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Effect of tricomplex fixation on lung tissue
© 1972 by Academic Press, Inc.
122
J. ULTRASTRUCTURERESEARCH40, 122-131 (1972)
Effect of Tricomplex Fixation on Lung Tissue JOAN GIL1
Anatomisehes Institut der Universitiit Bern, 3012 Bern, Switzerland JReeeived November 30, 1971 The effect of tricomplex fixation on lungs fixed through vascular perfusion or through instillation of fixatives into the airways has been studied. To test the specificity of electron dense precipitates for phospholipids, some preparations were treated with tricomplex fixatives after lipid extraction with chloroformmethanol in a Soxhlet apparatus. The same experiment was repeated with red blood cells taken from the inferior vena cava of rats. It is concluded that precipitates reported in lung tissue after tricomplex flocculation cannot be regarded as specific for surfactant phospholipids, since they appear in various locations of several cell types including the interior of red blood cells; their occurrence is furthermore not influenced by lipid extraction prior to the application of tricomplex fixatives. The morphological appearance of alveolar surfactant in electron micrographs of lung tissue has been the object of some recent discussion. Using a perfusion-fixation technique, we had succeeded in demonstrating a lining layer of lung alveoli, which we supposed to be closely related to or to contain the surface-active material (I4). The validity of this approach and interpretation has been discussed elsewhere (6, 8). In the meantime, application of the freeze-etching preparation technique (13) has confirmed the existence of this lining layer in the form described; in particular, that study has shown that this layer is not an artifact due to perfusion or chemical fixation. The finding is also reproducible with other techniques which allow a preservation of the air-liquid interface, such as fixation from the pleural side (5, 10). In lungs fixed with techniques which lead to a destruction of the air-liquid interface, findings have been presented which seem to be in conflict with our description of the lining layer. The present report is concerned with those studies using a tricomplex flocculation technique on lung tissue. This technique had been introduced by Elbers et al. (4) to study micelles of saturated lipids by electron microscopy. Dermer (2, 3) has used it to fix lungs in the hope to demonstrate the presence of the saturated surfactant phospholipids; he has described electron dense material located z Author's present address: Webb-Waring Institute and Department of Biochemistry, University of Colorado School of Medicine, Denver CO 80220.
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in several places of the lung p a r e n c h y m a , which he interpreted to specifically represent the alveolar surfactant. Several observations a n d some theoretical considerations led us to suspect t h a t this finding c o u l d n o t be r e g a r d e d as specific. W e therefore a p p l i e d t r i c o m p l e x fixatives to the lung b y tracheal instillation a n d vascular perfusion in o r d e r to system a t i c a l l y examine their effects on lung tissue. To check whether any finding could be r e g a r d e d as specific for surfactant a n d / o r for p h o s p h o l i p i d s , two tests were carried out: (a) Lungs fixed by instillation of t r i c o m p l e x solutions i n t o the t r a c h e a were e x a m i n e d before a n d after lipid extraction. (b) T h e same effects were investigated in erythrocytes t a k e n f r o m the vena cava of a rat.
MATERIAL AND METHODS This study comprises three series of experiments. First, the effect of tricomplex flocculation was examined on rat lungs fixed by perfusion through the pulmonary artery and through instillation into the airways; the second series included studies on the effect of tricomplex fixation after lipid extraction. Finally, in the third series, the effect of tricomplex fixation on erythrocytes before and after lipid extraction was examined. A. As a control, rat lungs were perfused through the pulmonary artery with Ringer, glutaraldehyde, osmium, and uranyl acetate at constant pressure conditions according to our previously described standard technique (8, 9). B. The effect of tricomplex fixation on rat lungs fixed through vascular perfusion was tested in the following manner: Rat lungs were perfused for 2 minutes with Ringer solution containing heparin and papaverine sulfate, followed by 1.5% isotonic glutaraldehyde in phosphate buffer for 12 minutes; subsequently tricomplex fixatives [Pb(NO)8 and K~Fe(CN)6 both 0.05 M in distilled water] were perfused during 15 minutes. Lungs were removed from the chest and diced; tissue blocks were refixed in 1% OsO~ in s-collidine buffer for 1.5-2 hours. The specimens were embedded in Epon 812. C. F o r this experiment, dog lungs which had been fixed for other purposes by instillation of 2.5 % glutaraldehyde into the airways after pneumothorax were used. Pieces of these lungs were diced into small blocks and washed overnight in isotonic buffer. The next day, the material was divided into two groups: (a) Blocks were treated with tricomplex fixatives (composition as above) for 30 minutes, refixed in osmium tetroxide, dehydrated, and em-
FIG. 1. Rat lung fixed by standard perfusion technique with glutaraldehyde, osmium tetroxide, and uranyl acetate. Capillaries (C) are flushed out; a depression between two capillaries is filled with an extracellular lining (L) which contains osmiophilic figures. A, alveolar space, x 7 900. FIG. 2. Rat lung perfused with glutaraldehyde and tricomplex fixatives. The lining layer is poorly preserved; it is transformed into disrupted myelin figures (arrows). The capillary on the left contains a dense precipitate (open arrow). These precipitates were relatively rare in this series of preparations. Notice the appearance of light areas in cell nuclei, x 6 400. FIG. 3. Same preparation as in Fig. 2. Dark precipitates (arrows) occur in various regions of the tissue, mainly in capillaries and interstitium. Notice the structural changes of nuclei, x 4 550. FIG. 4. Same preparation as in Fig. 3. Alveolar macrophage sitting in deep cleft between capillaries. Note the granular structures seen surrounding the light space in the nucleus. × 11 200.
