Pulmonary Neutral Fat Embolism in Dogs Danielle Jacobovitz-Derks, MD, and Christian M. Derks, MD
Twenty-two adult dogs were each given a single, 30-minute injection of 1.5 ml/kg bodyweight of pure triolein, and their pulmonarv, hepatic, renal, and cerebral morphology was observed for 1, 2, 3, 4, 6, 15, 24, and 48 hours; 3, 4, and 5 days; 1 and 2 weeks; and 1 month after the injection. A picture of massive capillary occlusion by lipid droplets was followed by rapidlv resolvable inflammatorv pneumopathv of granulomatous type, leaving a normal lung at the end of the experiment. The cleaning of the capillaries may be attributed to the mechanical action of the blood flow and to the inflammatorn reaction with evacuation of necrotic cells via the bronchial route. Transient pulmonary edema is attributed to increased pulmonarv arterial pressure. There was no intravascular coagulation. The few pulmonary lesions observed after the triolein injection suggest that the chemical theorv of neutral fat hvdrolvsis by pulmonary lipase and the toxicity of free fattv acids that are released should be reconsidered. (Am J Pathol 95:29-42, 1979)
ALTHOUGH a considerable number of studies are published everv year on the subject of fat embolism, there are still many aspects of this problem that remain obscure. The origin of embolized fat in the lung after large bone fracture is still a matter of controversv. Some authors 1-3 believe that the embolic material comes from the fractured bone itself. The discovery of bone marromv emboli in the pulmonary capillaries strongly supports that theory2'3 Other authors,44 without rejecting the first theory, maintain that the quantity of fat in a large bone is not sufficient to account for the entire embolic mass and that a change in the physicochemical properties of the blood causes lipid precipitation in the capillaries. Under clinical and experimental conditions, the composition of these fat emboli is 10 to 30% esterified or nonesterified cholesterol; the rest is neutral fat. The composition of lipids in the bones is approximately 1 % cholesterol and 995% neutral fat.5 Another important question is whether the fat emboli are toxic to the lung. Peltier 7 proposes a two-stage interpretation: after fracture, the neutral fat is embolized and causes capillanr obstruction (physical stage); then the pulmonary lipases h drol se the neutral fat and release free fatty From Laboratoire d'Anatomie Pathologique. et Institut de Recherches Interdisciplinaires en Biologie Humaine et Nuclaire. Institut de Recherches Cardiologiques, Faculte de \l&decine. U.niv ersite Libre de Bruxelles, Brussels, Belgium. Supported in part under contract of the Mlinist&re de la Politique Scientifique mvithin the framew-ork of the Association Euratom-U.nisersitv of Brussels-U.niversity of Pisa, and in part bv a grant from the Fonds de la Recherche Scientifique \ledicale. Xccepted for publication November 17. 1975. Address reprint requests to Dr. D Jacobov itz-Derks. Laboratorie d'Anatomie Pathologique. Faculte de \ledecine. 97. rue aux Laines. B-1000 Brussels. Belgium. 0002-9440/79/0411 -0029$01.00 29 © American Association of Pathologists
30
JACOBOVITZ-DERKS AND DERKS
American Journal of Pathology
acids which are extremely toxic for the lung (chemical stage). Other authors 8 stress the toxicity of the platelet aggregates, the formation of pulmonary capillary thrombi, and red cell sludge. Some insight into this problem may be gained from comparing the pulmonary and systemic lesions obtained experimentally by intravenous injection of neutral fat with the lesions observed in human pathologic conditions in which fat embolism is generally accompanied by slight or severe transient hypotension and marked stress. Such a comparison provides an indication of the importance of the embolism in a complex clinical picture. In this paper we report a study of the action of a pure neutral fat (triolein) on the lung, brain, liver, and kidney over given periods. The resulting lesions are compared with those obtained earlier by the FFA injection I and with those described in human disease.5'10 Materials and Methods Twenty-two healthy adult dogs, weighing between 13 and 24 kg, were each given a single intravenous injection of 1.5 ml/kg body weight of pure triolein over 30 minutes via a butterfly needle inserted into a back paw. For the duration of the injection, the dogs were kept harnessed, unanesthetized, in a standing position. Fourteen dogs were killed at 1, 2, 3, 4, 6, 15, 24, and 48 hours; 3, 4, and 5 days; 1 and 2 weeks; and 1 month after the injection; 1 other dog was killed at 4 days, another at 5 days, and 6 others at 1 month after the injection. Four normal dogs were given an injection of normal saline and were used as controls. At sacrifice, the dogs were anesthetized by an intravenous injection of 30 mg/kg of sodium pentobarbital and were killed by an intravenous injection of 30 ml of saturated KCI solution. Autopsy was performed immediately. The left lung was used for histologic examination and the right lung was weighed fresh and then was dried for calculation of the wet/dry lung weight ratio." Samples of liver, kidney, brain, and lung were taken from different sites (summit, base, anterior, and posterior regions). From the 7 dogs killed at 1 month, samples of intertracheobronchial lymph nodes and pulmonary hilar nodes were also taken. For light microscopy, fragments of the various organs, fixed in Bouin's fluid, were embedded in paraffin and stained with hematoxylin and eosin; others, fixed in formalin and embedded in gelatin, were cut with a freezing microtome and stained with Sudan IV red. Only the lung samples were examined by electron microscopy. For this purpose, small cubes were fixed for 2 hours in a 4% glutaraldehyde solution buffered at pH 7.4 following the technique of Millonig,12 then rinsed overnight in Millonig's buffer complemented with 5.4 g/liter of glucose. They were postfixed in 2% osmium tetroxide in the same buffer for 90 minutes, then dehydrated in the alcohol and propylene oxide, and embedded in Epon. Semi-thin sections cut with an LKB UM III Ultrotome, using glass knives, were stained with toluidine blue. Thin sections were prepared with a diamond knife, stained with uranyl acetate and lead citrate, then coated with a carbon film, and examined under a Siemens Elmiskop 101 electron microscope at 80 kV.
