Forensic Science International, ‘27 (1985) 81-92 Elsevier Scientific Publishers Ireland Ltd.

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STUDIES ON ASPHYXIA: ON THE CHANGES OF THE ALVEOLAR WALLS OF RATS IN THE HYPOXIC STATE

MASAHIKO MORITA, NORIKO TABATA and ATSUYO MAYA

Department of Legal Medicine, Sapporo Medical College, Nishi 17, Minami 1, Chuo-ku, Sapporo 060 (Japan)

(Received September 22,1983) (Revision received October 8, 1984) (Accepted December 7, 1984)

Summary

The morphological changes of the alveolar wall of adult rats in the hypoxic state were studied by light and electron microscopy.

The remarkable findings were the appearance of a large amount of lamellar, lattice- and thread-like structures together with a massive homogeneous substance on the surface of the alveoli which seemed to be closely connected with each other and with the surface of the cells lining the alveolus, especially in the 5%-group. The appearance of the above- mentioned structures with the homogeneous substance is considered to be the reaction of lung tissue to the decreased content of oxygen in the inhaled gas.

Key words: Asphyxia; Hypoxia; Alveolar wall; Surfactant

Introduction

Death from asphyxia occupies a considerably high percentage in the statis- tics of the causes of death. There is quite a lot of research on asphyxia but few of them use electron microscopy. Studies on the lung in hypoxic or anoxic state which should be regarded as the basic state of the asphyxia are especially rare. There are many ways to produce the asphyxic state, but the decreased amount of oxygen content in the inhaled air should be thought of as the basis of this kind of death.

This paper reports the results on the morphological changes of the alveolar wall, including epithelial cells, endothels of alveolar vessels and inner surface of the alveoli of rats in the hypoxic state.

Materials and Methods

Animal Twenty Wistar albino male rats weighing 200-300 g were used for this

study, 10 for the 5%group. 6 for the 10%group and the rest for control. At least two at each time point in each concentration of oxygen gas were

examined for this experiment.

0379~0738/85/$03.30 o 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Gas concentration Gas concentrations for the respiration of rats were 5% (5%~group) and

10% (lo%-group) oxygen with 95% and 90% nitrogen, respectively. Using a fluorometer, the gas was drawn into an observation box into

which the rats were put, and gas content was checked by gas chromato- graphy .

Perfusion technique The abdomens of the rats were opened to expose the inferior vena cava

through which flowed the fixation fluid of 4% formaldehyde and 1% glutaraldehyde in a 0.1 M cacodylate buffer solution with a pH of 7.4, from an irrigator hanging 80-100 cm higher than the plate on which rats were laid. The blood flowed out from the cut aperture distal to the point where the fixation fluid flowed in.

The abdomens of the rats of both 5%- and lo%-group were opened for the perfusion technique immediately after death which occurred without inter- ference about 60 minutes from the beginning of the experiment for the 5%- group.

A strike on the head with a blunt instrument was given to the rats of the lo%-group at the same time point as that at which the 5%-group died.

Preparation of tissue samples for microscopy Tissue samples were taken from the left lung and put into a chilled fixa-

tive, the same as used for the perfusion. Small pieces of the sample were processed according to the ordinary

method for electron microscopy. The ultra-thin sections for the electron microscope were stained doubly

with many1 acetate and lead citrate and then observed with a Hitachi H-500 type microscope.

Parts of the tissue blocks taken from the area near the small pieces used for the electron microscopy were embedded in paraffin and thinly sliced sections were examined by H & E, PAS, alcian blue and mucicarmine staining.

Results

Behavioral characteristics of rats until death 5%-group: All the rats of this group became still within 1 min after being

put into the observation box except for an occasional spasmodic movement such as jumping and scratching at the wall of the box. They dragged their hind legs as if paralysed when moving and could not stand in a normal position after 30 min from the beginning of the experiment.

They died after 1 h of the experiment, showing convulsions in all four legs followed by cheyne-stokes-like respiration.

1 O%-group: This group moved actively at the beginning of the experiment but took a crouching position with eyes half-open, and seemed to avoid

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useless movement except occasional scratching, streching and jumping at the wall of the observation box in the same way as those of the 5%group.

Morphological examination of the lung Gross appearances: In all the cases of both 5% and 10%group, the lung

was atelectatic and contained less air than the normal, but there were no differences in color.