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bedded. (b) A lipid extraction with chloroform:methanol (2:1) was performed in a Soxhlet apparatus for 5 hours. Thereafter, the blocks were treated with tricomplex fixatives for 30 minutes and refixed with osmium as in group a. D. Blood was withdrawn from the inferior vena cava of normal rats. To prevent clotting, heparin was added to the blood samples. These were centrifuged, and the blood cells were resuspended in isotonic, phosphate-buffered 1.5 % glutaraldehyde. After 1 hour the suspension was again centrifuged and blood cells were resuspended in isotonic buffer for washing. This material was divided into 2 groups: (a) Blood cells were treated with tricomplex fixatives, refixed in osmium, and embedded. (b) A lipid extraction with chloroform:methanol 2:1 in a Soxhlet apparatus was performed. Subsequently, the specimens were processed as for group a. All specimens were cut with diamond knives on LKB or Reichert ultramicrotomes. Sections were mounted on naked grids and examined in a Philips EM 300 electron microscope. RESULTS A. In rat lungs fixed by perfusion through the pulmonary artery with our standard technique (8, 9), an extracellular lining layer of lung alveoli can be consistently demonstrated (Fig. 1): it is characterized by a smooth surface and by its distribution in form of minute layers over bulging capillaries and deeper pools which even out depressions and crevices of the epithelial surface. The matrix of the base layer is amorphous; it may contain spheroidal or tubular myelin figures (15), and is often topped by a fine osmiopholic film. B. The results of perfusion with tricomplex fixatives following glutaraldehyde are shown in Figs. 2-4. In these preparations, the lining layer was less well preserved than in the above control experiment, possibly owing to the absence of osmium tetroxide perfusion. It appeared disrupted and exhibited an abundance of myelin figures. Sometimes thick black precipitates could be seen in several regions of the tissue, even within the flushed-out capillary space (Fig. 2). The location of these precipitates was not specific but rather random. A striking feature of these preparations was the appearance of the nuclei (Figs. 2-4): they often contained light areas surrounded by dark filamentous or granular material. C. The application of tricomplex fixation to dog lungs prefixed by instillation of glutaraldehyde into the airways showed results very similar to those reported by Dermer (2, 3): dense precipitates (Fig. 5) could be demonstrated in various regions of the lung tissue, including blood plasma, erythrocytes, and the cytoplasm of several cell types. These precipitates were also found, although seldom, on the alveolar epithelial surface. In these same lungs, very similar results could be recorded after lipid extraction with chloroform:methanol 2:1 (Fig. 6). That the lipid extraction was successful was demonstrated (a) by the emptiness of lamellated bodies of alveolar cells type II,
LUNG TRICOMPLEX FIXATION
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Fie. 5. Dog lung fixed by instillation of glutaraldehyde into the trachea and treated with tricomplex fixation. Many granules are seen on the surface of alveolar epithelium (A) and erythrocytes (E) as well as within the air-blood barrier and in red cells, x 30 000. Fr~. 6. Dog lung fixed in glutaraldehyde treated with tricomplex fixative and osmium tetroxide after extraction of lipids in the Soxhlet apparatus. A dense coat with a thick granule is seen "on the alveolar surface (A). The erythrocyte (E) exhibits some dense precipitates in its interior. × 56 000.