Results
The dogs were calm and nondyspneic at the time of the injection and appeared well at sacrifice. None of them died as a result of the treatment.
Vol. 95, No. 1 April 1979
NEUTRAL FAT EMBOLISM
31
MacocpcExamination
In the treated dogs, macroscopic examination of the lungs revealed moderate edema and congestion, associated with subpleural focal hemorrhage, mainly in the inferior lobes. These lesions were observed by the fourth hour. They reached maximum intensity approximately the fourth to fifth day. Liver, kidney, and brain were normal on macroscopic examination. In the control group, macroscopic autopsy of brain and organs proved normal. The wet/dry lung weight ratio of the normal dogs was 3.7 ± 0.3 (mean ± SD). The values of this ratio in the treated dogs are shown in Table 1. Thev were variably increased in the dogs killed between 1 hour and 5 days after the injection.
Lung (Table 1)
In accordance with Staub et al,'3 a distinction was made between pulmonary edema characterized by peribronchovascular cuff and edema with alveolar flooding. During the first 6 hours after the triolein injection, the lesions consisted mainly of capillary congestion, peribronchovascular and alveolar edema (Figure 1), and occasional alveolar hemorrhage in the subpleural regions. In the perivascular cuffs we found dilated lymphatics filled with eosinophilic fluid. At this stage there was no inflammatory infiltrate or macrophagic reaction. Sudan staining revealed a shower of lipid emboli scattered throughout the pulmonary capillaries (Figure 2) and in the segmental arteries. After the 15th hour, polymorphonuclear margination was observed in the pulmonary capillaries, and in the next 48 hours the polymorphonuclear leukocytes infiltrated the alveolar edema (Figure 3). After the 48th hour the number of lipid emboli had declined. Congestion and edema were unchanged and small subpleural hemorrhages were found. Three and four days after the injection, the pulmonary tissue was constellated with numerous granulomas composed of polymorphonuclear leukocytes and nonsudanophilic macrophages (Figures 4 and 5). After the first week, edema and congestion were discrete and alveolar hemorrhage had disappeared. The alveoli were distinctly clearer and Sudan staining failed to show any lipid emboli. One month after the injection, the pulmonary parenchyma presented a
32
JACOBOVITZ-DERKS AND DERKS
American Journal of Pathology
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Vol. 95, No. 1 April 1979
NEUTRAL FAT EMBOLISM
33
normal appearance which, apart from a few discrete lesions of edema and congestion, was undistinguishable from that of the lungs of the control animals. Liver, Kidney, Brain, and Nodes (Table 2)
In the treated dogs, Sudan staining revealed lipid droplets in the kidneys, at the glomerular and tubular levels, mainly during the 24 first hours of the trial, whereas Sudan-stained droplets were rare in the control dogs. There was no significant difference between the livers of control and treated dogs. In the brains of the treated dogs, lipid emboli predominated in the gray matter during the first 5 days; they were less frequent in the white matter. In the control animals a few lipid emboli were found in the white matter, but number was insignificant. No Sudan-stained material was found in the intertracheobronchial and pulmonary hilar nodes taken from the dogs killed at 1 month. Electron Microscopy (Table 1)
The appearance of the pulmonary lesions revealed by electron microscopy closely resembled that observed under the light microscope. Within the first few hours up to the third day, edematous thickening of the alveolocapillary wall and the formation of endothelial blebs were observed. The vacuoles of pinocytosis were more numerous than in the controls and contained lipid inclusions (Figure 6). These lesions were visible in capillaries free of lipid emboli. Epithelial cells of Type I and II were normal. During the first 24 hours, most of the capillaries were obstructed by lipid emboli (Figure 7). Some very large emboli plugged the capillary lumina completely, with the result that the vessels were considerably dilated and the vessel walls were molding around the embolic material. The endothelial cells of these capillaries were elongated but showed no sign of degeneration. After the fifth hour, the number of normal circulating polymorphonuclear leukocytes was higher than in the controls, with a peak occurring between the 24th and 48th hours. During this period, the alveoli were free and there was no accumulation of macrophages. After the 48th hour, occasional macrophages infiltrated the interstitial part of the septum. Between the third and the fifth days, electron microscopy confirmed the interstitial and, above all, alveolar localization of macrophage accumulation in the granulomatous lesions. By the end of the first week after the injection the alveoli were clear,
34
JACOBOVITZ-DERKS AND DERKS
American Journal of Pathology
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Vol. 95, No. 1 April 1979
NEUTRAL FAT EMBOLISM
35