Light microscopic examination: At higher magnification, there was found an extremely faint, pinkish homogeneous substance on the surface of alveoli of the 5%group in the H & E stained section, but this substance being negative by mucicarmine, alcian blue and PAS staining (Figs. lA, B).

But in the 10%group rats, the appearance of the homogeneous substance was not observed on the alveolar surface and the atelectatic changes were the only remarkable findings.

EEectron microscopic examination: In the normal rat lungs, there were found several great pneumocytes (Type II cell) which were releasing a linear or thread-like component of the content of the lamellar body trailing into the alveolar lumen (Figs. 2A,B).

Fig. 1. Light micrograph of lung of 5%~group rat. In both pictures, arrows and the arrow- head indicate the faintly stained homogenous substance. A: H & E staining, 800X ; B: PAS staining, 2000x. In all the following photomicrographs, the thick bar indicates 1 pm length except the thin bars in Figs. 8 and 11 which are 0.1 pm. Figures 3-11 relate to the 5%-group and Figs. 12 and 13 relate to the lO%-group.

Fig. 2. Electron micrograph of a normal rat lung. In both figures, arrows show the of a lamellar body trailing into the alveolar lumen. (A) 7200~ ; (B) 3500x .

content

In the 5%group, the great pneumocytes were often observed with their lamellar bodies fused together and thus the mitochondria were occasionally surrounded by large lamellar bodies in the cytoplasm. Several great pneumo- cytes had been destroyed and had discharged their cytoplasm with the fused lamellar bodies into the alveolar space (Fig. 3). Lamellar, except those mentioned above, and lattice-like structures were seen in the alveolar space with or without the homogeneous substance in various amounts, arranged randomly at an arbitrary width. They were observed attached or connected with each other.

In the 5%group rat lungs, the substance observed as homogeneous and faintly pink-coloured by light microscopy, was of medium electron density and filled the recesses of the alveoli (Figs. 4 and 5) or thinly covered the alveolar surface (Fig. 6). The inner surface of the substance was delineated clearly as a somewhat curved line (Fig. 4). At or near the centre of several alveoli, there was seen a round encircled space through which the air was thought to pass.

The lamellar, lattice-formed or tubular myelin and thread-like or membra- nous structures were observed frequently in the homogeneous substance, and the majority of lamellar and lattice-like structures were found at the surface of the substance near to the air passage. But, they were often observed very near to the cell surface as well, attached to the cell membrane (Fig. 7).

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Fig. 3. Pneumocyte II of the 5%-group, showing degeneration and destruction of the cytoplasm (asterisks). 8600x .

In the typically formed lattice-like structure (Fig. lo), there was occasion- ally found in each square a small medium electron-dense dot like a core (Fig. 11).

Another structural characteristic of the homogeneous substance was that it seemed to have some close connection with the lamellar or lattice-like structure (Figs. 8 and 9).

The density of the cytoplasm of the pneumocytes lining the alveoli diminished to where it was pale and thus was ascertained with ease by the contrast to the medium density of both the homogeneous substance and the basement membrane.

The membrane of the cells lining the alveoli showed bleb formation, especially in the 5%-group (Figs. 4, 5 and 8).

The width of the lamellae and the lattice-like structure was typically lo-30 nm and the interval between each was typically 50-70 nm between the centre of each width.

In the 10%group rat lungs, the substance which was observed on the alveolar surface of 5%group rats was occasionally found but a majority of them was not dense but grayish debris-like (Fig. 13).

Lattice-like structure was also found in a groove in a rare case (Fig. 12).

Fig. 4. Lattice-like structures are observed at the surface of a large amount of the ele dense homogeneous substance. The air is thought to pass through the lumen. 8600x

ctron-

Fig. 5. The substance fills a recess of the alveolar lining, 8700X. In both Figs. 4 and 5, the density of the cytoplasm of cells lining the alveoli has markedly decreased.

Fig. 6. The substance thinly covers the cells lining the alveolus. 8600X.

Fig. 7. Lattice-like structures are attached to the cell surface. 35,000X.

Fig. 8. A highly magnification of the homogeneous substance and the lattice-like struc- tures. A part of one structure is in the substance near to the air passage (upper right) and another is near to the cell surface (left).