128
GIL
(b) by the lack of myelin figures, and (c) by the disappearance of the black membrane traces. The question whether these precipitates were present to the same extent as in the group without lipid extraction cannot be clearly answered, since the finding was irregular and polymorphous in both groups. D. Erythrocytes from the inferior vena cava of a rat, treated with tricomplex fixatives after glutaraldehyde fixation, exhibited the same appearance as erythrocytes observed in the lung. In their matrix as well as on their surface, polymorphous black precipitates were observed (Fig. 7). These were also present in spaces between the single cells. Figure 8 shows a RBC fixed with glutaraldehyde which had undergone a lipid extraction before application of tricomplex fixatives. As seen in this micrograph, similar coatings and precipitates were seen on and in most erythrocytes.
DISCUSSION It has recently been claimed that the alveolar surfactant system can be demonstrated electron microscopically only in lungs fixed by a tricomplex flocculation technique (2, 3), whereas with conventional fixation techniques it should theoretically be impossible to preserve its main constituent, dipalmitoyl lecithin, a saturated phospholipid (1, 11). Dermer (2, 3) has at the same time claimed that electron dense precipitates found in the lung after tricomplex fixation are specific for the presence of surfactant. Since this suggestion meets with great interest because of its potential usefulness in pathology, we have reexamined the significance of these precipitates. The tricomplex flocculation technique had been introduced by Elbers et al. (4) in order to overcome the shortcoming of oxidizing fixatives, such as osmium tetroxide or potassium permanganate, that these cannot be used to fix fully saturated lipids. The technique was thought to be a tool for the in vitro study of phospholipid micelles by electron microscopy, irrespective of whether they consist of saturated or unsaturated compounds. The effect of tricomplex fixation consists in a flocculation due to complexing of neighboring phosphatide amphoions by means of links formed between suitable ion pairs and polar groups of phospholipids. In model systems this results in the formation of multilayered micelles with clearly recognizable periodicity due to the specific incorporation of heavy metal atoms at the polar groups of the FI~. 7. Red blood cell taken from the inferior vena cava of a rat, fixed in glutaraldehyde, and refixed with tricomplex fixative and osmium tetroxide. Note the coarse precipitates both on the surface and in the interior. Such precipitates could also be demonstrated in variable amounts in the lungs presented in Figs. 2-6. x 22 900. FI~. 8. Red blood cell from the vena cava, fixed in glutaraldehyde. After lipid extraction, tricomplex fixatives and osmium tetroxide were applied. Note the precipitates on the cell surface and in the matrix, x 59 000.
LUNG
9 -- 7 2 1 8 3 3
J . Ultrastructure Research
TR1COMPLEX
FIXATION
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130
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phospholipid molecules. Since phospholipids in aqueous media have a tendency to pair off hydrophobic and hydrophilic ends (12), this reaction can only be regarded as specific for phospholipids if it results in the formation of lamellae at a spacing of the order of 4-5 nm. The dense granules and precipitates found in the present study after tricomplex fixation did not exhibit any internal repeat period. Under these circumstances, the possibility cannot be ruled out that the reagents may have produced unspecific precipitates with other, as yet unidentified, tissue components. If phospholipids were present in a form that flocculation in the sense of this reaction was possible, the chloroform-methanol treatment should have extracted them. But after lipid extraction, precipitates were found which were identical, or at least very similar, to those observed in the unextracted tissue. This would speak against the possibility that these amorphous precipitates represent phospholipids; the reaction product must hence be regarded as unspecific. This view is strongly supported by the ubiquity of these precipitates, especially their presence within the cytoplasm of red blood cells, and by their polymorphism. These findings lead to the conclusion that no statements about the chemical nature of the reaction product of tricomplex flocculation in the lungs can be made and, specifically, that no relationship of any kind has as yet been demonstrated between these precipitates and pulmonary surfactant phospholipids. To some extent, there appears to be a certain similarity between these dense precipitates and an artifact due to the use of phosphate-buffered osmium after prefixation with glutaraldehyde (7). With uranyl acetate used as block stain and with Dalton's chrome-osmium fixative, similar findings can sometimes be observed in the lung, as well as in and on erythrocytes (6, 7 and unpublished observations). Considering the observations made on isolated erythrocytes, one would be tempted to speculate that these reactions take place when the reagents come into direct primary contact with free cell surfaces, a condition that is unusually well met in lung tissue or in isolated blood cells. We can, however, certainly arrive at the conclusion that precipitates formed on the alveolar surface of the lung after treatment with tricomplex fixatives are unspecific and may not be claimed to be related to surfactant phospholipids. The author is deeply indebted to Dr Ewald R. Weibel for his encouragement and help. Assistance was received from Miss G. Reber, Mrs R. Bachmann and Mr K. Babl. This work was supported by Grant No. 3.5.68 of the Swiss National Science Foundation. REFERENCES 1. CLEMENTS,J. A., Amer. Rev. Resp. Dis. 101, 984 (1970). 2. DERMER,G. B., J. Ultrastruet. Res. 27, 88 (1969). 3. - ibid. 31, 229 (1970).