and by the end of the first month the morphology of the lungs of the treated dogs was entirely similar to that of the controls. Throughout the course of this pulmonary affection there was no sign of Type II cell hyperplasia nor any evidence of intravascular coagulation. Discussion One of the most striking findings of this investigation was the insignificance of the observed pulmonary lesions compared with the quantity of triolein embolized. A comparison of the pulmonary morphologic picture after oleic acid embolism with that produced by triolein embolism shows that the former is essentially characterized by cell necrosis and hemorrhage, leading to fibrosis, whereas the latter is characterized by lesions of edema and congestion, accompanied by massive obstruction of the pulmonary capillaries by lipid droplets. The dose of triolein received by the animals was such that if neutral fat had been hydrolyzed by the pulmonary and circulating lipases, as Peltier suggests,7 only 3% of the injected quantity would have needed to be transformed into FFA to produce a picture of fibrosing lung disease such as that observed after oleic acid embolism.9 Peltier 14 suggests that the secretion of pulmonary lipase is specifically stimulated after neutral fat embolism. This theory is supported by some authors 15 but disputed by others, in particular by Soloway et al,16 who observed no increase in serum lipase during the 7 days immediately following triolein embolism in the rabbit. The circulating lipase values published by Peltier 17 in cases of fat embolism in humans lie within the normal range. Similarly, when Jacks et al is observed the increase in FFA and circulating lipase in blood returning from the left heart after pulmonary stenosis, they concluded that anoxia and hypoxia played a considerable part in this increase in FFA. Hausberger et al 19 demonstrated that after embolism by mineral oil, this inert lipid remained in the lung throughout the duration of their experiment, ie, 20 days, and that FFA could not be attributed to lysis of embolized lipids. The authors concluded that there must therefore be another source of FFA in the picture of fat embolism and that FFA cannot be regarded as responsible for the pulmonary embolism lesions. Pulmonary edema can be induced either by injecting toxins which increase capillary permeability or by increasing pulmonary capillary pressure. A physiologic study " of anesthetized dogs ventilated with air by a constant-volume respirator after an intravenous injection of 1.5 ml/kg of pure triolein shows an average pressure of 31 ± 4 mmHg in the pul-
36
JACOBOVITZ-DERKS AND DERKS
American Journal of Pathology
monary artery 15 minutes after the end of the injection and 27 ± 3 mmHg at 2 hours after the injection. No capillary cell lesions suggested hyperpermeability of toxic origin. Thus, in our study, the pulmonary edema seemed to be mainly due to the hemodynamic factor. This edema was resorbed after 4 days. Another important fact is the lack of intravascular coagulation. Soloway et al 21 observed the same phenomenon after injection of triolein in the rabbit. However, other authors 22,23 using homogenates of fat tissue as emboli observed a typical picture of intravascular coagulation. The homogenate contained tissue thromboplastins which were very probably responsible for the hypercoagulability. Triolein behaves as an inert substance, ie, it possesses no electronegative radicals to permit the release of heparin, which is partly responsible for the clearance of neutral fat in humans.24 It has little affinity for the carrier plasma proteins; Gustafson and Kerstell 25 have shown that after embolism in humans there is no change in the distribution of circulating lipoproteins, which indicates that they take no part in the transport of embolized lipids. The size and shape of the embolized corpuscles are important: in the case of triolein, the corpuscles are small and regularly shaped, encountering no obstacle to continuous circulation. The mechanism of pulmonary capillary cleaning is a complex phenomenon in which the inflammatory reaction and the positive mechanical action of pulmonary blood flow play varying roles. Riedel 26 injected oil in a systemic artery and found that most of it made its way to the lung. This may be explained by the low pressure of pulmonary circulation and by the lack of support around the pulmonary capillaries. In contrast, in the systemic capillaries, the blood pressure pushes the fat and flushes the capillary. This purely mechanical cleaning action no doubt also takes place in the lung, but more slowly, with the result that smaller quantities of lipids are cleared. During embolism and the hours immediately following the injection, pressure in the pulmonary artery is high, which would facilitate the cleaning of the pulmonary capillaries. It is during these first few hours that the quantity of fat found in the kidney and brain is highest. Soloway et al 27 demonstrated that 80% of the embolized triolein remained in the lungs of normotensive rabbits but that approximately 98% of embolized triolein stayed in the lungs of rabbits in a state of shock. Shaffer et al 28 have shown that radiolabeled triolein leaks slowly from the pulmonary circulation to the systemic circulation. Armin et al 2 found a proportional relation between the quantity of lipid injected and the peak of peripheral emboli. The inflammatory reaction undoubtedly plays a significant role in pulmonary cleaning. The macrophages are loaded with lipid inclusions and
Vol. 95, No. 1 April 1979
NEUTRAL FAT EMBOLISM
37