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Fig. 9. Grayish debris-like substance seems to have a close connection with the lattice-like and/or membranous structures. 8600x .

Fig. 10. An electron-dense dot can be seen in each square of the lattice-like structure. 16,000x.

Fig. 11. A higher magnification of Fig. 10. The diamond-shaped dots in each square are easily perceived. 45,000~.

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Fig. 12. Lattice-like structure is occasionally observed in a groove or recess. 3600~ .

Fig. 13. Grayish debris-like substance is scarcely observed in the alveolar lumen of lO%- group rats. AL: alveolar lumen, CL: capillary lumen. 3000x.

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Discussion

Many articles [l-28] have been published on the substance on the surface of the cells lining the alveolus, which is called “surfactant”.

The lamellar and lattice-like structures are the main morphological compo- nents of the surfactant which is thought to be the discharge from the great pneumocyte [l-5].

The surfactant is closely related to respiration [6-121, that is the intake of oxygen through the surface of the cells lining the alveoli into the blood to the erythrocytes.

In the forensic field, there are many cases in which cause of death is con- sidered to be by asphyxia, whatever the mode of death should be, and almost all cases are resolved by macroscopic observations of the outside of the body and the findings in the organs.

The authors expected to find the morphological changes of cell compo- nents attributable to oxygen deficiency. But contrarily, the morphological changes were not so remarkable except in the destruction of the cytoplasm of the great pneumocytes, releasing cell organelles together with a large amount of the fused lamellar bodies into the alveolar lumen, and the bleb formation of both the cells lining the alveoli and endothels of the alveolar vessels.

But the unexpected findings in this experiment were the appearance of the lamellar and/or lattice-like structures and tubular myelin with a large amount of the homogeneous electron-dense substance manifest especially in the 5%group.

Whereas, in the 10%group, the changes of cellular components were not severe and the appearance of the homogeneous substance, like that of the 5%group, was not descernible but the lattice-like structure was observed occasionally in a small amount.

Many studies [13-151 have revealed that the lamellar bodies convert to the lattice-like structures after the discharge from the great pneumocytes, and then to the monolayer of lecithin which covers the surface of the alveoli.

Some investigators [16-201 have reported that the surfactant in respira- tory distress disease is deficient and others [21] on the relationship to the environmental gaseous conditions. But, as the surfactant is regarded as the important factor in taking oxygen into the body at the alveoli in the lungs, an increase in theamount of the surfactant in the hypoxic state should rather be expected.

The appearance of a large amount of lamellar, lattice- and thread-like structures with massive homogeneous substance in the alveoli is easily ascer- tainable and seems to be attributable to the reaction of lung tissue to the hypoxic state.

Many investigators [22--281 have studied the biochemical features of the surfactant, and the authors think the analysis of the alveolar lavage of rats obtained under the same conditions as in this experiment is necessary to

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confirm biochemically the substance which appeared in the alveoli to be the “surfactant”.

Acknowledgement

The authors wish to express their cordial thanks to Professor M. Mori of the Department of Pathology and to Professor T. Akino of the Department of Biochemistry for their helpful advice and suggestions on the surfactant, from each of their viewpoints, and to Miss Emiko Suzuki for her technical assistance in preparing the ultra-thin sections for the electron microscopy, and to Mr. Seiji Ohtani for his assistance in preparing sections for the light microscopy.

References

1 E.R. Weibel, G.S. Kistler and G. Tondry, A stereologic electron microscope study of “tubular myelin figures” in alveolar fluids of rat lungs. 2. Zellforsch. Mikrosk. Anat., 69 (1966) 418-427.

2 M.C. Williams, Conversion of lamellar body membranes into tubular myelin in alveoli of fetal rat lungs. J. Cell Biol., 72 (1977) 260-277.

3 K.H. O’Hare and M.N. Sheridan, Electron microscopic observations on the morpho- genesis of the albino rat lung, with special reference to pulmonary epithlial cells. Am. J. Anat., 127 (1970) 181-206.

4 S.P. Sorokin, A morphologic and cytochemical study on the great alveolar cell. J. Histochem. Cytochem., 14 (1967) 884-897.

5 C.N. Sun, Lattice structures and osmiophilic bodies in the developing respiratory tissue of rats. J. Ultrastruct., Res., 15 (1966) 380-388.

