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Pulmonary Changes After Temporary Ischemia of Varying Duration
Beitr. Path. Bd. 149,270-279 (1973)
Senckenbergisches Zentrum der Pathologie der Universitat Frankfurt am Main, Abteilung I (Leiter: Prof. Dr. W. ROTTER)
Pulmonary Changes After Temporary Ischemia of Varying Duration Lungenveranderungen nach abgestufter temporarer Ischamie M.
AMTHOR
With 4 Figures' Received January 23, 1973 . Accepted April 5, 1973
Summary In 84 white female Wistar rats, the upper (or middle) lobe of the right lung was subjected to temporary ischemia of 5, 10, 15, 30, 90, 300 and 360 minutes. Paraplast®embedded sections were examined I, 2, 3, 5, 7, and 10 days after the procedure. We found an intraalveolar extravasate lasting for 3 days. The protein and erythrocyte content of the extravasate depends upon the duration of the preceding temporary ischemia. The appearance of the extravasate is considered to be due to postischemic insufficiency of the terminal vascular system with an increase in permeability according to the duration of the ischemia. The extravasate phase is followed by an atelectatic phase, the duration of which correlates well with the duration of the preceding temporary ischemia. In our opinion this is due to a destruction of the surfactant by the intraalveolar extravasate followed by delayed replacement of the surfactant because of a postischemic insufficiency of the surfactant-producing pneumocytes II. Following temporary ischemia, necroses can be observed as follows: interalveolar septa (10 - I 5 min.), bronchial epithelium (30 -90 min.), smooth muscles (300 - 360 min.). The time of temporary ischemia after which necroses of the septa occurred was not only extremely short but also equal in length for all component cell types. Consequently, the assumption is made that a viscosity increase following a hemoconcentration caused by an extravasation of plasma led to a circulatory restriction, or stasis, which in turn prolonged the state of ischemia. After reinflation, enlargement of the alveolar air spaces was observed. This appears to be due to necrosis in a number of alveolar walls.
Experiments dealing with temporary ischemia can provide essential information about tissue susceptibility to lack of oxygen and energy supply (for references, see ROTTER, 1959; ZIMMERMANN, 1959)' One finds, however,
Pulmonary Changes After Temporary Ischemia' 27 1
only sporadic work related to temporary ischemia of the lung, although this would appear to be especially important in operations involving ischemia and transplantations. These previous investigations have dealt with monkeys, dogs and sheep (BLADES et aI., 1953; BORRIE and LICHTER, 1964; CONNAUGHTON et aI., 1962; DAVIS et aI., 1952; EISENBACH, 1968; HUBER et aI., 1967). Smaller animals, however, in spite of a few pecularities of the pulmonary anatomy (McLAUGHLIN et aI., 1966) would be time saving and offer technical advantages, thus allowing larger series which can compensate for the considerable individual differences in reaction to be expected. However, a special device for insufflation anesthesia of small animals is necessary (AMTHOR, 1972). The following work was carried out to investigate the specific reversible and irreversible pulmonary changes following temporary ischemia of varying duration.
Material and Methods Eighty-four conventional white, female Wi star rats with an average weight of 2II g (167 g - 250 g) were divided into 7 groups, each consisting of 12 animals in which the upper lobe (in a few cases the middle lobe) of the right lung was subjected to temporary ischemia of 5,10,15,30,90,300, and 360 minutes. All animals were given food (pellets) and water ad libitum. The operations were performed under insufflation anesthesia with a mixture of ether and oxygen (barbiturates and chloral hydrate had proved unsuitable for our purposes). After the thorax was opened at the 4 th or 5 th intercostal space, the upper lobe of the right lung was pinched off at the hilum with a small clamp. In animals with a very small upper lobe, the middle lobe was used. (Animals with spontaneous atelectasis or pneumonia were not used.) During the temporary ischemia, the open thorax was covered with a dressing moistened with a warm solution of isotonic sodium chloride. After completion of ischemia, the thorax was closed with one Z-type suture with metal pins adapting the skin. A draining tube was not needed. Periods of ischemia of 90, 300, and 360 minutes were achieved by operating in two sessions. For this purpose, we used a small household rubber band which had been pulled through a 3 mm long, very narrow glass tube, thus forming a loop on each end. One loop was laid around the lobe, then the rubber band was stretched by pulling on the free loop and fixed in this position by a small metal pin. Complete circulation arrest to the operated lobe was demonstrated in two operated animals by intracardial injections of lissamin-green (10% solution, I ml/ 100 g body weight) and in one animal by intracardial injection of acridine-orange (0,5 mg /100 g body weight). After installation of the tourniquet, the thorax and skin were temporarily closed. Shortly before ischemia was discontinued, a rethoracotomy was performed and the tourniquet removed by cutting the rubber band below the pin. Then the thorax and skin were closed. All animals received chloramphenicol (Catilan®-Hoechst 10 mg/lOo g body weight) on the day of the operation and on the 2nd, 3 rd, 5 th, 7th, and 9th postoperative day to prevent pneumonia which had been observed in preliminary experiments.