the necrotic cells are evacuated via the bronchi. An earlier clinical test consisted in looking for lipids in the expectorations of patients Xwith suspected fat embolism. The lymphatic channels seem to be ruled out because no lipids have been found at anx time in the pulmonary lynmphatics or intertracheobronchial or hilar nodes. Triolein behaves as an inert substance and reacts like lvcopodium spores, which are also evacuated after embolism through the intermediary of the inflammatory reaction via the bronchial route." The clinical picture of human fat embolism and the one observed in this experiment differ in various ways. In humans, the congestion never persists as long as in the experimental animals and there is no inflammatorv reaction in the embolized zones."5 The tolerated doses of vegetable oil varies from one study to another and probably depends on the technique of injection. The tolerated dose seems to be 1 to 3 ml/kg,8 which would correspond to 210 ml of embolized fat in a 70-kg man. But in human pathologic conditions it is known that small quantities of lipids may give rise to an alarming clinical picture. Consequently, in traumatic fat embolism it is essential not to overlook the respective roles of hypovolemia, ischemia, the tissue lesion itself, the release of thromboplastin, and associated vascular coagulation. The combination of all these factors plays a role as important as, or more important than, the embolism itself. References 1. Gauss H: The pathology of fat embolism. Arch Surg 9:593-605, 1924 2. Armin J, Grant RT: Observations on gross pulmonary fat embolism in man and in the rabbit. Clin Sci 10:441-469, 1951 3. Schinella RA: Bone marrow emboli: Their fate in the vasculature of the human lung. Arch Pathol 95:386-391, 1973 4. Scriba J: Untersuchungen uber die Fettembolie. Deutsche Ztschr Chir 12:118-219, 1879 a. Lequire VS, Shapiro JL, Lequire CB, Cobb CA, Fleet %'F: A study of the pathogenesis of fat embolism based on human necropsy material and animal experiments. Am J Pathol 35:999-1015, 1959 6. Lehman EP, Moore RMI: Fat embolism including experimental production without trauma. Arch Surg 14:621-662, 1927 7. Peltier LF: Fat embolism. III. The toxic properties of neutral fat and free fatty acids. Surgery 40:665-670, 1956 8. Bergentz S-E: Studies on the genesis of posttraumatic fat embolism. Acta Chir Scand 282(Suppl):1-72, 1961 9. Derks CM, Jacobovitz-Derks D: Embolic pneumopathy induced by oleic acid: A sy-stematical morphological study. Am J Pathol 87:143-158, 1977 10. Saldeen T: Fat embolism and signs of intravascular coagulation in a posttraumatic autopsy material. J Trauma 10:273-286, 1970 11. Guy-ton AC, Lindsey AW: Effect of elevated left atrial pressure and decreased plasma protein concentration in the development of pulmonarn edema. Circ Res 7:649-657, 1959
38
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American Journal of Pathology
12. Millonig G: Further observations on a phosphate buffer for osmium solutions. Fifth International Conference on Electron Microscopy. New York, Academic Press, 1962, p8 13. Staub NC, Nagano H, Pearse ML: Pulmonary edema in dogs, especially the sequence of fluid accumulation in lungs. J Appl Physiol 22:227-240, 1967 14. Peltier LF, Scott JR: Fat embolism: Changes in the level of blood lipase following the intravenous injection of neutral fat, fatty acids and other substances into dogs. Surgery 42:541-547, 1957 15. Fonte DA, Hatsberger FX: Pulmonary free fatty acids in experimental fat embolism. J Trauma 11:668-672, 1971 16. Soloway HB, Robinson EF, Sleeman HK, Huyser KL, Hufnagel HV: Resolution of experimental fat embolism. Arch Pathol 90:230-234, 1970 17. Peltier LF, Adler F, Lai SP: Fat embolism. The significance of an elevated serum lipase after trauma to bone. Am J Surg 99:821-826, 1960 18. Jacks ML, Reed WA, Allbritten FF: Alterations in plasma free fatty acids during extracorporeal circulation. Surg Forum 15:279-281, 1964 19. Hausberger FX, Phelan TI: Pulmonary free fatty acids in experimental mineral oil embolism. J Trauma 14:950-954, 1974 20. Halasz NA, Marasco JP: An experimental study of fat embolism. Surgery 41:921929, 1957 21. Soloway HB, Robinson EF: The coagulation mechanism in experimental pulmonary fat embolism. J Trauma 12:630-631, 1972 22. Saldeen T: The importance of intravascular coagulation and inhibition of the fibrinolytic system in experimental fat embolism. J Trauma 10:287-298, 1970 23. Cotev S, Rosenmann E, Eyal Z, Weinberg H, Shafrir E, Davidson JT: The role of hypovolemic stress in the production of fat embolism in rabbits. I. Morphological alterations of the lungs. Chest 69:523-528, 1976 24. Robinson DS, Harris PM, Ricketts CR: The production of lipolytic activity of rat plasma after the intravenous injection of dextran sulphate. Biochem J 71:286-292, 1959 25. Gustafson A, Kerstell J: Serum lipoprotein pattern in fat embolism in the dog. Acta Med Scand 449(Suppl):19-23, 1969 26. Riedel B: Zur fettembolie. Deutsche Ztschr Chir 8:571-596, 1877 27. Soloway HB, Robinson EF, Hufnagel HV, Huyser KL: Experimental fat embolism: Initial distribution of fat emboli labeled with 1311 in normotensive and hypotensive rabbits. Arch Pathol 88:171-174, 1969 28. Shaffer JW, Sealy WC, Seaber AV, Hagen PO, Goldner JL: Effects and distribution of acute fat embolism in spontaneously breathing dog using radioactive carbon triolein. Surg Gynecol Obstet 141:387-393, 1975 29. Vales 0: Embolie pulmonaire experimentale chez le lapin par injection de licopode. CR Soc Biol 162:1855-1856, 1969
Acknowledgments The authors are greatly indebted to Prof. P. Dustin for his critical advice and to Mrs. R. Menu for her excellent technical assistance.