LUNG TRICOMPLEXFIXATION
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4. ELBERS,P. F., VERVERGAERT,P. M. J. T. and DEMEL,R., J. Cell Biol. 24, 23 (1965). 5. FINLEY, T. N., PRATT, S. A., LADMANN, A. J., BREWEER, L. and McKAY, M. B., J. Lipid Res. 9, 357 (1968). 6. GIL, J., Arch. Int. Med. 127, 896 (1971). 7. GIL, J. and WEmEL, E. R., J. Ultrastruct. Res. 25, 331 (1968). 8. - - - - Resp. Physiol. 8, 13 (1969/70). 9. - J. Microsc. in press (1972). 10. KIKKAWA,Y., Anat. Rec. 167, 389 (1970). 11. SCARVELLI,E. M. The Surfactant System of the Lung. Lea and Febiger, Philadelphia, 1968. 12. STOECKENIUS,W., ,J. Cell Biol. 12, 221 (1962). 13. UNTERSEE,P., GIL, J. and WEIBEL,E. R., Resp. Physiol. 13, 171 (1971). 14. WEIBEL,E. R. and GIL, J., Resp. Physiol. 4, 42 (1968). 15. WEIBEL, E. R., KISTLER, G. and TOENDURY, G., Z. Zellforseh. Mikrosk. Anat. 69, 418 (1966).
122
J. ULTRASTRUCTURERESEARCH40, 122-131 (1972)
Effect of Tricomplex Fixation on Lung Tissue JOAN GIL1
Anatomisehes Institut der Universitiit Bern, 3012 Bern, Switzerland JReeeived November 30, 1971 The effect of tricomplex fixation on lungs fixed through vascular perfusion or through instillation of fixatives into the airways has been studied. To test the specificity of electron dense precipitates for phospholipids, some preparations were treated with tricomplex fixatives after lipid extraction with chloroformmethanol in a Soxhlet apparatus. The same experiment was repeated with red blood cells taken from the inferior vena cava of rats. It is concluded that precipitates reported in lung tissue after tricomplex flocculation cannot be regarded as specific for surfactant phospholipids, since they appear in various locations of several cell types including the interior of red blood cells; their occurrence is furthermore not influenced by lipid extraction prior to the application of tricomplex fixatives. The morphological appearance of alveolar surfactant in electron micrographs of lung tissue has been the object of some recent discussion. Using a perfusion-fixation technique, we had succeeded in demonstrating a lining layer of lung alveoli, which we supposed to be closely related to or to contain the surface-active material (I4). The validity of this approach and interpretation has been discussed elsewhere (6, 8). In the meantime, application of the freeze-etching preparation technique (13) has confirmed the existence of this lining layer in the form described; in particular, that study has shown that this layer is not an artifact due to perfusion or chemical fixation. The finding is also reproducible with other techniques which allow a preservation of the air-liquid interface, such as fixation from the pleural side (5, 10). In lungs fixed with techniques which lead to a destruction of the air-liquid interface, findings have been presented which seem to be in conflict with our description of the lining layer. The present report is concerned with those studies using a tricomplex flocculation technique on lung tissue. This technique had been introduced by Elbers et al. (4) to study micelles of saturated lipids by electron microscopy. Dermer (2, 3) has used it to fix lungs in the hope to demonstrate the presence of the saturated surfactant phospholipids; he has described electron dense material located z Author's present address: Webb-Waring Institute and Department of Biochemistry, University of Colorado School of Medicine, Denver CO 80220.
