The origin and significance of the membranebound blebs and granules frequently seen in capillary lumen, especially in 4 and 8 hour biopsies is difficult to determine. Endothelial cells, platelets, leucocytes and erythrocytes apparently all contribute to this cell debris. Some of these structures may perhaps also have originated in the ischemic liver. Similar vesicles have been described in pulmonary oedema produced by ischaemia and a number of toxic agents (1 2, 33). It is suggested that vesicle formation is the result of direct injury to endothelial cells, and Shultz (34) assumed that such vesicles were the result of enlargementof intracellular vacuoles with cellular rupture and release of large membrane-bound blebs into the capillary lumen. Since in our material a few blebs also were recognized in 0 hour biopsies and controls, we believe, however, that they were partly fixation artefacts, or derived from senescent cells not influenced by the experimental procedure. This work was supported by grants from the Agricultural Research Council of Norway and Jahre's foundation. The skilful technical assistance of Mrs. Anne-Grerhe Nordahl is gratefully acknowledged.

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