1
2
Figure 1-Six hours after triolein injection. Edema characterized by a mainly perivascular cuff. Note the intravascular lipid emboli and dilated lymphatics (arrows) filled with eosinophilic fluid. Fgure 2-Six hours after triolein injection. Many Alveolar edema is discrete. (H&E, X 75)
capillaries are obstructed by lipid emboli; some are considerably dilated. Semithin section, Eponembedded. (x 630)
3
4
A~~~~~~~~~~~
Figure 3-Fifteen hours after triolein injection. Margination of polymorphonuclear leukocytes. (H&E, X 100) Figure 4-Four days after triolein injection. Pulmonary tissue is scattered with granulomas, tending to converge. (H&E, X 75)
5
6
Fge 5-Marked increase in size of lesions, showing that the granulomas
are
composed of
and macrophages. (H&E, x 475) Fge 6-Six hours after triolein injection. Endothelial cells containing lipid vacuoles. The vesicles due to pinocytosis are
polymorphonuclear leukocytes numerous. (x 19,200)
x~~~~~~~~~~~~~~~~~~
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Figure 7-Six hours after triolein injection. Several capillaries are obstructed by lipid droplets without any lesion of endothelial cells. The alveoli are clear. (X 8400)
Twenty-two adult dogs were each given a single, 30-minute injection of 1.5 ml/kg bodyweight of pure triolein, and their pulmonarv, hepatic, renal, and cerebral morphology was observed for 1, 2, 3, 4, 6, 15, 24, and 48 hours; 3, 4, and 5 days; 1 and 2 weeks; and 1 month after the injection. A picture of massive capillary occlusion by lipid droplets was followed by rapidlv resolvable inflammatorv pneumopathv of granulomatous type, leaving a normal lung at the end of the experiment. The cleaning of the capillaries may be attributed to the mechanical action of the blood flow and to the inflammatorn reaction with evacuation of necrotic cells via the bronchial route. Transient pulmonary edema is attributed to increased pulmonarv arterial pressure. There was no intravascular coagulation. The few pulmonary lesions observed after the triolein injection suggest that the chemical theorv of neutral fat hvdrolvsis by pulmonary lipase and the toxicity of free fattv acids that are released should be reconsidered. (Am J Pathol 95:29-42, 1979)
ALTHOUGH a considerable number of studies are published everv year on the subject of fat embolism, there are still many aspects of this problem that remain obscure. The origin of embolized fat in the lung after large bone fracture is still a matter of controversv. Some authors 1-3 believe that the embolic material comes from the fractured bone itself. The discovery of bone marromv emboli in the pulmonary capillaries strongly supports that theory2'3 Other authors,44 without rejecting the first theory, maintain that the quantity of fat in a large bone is not sufficient to account for the entire embolic mass and that a change in the physicochemical properties of the blood causes lipid precipitation in the capillaries. Under clinical and experimental conditions, the composition of these fat emboli is 10 to 30% esterified or nonesterified cholesterol; the rest is neutral fat. The composition of lipids in the bones is approximately 1 % cholesterol and 995% neutral fat.5 Another important question is whether the fat emboli are toxic to the lung. Peltier 7 proposes a two-stage interpretation: after fracture, the neutral fat is embolized and causes capillanr obstruction (physical stage); then the pulmonary lipases h drol se the neutral fat and release free fatty From Laboratoire d'Anatomie Pathologique. et Institut de Recherches Interdisciplinaires en Biologie Humaine et Nuclaire. Institut de Recherches Cardiologiques, Faculte de \l&decine. U.niv ersite Libre de Bruxelles, Brussels, Belgium. Supported in part under contract of the Mlinist&re de la Politique Scientifique mvithin the framew-ork of the Association Euratom-U.nisersitv of Brussels-U.niversity of Pisa, and in part bv a grant from the Fonds de la Recherche Scientifique \ledicale. Xccepted for publication November 17. 1975. Address reprint requests to Dr. D Jacobov itz-Derks. Laboratorie d'Anatomie Pathologique. Faculte de \ledecine. 97. rue aux Laines. B-1000 Brussels. Belgium. 0002-9440/79/0411 -0029$01.00 29 © American Association of Pathologists
30
JACOBOVITZ-DERKS AND DERKS
American Journal of Pathology
acids which are extremely toxic for the lung (chemical stage). Other authors 8 stress the toxicity of the platelet aggregates, the formation of pulmonary capillary thrombi, and red cell sludge. Some insight into this problem may be gained from comparing the pulmonary and systemic lesions obtained experimentally by intravenous injection of neutral fat with the lesions observed in human pathologic conditions in which fat embolism is generally accompanied by slight or severe transient hypotension and marked stress. Such a comparison provides an indication of the importance of the embolism in a complex clinical picture. In this paper we report a study of the action of a pure neutral fat (triolein) on the lung, brain, liver, and kidney over given periods. The resulting lesions are compared with those obtained earlier by the FFA injection I and with those described in human disease.5'10 Materials and Methods Twenty-two healthy adult dogs, weighing between 13 and 24 kg, were each given a single intravenous injection of 1.5 ml/kg body weight of pure triolein over 30 minutes via a butterfly needle inserted into a back paw. For the duration of the injection, the dogs were kept harnessed, unanesthetized, in a standing position. Fourteen dogs were killed at 1, 2, 3, 4, 6, 15, 24, and 48 hours; 3, 4, and 5 days; 1 and 2 weeks; and 1 month after the injection; 1 other dog was killed at 4 days, another at 5 days, and 6 others at 1 month after the injection. Four normal dogs were given an injection of normal saline and were used as controls. At sacrifice, the dogs were anesthetized by an intravenous injection of 30 mg/kg of sodium pentobarbital and were killed by an intravenous injection of 30 ml of saturated KCI solution. Autopsy was performed immediately. The left lung was used for histologic examination and the right lung was weighed fresh and then was dried for calculation of the wet/dry lung weight ratio." Samples of liver, kidney, brain, and lung were taken from different sites (summit, base, anterior, and posterior regions). From the 7 dogs killed at 1 month, samples of intertracheobronchial lymph nodes and pulmonary hilar nodes were also taken. For light microscopy, fragments of the various organs, fixed in Bouin's fluid, were embedded in paraffin and stained with hematoxylin and eosin; others, fixed in formalin and embedded in gelatin, were cut with a freezing microtome and stained with Sudan IV red. Only the lung samples were examined by electron microscopy. For this purpose, small cubes were fixed for 2 hours in a 4% glutaraldehyde solution buffered at pH 7.4 following the technique of Millonig,12 then rinsed overnight in Millonig's buffer complemented with 5.4 g/liter of glucose. They were postfixed in 2% osmium tetroxide in the same buffer for 90 minutes, then dehydrated in the alcohol and propylene oxide, and embedded in Epon. Semi-thin sections cut with an LKB UM III Ultrotome, using glass knives, were stained with toluidine blue. Thin sections were prepared with a diamond knife, stained with uranyl acetate and lead citrate, then coated with a carbon film, and examined under a Siemens Elmiskop 101 electron microscope at 80 kV.