LUNG TRICOMPLEX FIXATION
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in several places of the lung p a r e n c h y m a , which he interpreted to specifically represent the alveolar surfactant. Several observations a n d some theoretical considerations led us to suspect t h a t this finding c o u l d n o t be r e g a r d e d as specific. W e therefore a p p l i e d t r i c o m p l e x fixatives to the lung b y tracheal instillation a n d vascular perfusion in o r d e r to system a t i c a l l y examine their effects on lung tissue. To check whether any finding could be r e g a r d e d as specific for surfactant a n d / o r for p h o s p h o l i p i d s , two tests were carried out: (a) Lungs fixed by instillation of t r i c o m p l e x solutions i n t o the t r a c h e a were e x a m i n e d before a n d after lipid extraction. (b) T h e same effects were investigated in erythrocytes t a k e n f r o m the vena cava of a rat.
MATERIAL AND METHODS This study comprises three series of experiments. First, the effect of tricomplex flocculation was examined on rat lungs fixed by perfusion through the pulmonary artery and through instillation into the airways; the second series included studies on the effect of tricomplex fixation after lipid extraction. Finally, in the third series, the effect of tricomplex fixation on erythrocytes before and after lipid extraction was examined. A. As a control, rat lungs were perfused through the pulmonary artery with Ringer, glutaraldehyde, osmium, and uranyl acetate at constant pressure conditions according to our previously described standard technique (8, 9). B. The effect of tricomplex fixation on rat lungs fixed through vascular perfusion was tested in the following manner: Rat lungs were perfused for 2 minutes with Ringer solution containing heparin and papaverine sulfate, followed by 1.5% isotonic glutaraldehyde in phosphate buffer for 12 minutes; subsequently tricomplex fixatives [Pb(NO)8 and K~Fe(CN)6 both 0.05 M in distilled water] were perfused during 15 minutes. Lungs were removed from the chest and diced; tissue blocks were refixed in 1% OsO~ in s-collidine buffer for 1.5-2 hours. The specimens were embedded in Epon 812. C. F o r this experiment, dog lungs which had been fixed for other purposes by instillation of 2.5 % glutaraldehyde into the airways after pneumothorax were used. Pieces of these lungs were diced into small blocks and washed overnight in isotonic buffer. The next day, the material was divided into two groups: (a) Blocks were treated with tricomplex fixatives (composition as above) for 30 minutes, refixed in osmium tetroxide, dehydrated, and em-
FIG. 1. Rat lung fixed by standard perfusion technique with glutaraldehyde, osmium tetroxide, and uranyl acetate. Capillaries (C) are flushed out; a depression between two capillaries is filled with an extracellular lining (L) which contains osmiophilic figures. A, alveolar space, x 7 900. FIG. 2. Rat lung perfused with glutaraldehyde and tricomplex fixatives. The lining layer is poorly preserved; it is transformed into disrupted myelin figures (arrows). The capillary on the left contains a dense precipitate (open arrow). These precipitates were relatively rare in this series of preparations. Notice the appearance of light areas in cell nuclei, x 6 400. FIG. 3. Same preparation as in Fig. 2. Dark precipitates (arrows) occur in various regions of the tissue, mainly in capillaries and interstitium. Notice the structural changes of nuclei, x 4 550. FIG. 4. Same preparation as in Fig. 3. Alveolar macrophage sitting in deep cleft between capillaries. Note the granular structures seen surrounding the light space in the nucleus. × 11 200.