6 E.R. Weibel, Morphological basis of alveolar-capillary gas exchange. Physiol. Reu., 53 (1973) 419-495.

7 J.A. Clements, J. Nellenbogen and H.J. Trahan, Pulmonary surfactant and evolution of the lungs. Science, 169 (1970) 603.

8 M. Wittner and R.M. Rosenbaum, Pathophysiology of pulmonary oxygen toxicity. In I.W. Brown, Jr. and B.G. Cox (eds.), Proc. Third Int. Conf Hyperbaric Med., NAS/NRC Publ. 1404, Washington, D.C., 1966, p. 179.

9 T.E. Morgan, T.N. Finley, G.L. Huber and H. Fialkow, Alterations in pulmonary surface active lipids during exposure to increased oxygen tension. J. Clin. Invest., 44 (1965) 1737.

10 D.L. Beckman and H.S. Weiss, Hyperoxia compared to surfactant washout on pul- monary compliance in rats. J. Appl. Physiol., 26 (1969) 700.

11 H. Gilder and C.K. McSherry, Mechanisms of oxygen inhibition of pulmonary sur- factant synthesis. Surgery, 76 (1974) 72.

12 G. Polgar, W. Antagnoli, L.W. Ferrigan, E.A. Martin and W.P. Gregg, The effect of chronic exposure to 100% oxygen in newborn mice. Am. J. Med. Sci., 252 (1966) 580.

13 J. Goerke, Lung surfactant. Biochim. Biophys. Acta, 344 (1974) 241-261. 14 J. Gill and O.K. Reiss, Isolation and characterization of lamellar bodies and tubular

myelin from rat lung homogenates. J. Cell Biol., 58 (1973) 152-171. 15 G.B. Dermer, The fixation of pulmonary surfactant for electron microscopy. II.

Transport of surfactant through the air-blood barrier. J. Ultrastruct., 31 (1970) 229.

16 J.A. Clements, Surfactant in pulmonary disease. N. Engl. J. Med., 274 (1965) 1336.

92

17 F.H. Adams, T. Fujiwara, C.C. Emmanouilides and N. Raiha, Lung phospholipids of human fetuses and infants with and without hyaline membrane disease. J. Pediatr., 77 (1970) 333.

18 R. Schober, K.G. Bensch, J.C. Kosek and W.H. Northway, On the origin of the mem- branous intraalveolar material in pulmonary alveolar proteinosis. Exp. Mo2. Pathol., 21 (1974) 246.

19 P.M. Farrell and M.E. Avery, Hyaline membrane disease. Am. Reu. Respir. Dis., 111 (1975) 657.

20 T. Akino, G. Okano and K. Ohno, Alveolar phospholipids in pulmonary alveolar proteinosis. Tohohu J. Exp. Med., 126 (1978) 51.

21 Y. Alarie, M.A. Choby and W.E. Poel, Alveolar instability following administration of fluorocarbon. Toxicol. Appl. Pharmacol., 22 (1972) 306.

22 D. Newman and A. Naimark, Palmitate-‘C uptake by rat lung: effect of altered gas tensions. Am. J. Physiol., 214 (1968) 305.

23 T.E. Morgan, Biosynthesis of pulmonary surface-active lipid. Arch. Irztern. Med., 127 (1971) 401.

24 J.A. Clements, Comparative lipid chemistry of lungs, Arch, Intern. Med., 127 (1971) 387.

25 M. Abe and T. Akino, The conversion of lysolecithin th lecithin in supernatant fractions of various rat tissues: on the distribution of the so-called Marinettl’s enzyme. Tohohu J. Exp. Med., 110 (1973) 167.

26 G. Okano and T. Akino, Changes in the structual and metabolic heterogeneity of phosphatidylcholines in the developing rat lung. Biochim. Biophys. Acta, 528 (1978) 373.

27 G. Okano, T. Kawamoto and T. Akino, Comparison of molecular structure of glycero- lipids in rat lung. Biochim. Biophys. Acta, 528 (1978) 385.

28 R.D. Mavis, J.N. Finkelstein and BP. Hall, Pulmonary surfactant synthesis, A highly active microsomal phosphatidate phosphohydrolase in the lung. J. Lipid Res., 19 (1978) 467.