Results
The dogs were calm and nondyspneic at the time of the injection and appeared well at sacrifice. None of them died as a result of the treatment.
Vol. 95, No. 1 April 1979
NEUTRAL FAT EMBOLISM
31
MacocpcExamination
In the treated dogs, macroscopic examination of the lungs revealed moderate edema and congestion, associated with subpleural focal hemorrhage, mainly in the inferior lobes. These lesions were observed by the fourth hour. They reached maximum intensity approximately the fourth to fifth day. Liver, kidney, and brain were normal on macroscopic examination. In the control group, macroscopic autopsy of brain and organs proved normal. The wet/dry lung weight ratio of the normal dogs was 3.7 ± 0.3 (mean ± SD). The values of this ratio in the treated dogs are shown in Table 1. Thev were variably increased in the dogs killed between 1 hour and 5 days after the injection.
Lung (Table 1)
In accordance with Staub et al,'3 a distinction was made between pulmonary edema characterized by peribronchovascular cuff and edema with alveolar flooding. During the first 6 hours after the triolein injection, the lesions consisted mainly of capillary congestion, peribronchovascular and alveolar edema (Figure 1), and occasional alveolar hemorrhage in the subpleural regions. In the perivascular cuffs we found dilated lymphatics filled with eosinophilic fluid. At this stage there was no inflammatory infiltrate or macrophagic reaction. Sudan staining revealed a shower of lipid emboli scattered throughout the pulmonary capillaries (Figure 2) and in the segmental arteries. After the 15th hour, polymorphonuclear margination was observed in the pulmonary capillaries, and in the next 48 hours the polymorphonuclear leukocytes infiltrated the alveolar edema (Figure 3). After the 48th hour the number of lipid emboli had declined. Congestion and edema were unchanged and small subpleural hemorrhages were found. Three and four days after the injection, the pulmonary tissue was constellated with numerous granulomas composed of polymorphonuclear leukocytes and nonsudanophilic macrophages (Figures 4 and 5). After the first week, edema and congestion were discrete and alveolar hemorrhage had disappeared. The alveoli were distinctly clearer and Sudan staining failed to show any lipid emboli. One month after the injection, the pulmonary parenchyma presented a
32
JACOBOVITZ-DERKS AND DERKS
American Journal of Pathology
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Vol. 95, No. 1 April 1979
NEUTRAL FAT EMBOLISM
33
normal appearance which, apart from a few discrete lesions of edema and congestion, was undistinguishable from that of the lungs of the control animals. Liver, Kidney, Brain, and Nodes (Table 2)
In the treated dogs, Sudan staining revealed lipid droplets in the kidneys, at the glomerular and tubular levels, mainly during the 24 first hours of the trial, whereas Sudan-stained droplets were rare in the control dogs. There was no significant difference between the livers of control and treated dogs. In the brains of the treated dogs, lipid emboli predominated in the gray matter during the first 5 days; they were less frequent in the white matter. In the control animals a few lipid emboli were found in the white matter, but number was insignificant. No Sudan-stained material was found in the intertracheobronchial and pulmonary hilar nodes taken from the dogs killed at 1 month. Electron Microscopy (Table 1)
The appearance of the pulmonary lesions revealed by electron microscopy closely resembled that observed under the light microscope. Within the first few hours up to the third day, edematous thickening of the alveolocapillary wall and the formation of endothelial blebs were observed. The vacuoles of pinocytosis were more numerous than in the controls and contained lipid inclusions (Figure 6). These lesions were visible in capillaries free of lipid emboli. Epithelial cells of Type I and II were normal. During the first 24 hours, most of the capillaries were obstructed by lipid emboli (Figure 7). Some very large emboli plugged the capillary lumina completely, with the result that the vessels were considerably dilated and the vessel walls were molding around the embolic material. The endothelial cells of these capillaries were elongated but showed no sign of degeneration. After the fifth hour, the number of normal circulating polymorphonuclear leukocytes was higher than in the controls, with a peak occurring between the 24th and 48th hours. During this period, the alveoli were free and there was no accumulation of macrophages. After the 48th hour, occasional macrophages infiltrated the interstitial part of the septum. Between the third and the fifth days, electron microscopy confirmed the interstitial and, above all, alveolar localization of macrophage accumulation in the granulomatous lesions. By the end of the first week after the injection the alveoli were clear,
34
JACOBOVITZ-DERKS AND DERKS
American Journal of Pathology
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Vol. 95, No. 1 April 1979
NEUTRAL FAT EMBOLISM
35