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bedded. (b) A lipid extraction with chloroform:methanol (2:1) was performed in a Soxhlet apparatus for 5 hours. Thereafter, the blocks were treated with tricomplex fixatives for 30 minutes and refixed with osmium as in group a. D. Blood was withdrawn from the inferior vena cava of normal rats. To prevent clotting, heparin was added to the blood samples. These were centrifuged, and the blood cells were resuspended in isotonic, phosphate-buffered 1.5 % glutaraldehyde. After 1 hour the suspension was again centrifuged and blood cells were resuspended in isotonic buffer for washing. This material was divided into 2 groups: (a) Blood cells were treated with tricomplex fixatives, refixed in osmium, and embedded. (b) A lipid extraction with chloroform:methanol 2:1 in a Soxhlet apparatus was performed. Subsequently, the specimens were processed as for group a. All specimens were cut with diamond knives on LKB or Reichert ultramicrotomes. Sections were mounted on naked grids and examined in a Philips EM 300 electron microscope. RESULTS A. In rat lungs fixed by perfusion through the pulmonary artery with our standard technique (8, 9), an extracellular lining layer of lung alveoli can be consistently demonstrated (Fig. 1): it is characterized by a smooth surface and by its distribution in form of minute layers over bulging capillaries and deeper pools which even out depressions and crevices of the epithelial surface. The matrix of the base layer is amorphous; it may contain spheroidal or tubular myelin figures (15), and is often topped by a fine osmiopholic film. B. The results of perfusion with tricomplex fixatives following glutaraldehyde are shown in Figs. 2-4. In these preparations, the lining layer was less well preserved than in the above control experiment, possibly owing to the absence of osmium tetroxide perfusion. It appeared disrupted and exhibited an abundance of myelin figures. Sometimes thick black precipitates could be seen in several regions of the tissue, even within the flushed-out capillary space (Fig. 2). The location of these precipitates was not specific but rather random. A striking feature of these preparations was the appearance of the nuclei (Figs. 2-4): they often contained light areas surrounded by dark filamentous or granular material. C. The application of tricomplex fixation to dog lungs prefixed by instillation of glutaraldehyde into the airways showed results very similar to those reported by Dermer (2, 3): dense precipitates (Fig. 5) could be demonstrated in various regions of the lung tissue, including blood plasma, erythrocytes, and the cytoplasm of several cell types. These precipitates were also found, although seldom, on the alveolar epithelial surface. In these same lungs, very similar results could be recorded after lipid extraction with chloroform:methanol 2:1 (Fig. 6). That the lipid extraction was successful was demonstrated (a) by the emptiness of lamellated bodies of alveolar cells type II,
LUNG TRICOMPLEX FIXATION
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Fie. 5. Dog lung fixed by instillation of glutaraldehyde into the trachea and treated with tricomplex fixation. Many granules are seen on the surface of alveolar epithelium (A) and erythrocytes (E) as well as within the air-blood barrier and in red cells, x 30 000. Fr~. 6. Dog lung fixed in glutaraldehyde treated with tricomplex fixative and osmium tetroxide after extraction of lipids in the Soxhlet apparatus. A dense coat with a thick granule is seen "on the alveolar surface (A). The erythrocyte (E) exhibits some dense precipitates in its interior. × 56 000.
128
GIL
(b) by the lack of myelin figures, and (c) by the disappearance of the black membrane traces. The question whether these precipitates were present to the same extent as in the group without lipid extraction cannot be clearly answered, since the finding was irregular and polymorphous in both groups. D. Erythrocytes from the inferior vena cava of a rat, treated with tricomplex fixatives after glutaraldehyde fixation, exhibited the same appearance as erythrocytes observed in the lung. In their matrix as well as on their surface, polymorphous black precipitates were observed (Fig. 7). These were also present in spaces between the single cells. Figure 8 shows a RBC fixed with glutaraldehyde which had undergone a lipid extraction before application of tricomplex fixatives. As seen in this micrograph, similar coatings and precipitates were seen on and in most erythrocytes.
DISCUSSION It has recently been claimed that the alveolar surfactant system can be demonstrated electron microscopically only in lungs fixed by a tricomplex flocculation technique (2, 3), whereas with conventional fixation techniques it should theoretically be impossible to preserve its main constituent, dipalmitoyl lecithin, a saturated phospholipid (1, 11). Dermer (2, 3) has at the same time claimed that electron dense precipitates found in the lung after tricomplex fixation are specific for the presence of surfactant. Since this suggestion meets with great interest because of its potential usefulness in pathology, we have reexamined the significance of these precipitates. The tricomplex flocculation technique had been introduced by Elbers et al. (4) in order to overcome the shortcoming of oxidizing fixatives, such as osmium tetroxide or potassium permanganate, that these cannot be used to fix fully saturated lipids. The technique was thought to be a tool for the in vitro study of phospholipid micelles by electron microscopy, irrespective of whether they consist of saturated or unsaturated compounds. The effect of tricomplex fixation consists in a flocculation due to complexing of neighboring phosphatide amphoions by means of links formed between suitable ion pairs and polar groups of phospholipids. In model systems this results in the formation of multilayered micelles with clearly recognizable periodicity due to the specific incorporation of heavy metal atoms at the polar groups of the FI~. 7. Red blood cell taken from the inferior vena cava of a rat, fixed in glutaraldehyde, and refixed with tricomplex fixative and osmium tetroxide. Note the coarse precipitates both on the surface and in the interior. Such precipitates could also be demonstrated in variable amounts in the lungs presented in Figs. 2-6. x 22 900. FI~. 8. Red blood cell from the vena cava, fixed in glutaraldehyde. After lipid extraction, tricomplex fixatives and osmium tetroxide were applied. Note the precipitates on the cell surface and in the matrix, x 59 000.