and by the end of the first month the morphology of the lungs of the treated dogs was entirely similar to that of the controls. Throughout the course of this pulmonary affection there was no sign of Type II cell hyperplasia nor any evidence of intravascular coagulation. Discussion One of the most striking findings of this investigation was the insignificance of the observed pulmonary lesions compared with the quantity of triolein embolized. A comparison of the pulmonary morphologic picture after oleic acid embolism with that produced by triolein embolism shows that the former is essentially characterized by cell necrosis and hemorrhage, leading to fibrosis, whereas the latter is characterized by lesions of edema and congestion, accompanied by massive obstruction of the pulmonary capillaries by lipid droplets. The dose of triolein received by the animals was such that if neutral fat had been hydrolyzed by the pulmonary and circulating lipases, as Peltier suggests,7 only 3% of the injected quantity would have needed to be transformed into FFA to produce a picture of fibrosing lung disease such as that observed after oleic acid embolism.9 Peltier 14 suggests that the secretion of pulmonary lipase is specifically stimulated after neutral fat embolism. This theory is supported by some authors 15 but disputed by others, in particular by Soloway et al,16 who observed no increase in serum lipase during the 7 days immediately following triolein embolism in the rabbit. The circulating lipase values published by Peltier 17 in cases of fat embolism in humans lie within the normal range. Similarly, when Jacks et al is observed the increase in FFA and circulating lipase in blood returning from the left heart after pulmonary stenosis, they concluded that anoxia and hypoxia played a considerable part in this increase in FFA. Hausberger et al 19 demonstrated that after embolism by mineral oil, this inert lipid remained in the lung throughout the duration of their experiment, ie, 20 days, and that FFA could not be attributed to lysis of embolized lipids. The authors concluded that there must therefore be another source of FFA in the picture of fat embolism and that FFA cannot be regarded as responsible for the pulmonary embolism lesions. Pulmonary edema can be induced either by injecting toxins which increase capillary permeability or by increasing pulmonary capillary pressure. A physiologic study " of anesthetized dogs ventilated with air by a constant-volume respirator after an intravenous injection of 1.5 ml/kg of pure triolein shows an average pressure of 31 ± 4 mmHg in the pul-
36
JACOBOVITZ-DERKS AND DERKS
American Journal of Pathology
monary artery 15 minutes after the end of the injection and 27 ± 3 mmHg at 2 hours after the injection. No capillary cell lesions suggested hyperpermeability of toxic origin. Thus, in our study, the pulmonary edema seemed to be mainly due to the hemodynamic factor. This edema was resorbed after 4 days. Another important fact is the lack of intravascular coagulation. Soloway et al 21 observed the same phenomenon after injection of triolein in the rabbit. However, other authors 22,23 using homogenates of fat tissue as emboli observed a typical picture of intravascular coagulation. The homogenate contained tissue thromboplastins which were very probably responsible for the hypercoagulability. Triolein behaves as an inert substance, ie, it possesses no electronegative radicals to permit the release of heparin, which is partly responsible for the clearance of neutral fat in humans.24 It has little affinity for the carrier plasma proteins; Gustafson and Kerstell 25 have shown that after embolism in humans there is no change in the distribution of circulating lipoproteins, which indicates that they take no part in the transport of embolized lipids. The size and shape of the embolized corpuscles are important: in the case of triolein, the corpuscles are small and regularly shaped, encountering no obstacle to continuous circulation. The mechanism of pulmonary capillary cleaning is a complex phenomenon in which the inflammatory reaction and the positive mechanical action of pulmonary blood flow play varying roles. Riedel 26 injected oil in a systemic artery and found that most of it made its way to the lung. This may be explained by the low pressure of pulmonary circulation and by the lack of support around the pulmonary capillaries. In contrast, in the systemic capillaries, the blood pressure pushes the fat and flushes the capillary. This purely mechanical cleaning action no doubt also takes place in the lung, but more slowly, with the result that smaller quantities of lipids are cleared. During embolism and the hours immediately following the injection, pressure in the pulmonary artery is high, which would facilitate the cleaning of the pulmonary capillaries. It is during these first few hours that the quantity of fat found in the kidney and brain is highest. Soloway et al 27 demonstrated that 80% of the embolized triolein remained in the lungs of normotensive rabbits but that approximately 98% of embolized triolein stayed in the lungs of rabbits in a state of shock. Shaffer et al 28 have shown that radiolabeled triolein leaks slowly from the pulmonary circulation to the systemic circulation. Armin et al 2 found a proportional relation between the quantity of lipid injected and the peak of peripheral emboli. The inflammatory reaction undoubtedly plays a significant role in pulmonary cleaning. The macrophages are loaded with lipid inclusions and
Vol. 95, No. 1 April 1979
NEUTRAL FAT EMBOLISM
37