LUNG
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FIXATION
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130
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phospholipid molecules. Since phospholipids in aqueous media have a tendency to pair off hydrophobic and hydrophilic ends (12), this reaction can only be regarded as specific for phospholipids if it results in the formation of lamellae at a spacing of the order of 4-5 nm. The dense granules and precipitates found in the present study after tricomplex fixation did not exhibit any internal repeat period. Under these circumstances, the possibility cannot be ruled out that the reagents may have produced unspecific precipitates with other, as yet unidentified, tissue components. If phospholipids were present in a form that flocculation in the sense of this reaction was possible, the chloroform-methanol treatment should have extracted them. But after lipid extraction, precipitates were found which were identical, or at least very similar, to those observed in the unextracted tissue. This would speak against the possibility that these amorphous precipitates represent phospholipids; the reaction product must hence be regarded as unspecific. This view is strongly supported by the ubiquity of these precipitates, especially their presence within the cytoplasm of red blood cells, and by their polymorphism. These findings lead to the conclusion that no statements about the chemical nature of the reaction product of tricomplex flocculation in the lungs can be made and, specifically, that no relationship of any kind has as yet been demonstrated between these precipitates and pulmonary surfactant phospholipids. To some extent, there appears to be a certain similarity between these dense precipitates and an artifact due to the use of phosphate-buffered osmium after prefixation with glutaraldehyde (7). With uranyl acetate used as block stain and with Dalton's chrome-osmium fixative, similar findings can sometimes be observed in the lung, as well as in and on erythrocytes (6, 7 and unpublished observations). Considering the observations made on isolated erythrocytes, one would be tempted to speculate that these reactions take place when the reagents come into direct primary contact with free cell surfaces, a condition that is unusually well met in lung tissue or in isolated blood cells. We can, however, certainly arrive at the conclusion that precipitates formed on the alveolar surface of the lung after treatment with tricomplex fixatives are unspecific and may not be claimed to be related to surfactant phospholipids. The author is deeply indebted to Dr Ewald R. Weibel for his encouragement and help. Assistance was received from Miss G. Reber, Mrs R. Bachmann and Mr K. Babl. This work was supported by Grant No. 3.5.68 of the Swiss National Science Foundation. REFERENCES 1. CLEMENTS,J. A., Amer. Rev. Resp. Dis. 101, 984 (1970). 2. DERMER,G. B., J. Ultrastruet. Res. 27, 88 (1969). 3. - ibid. 31, 229 (1970).
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4. ELBERS,P. F., VERVERGAERT,P. M. J. T. and DEMEL,R., J. Cell Biol. 24, 23 (1965). 5. FINLEY, T. N., PRATT, S. A., LADMANN, A. J., BREWEER, L. and McKAY, M. B., J. Lipid Res. 9, 357 (1968). 6. GIL, J., Arch. Int. Med. 127, 896 (1971). 7. GIL, J. and WEmEL, E. R., J. Ultrastruct. Res. 25, 331 (1968). 8. - - - - Resp. Physiol. 8, 13 (1969/70). 9. - J. Microsc. in press (1972). 10. KIKKAWA,Y., Anat. Rec. 167, 389 (1970). 11. SCARVELLI,E. M. The Surfactant System of the Lung. Lea and Febiger, Philadelphia, 1968. 12. STOECKENIUS,W., ,J. Cell Biol. 12, 221 (1962). 13. UNTERSEE,P., GIL, J. and WEIBEL,E. R., Resp. Physiol. 13, 171 (1971). 14. WEIBEL,E. R. and GIL, J., Resp. Physiol. 4, 42 (1968). 15. WEIBEL, E. R., KISTLER, G. and TOENDURY, G., Z. Zellforseh. Mikrosk. Anat. 69, 418 (1966).