the necrotic cells are evacuated via the bronchi. An earlier clinical test consisted in looking for lipids in the expectorations of patients Xwith suspected fat embolism. The lymphatic channels seem to be ruled out because no lipids have been found at anx time in the pulmonary lynmphatics or intertracheobronchial or hilar nodes. Triolein behaves as an inert substance and reacts like lvcopodium spores, which are also evacuated after embolism through the intermediary of the inflammatory reaction via the bronchial route." The clinical picture of human fat embolism and the one observed in this experiment differ in various ways. In humans, the congestion never persists as long as in the experimental animals and there is no inflammatorv reaction in the embolized zones."5 The tolerated doses of vegetable oil varies from one study to another and probably depends on the technique of injection. The tolerated dose seems to be 1 to 3 ml/kg,8 which would correspond to 210 ml of embolized fat in a 70-kg man. But in human pathologic conditions it is known that small quantities of lipids may give rise to an alarming clinical picture. Consequently, in traumatic fat embolism it is essential not to overlook the respective roles of hypovolemia, ischemia, the tissue lesion itself, the release of thromboplastin, and associated vascular coagulation. The combination of all these factors plays a role as important as, or more important than, the embolism itself. References 1. Gauss H: The pathology of fat embolism. Arch Surg 9:593-605, 1924 2. Armin J, Grant RT: Observations on gross pulmonary fat embolism in man and in the rabbit. Clin Sci 10:441-469, 1951 3. Schinella RA: Bone marrow emboli: Their fate in the vasculature of the human lung. Arch Pathol 95:386-391, 1973 4. Scriba J: Untersuchungen uber die Fettembolie. Deutsche Ztschr Chir 12:118-219, 1879 a. Lequire VS, Shapiro JL, Lequire CB, Cobb CA, Fleet %'F: A study of the pathogenesis of fat embolism based on human necropsy material and animal experiments. Am J Pathol 35:999-1015, 1959 6. Lehman EP, Moore RMI: Fat embolism including experimental production without trauma. Arch Surg 14:621-662, 1927 7. Peltier LF: Fat embolism. III. The toxic properties of neutral fat and free fatty acids. Surgery 40:665-670, 1956 8. Bergentz S-E: Studies on the genesis of posttraumatic fat embolism. Acta Chir Scand 282(Suppl):1-72, 1961 9. Derks CM, Jacobovitz-Derks D: Embolic pneumopathy induced by oleic acid: A sy-stematical morphological study. Am J Pathol 87:143-158, 1977 10. Saldeen T: Fat embolism and signs of intravascular coagulation in a posttraumatic autopsy material. J Trauma 10:273-286, 1970 11. Guy-ton AC, Lindsey AW: Effect of elevated left atrial pressure and decreased plasma protein concentration in the development of pulmonarn edema. Circ Res 7:649-657, 1959
38
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American Journal of Pathology
12. Millonig G: Further observations on a phosphate buffer for osmium solutions. Fifth International Conference on Electron Microscopy. New York, Academic Press, 1962, p8 13. Staub NC, Nagano H, Pearse ML: Pulmonary edema in dogs, especially the sequence of fluid accumulation in lungs. J Appl Physiol 22:227-240, 1967 14. Peltier LF, Scott JR: Fat embolism: Changes in the level of blood lipase following the intravenous injection of neutral fat, fatty acids and other substances into dogs. Surgery 42:541-547, 1957 15. Fonte DA, Hatsberger FX: Pulmonary free fatty acids in experimental fat embolism. J Trauma 11:668-672, 1971 16. Soloway HB, Robinson EF, Sleeman HK, Huyser KL, Hufnagel HV: Resolution of experimental fat embolism. Arch Pathol 90:230-234, 1970 17. Peltier LF, Adler F, Lai SP: Fat embolism. The significance of an elevated serum lipase after trauma to bone. Am J Surg 99:821-826, 1960 18. Jacks ML, Reed WA, Allbritten FF: Alterations in plasma free fatty acids during extracorporeal circulation. Surg Forum 15:279-281, 1964 19. Hausberger FX, Phelan TI: Pulmonary free fatty acids in experimental mineral oil embolism. J Trauma 14:950-954, 1974 20. Halasz NA, Marasco JP: An experimental study of fat embolism. Surgery 41:921929, 1957 21. Soloway HB, Robinson EF: The coagulation mechanism in experimental pulmonary fat embolism. J Trauma 12:630-631, 1972 22. Saldeen T: The importance of intravascular coagulation and inhibition of the fibrinolytic system in experimental fat embolism. J Trauma 10:287-298, 1970 23. Cotev S, Rosenmann E, Eyal Z, Weinberg H, Shafrir E, Davidson JT: The role of hypovolemic stress in the production of fat embolism in rabbits. I. Morphological alterations of the lungs. Chest 69:523-528, 1976 24. Robinson DS, Harris PM, Ricketts CR: The production of lipolytic activity of rat plasma after the intravenous injection of dextran sulphate. Biochem J 71:286-292, 1959 25. Gustafson A, Kerstell J: Serum lipoprotein pattern in fat embolism in the dog. Acta Med Scand 449(Suppl):19-23, 1969 26. Riedel B: Zur fettembolie. Deutsche Ztschr Chir 8:571-596, 1877 27. Soloway HB, Robinson EF, Hufnagel HV, Huyser KL: Experimental fat embolism: Initial distribution of fat emboli labeled with 1311 in normotensive and hypotensive rabbits. Arch Pathol 88:171-174, 1969 28. Shaffer JW, Sealy WC, Seaber AV, Hagen PO, Goldner JL: Effects and distribution of acute fat embolism in spontaneously breathing dog using radioactive carbon triolein. Surg Gynecol Obstet 141:387-393, 1975 29. Vales 0: Embolie pulmonaire experimentale chez le lapin par injection de licopode. CR Soc Biol 162:1855-1856, 1969
Acknowledgments The authors are greatly indebted to Prof. P. Dustin for his critical advice and to Mrs. R. Menu for her excellent technical assistance.
1
2
Figure 1-Six hours after triolein injection. Edema characterized by a mainly perivascular cuff. Note the intravascular lipid emboli and dilated lymphatics (arrows) filled with eosinophilic fluid. Fgure 2-Six hours after triolein injection. Many Alveolar edema is discrete. (H&E, X 75)
capillaries are obstructed by lipid emboli; some are considerably dilated. Semithin section, Eponembedded. (x 630)
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Figure 3-Fifteen hours after triolein injection. Margination of polymorphonuclear leukocytes. (H&E, X 100) Figure 4-Four days after triolein injection. Pulmonary tissue is scattered with granulomas, tending to converge. (H&E, X 75)
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