27 2 .
M. AM THOR
Two animals out of each group were sacrificed on the 1St, 2 nd, 3rd, 5 th, 7 th and loth day after the operation by decapitation under anesthesia while preserving the trachea. The right upper lobe (or middle lobe if used), the remaining right lung, and the left lung were removed separately. Fixation was accomplished by 4% formalin. The tissues were passed through graded alcohol, methylbenzoate and benzene and embedded in Paraplast®. Staining: hematoxylin and eosin (ROMEIS, 1968); connective tissue according to Domagk (ROMErs, 1968); elastic fibers according to Hart (BURCK, 1969); fibrin stain according to Ladewig (LADEWIG, 1938) and Mallory (PREECE, 1959); acid mucopolysaccharides (Chesa-Stain, BURCK, 1969), and PAS-reaction (ROMEIS, 1968). Controls:
The remaining lobes of the right lung and the left lung of the experimental animals were examined regularly. In addition, six animals were subjected to a sham operation with thoracotomy and artificial respiration of 35 minutes duration. Three animals were sacrificed on the first postoperative day, and three on the second day. Embedding and staining procedures were the same as in the other animals.
Results After temporary ischemia of varying duration quite uniform changes are found within the first days. The perivascular connective tissue of the great hilar vessels is interspersed with an extravasate enclosing several sometimes extremely dilated lymphatic vessels. Also, the interalveolar septa are broadened by an extravasate. The alveolar epithelial cells exhibit clear and finely vacuolated cytoplasm. The alveoli, like the bronchioles and the bronchi, are expanded containing a proteinaceous and hemorrhagic extravasate. The interstitial extravasate extending into the interalveolar septa and around the great vessels and the dilatation of the lymphatic vessels start to recede slowly after the 2 nd day; on the 5th day they can hardly be demonstrated, while they have disappeared completely on the 7th day. The intraalveolar extravasate vanishes fairly rapidly after the 2nd day. On the 3rd day it is found only in a few alveoli, while it has disappeared completely on the 5th day. However, while it disappeares completely without leaving any residues after a 5-minute period of ischemia, after interruption of the circulation for 10 minutes and longer great brownish red bar- or needle-type crystals as well as alveolar casts rich in fibrin and consisting of amorphous eosinophilic material sometimes including a few alveolar cells are seen after the 1st day. The alveoli not containing any extravasate are collapsed; the intraalveolar extravasate is immediately followed by atelectasis (Fig. I). After the 3rd to 5th day, the parenchyma expands increasingly, apparently depending upon the duration of the preceding temporary ischemia. After a 5-minute ischemia the whole alveolar parenchyma and the bronchi are com-
Puimonary Changes After Temporary Ischemia' 273 Slt1 day
'0
intraalveolar exlravasate
0
::'" .E c'" ~ u
E .~
emphysema
intt'rstltial extravasate dllatallon of Iymphalic vessels
I
inlraalveolar ext ravasate
m
sema
ateleclasls :
interstitial extrayasate dilatation of lymphatic vessels
intraalveolar extravasate
atelectasis
interstillel extravasate dilatation of lymphatic vessels
Fig.
1.
Sequence of the postischemic lesions.
number of animals
24
septa mloralveolallg bronchial epllhellum
12
smooth muscles
D
8
t isch 10' ,15' n= 2L
tisch. 30',90' n=2L
number of cases With thrombosIs
I iseh
300',360' n=24
Fig, 2. Number of cases with wide-range necrosis of the septa and of bronchial epithelium and smooth muscles. Bottom: cases with total necrosis following occlusive thrombosis of a large branch of the pulmonary artery.
274 . M. AMTHOR
Fig. 3. Extreme enlargement of the alveolar air spaces in the upper right lobe (right). The left lung of the same animal shows an inconspicuous parenchyma (left). 5 minutes of ischemia, 5 th day after the operation. Hematoxylin and eosin; X 85.
pletely expanded at this time. After 10, 15, and 30 minutes of ischemia, however, the lobe expands only partially; the findings on the loth day are comparable with those on the 5th day after 5 minutes of ischemia (Fig. 1). Finally, after an ischemia of at least 90 minutes, the whole lobe remains atelectatic up to the loth day. During the reexpansion of the lobe, a considerable enlargement of the alveolar parenchyma is frequently noticed (Fig. 3). The cause probably lies in several shortened and necrotic interalveolar septa. Wide-range necrosis of the lung tissues is not found, however, until after an ischemia of more than 5 minutes. The degree of necrosis increases with the duration of ischemia. Accordingly, if considerable individual differences in ischemic tolerance are taken into account, a conspicuous grading of the time of ischemia after which necrosis occured can be seen between interalveolar septa, the epithelium of the bronchi, and the smooth muscles of the vessels and the bronchi (Fig. z.). Thus, after a temporary ischemia of 10 and 15 minutes, the necroses are limited to the interalveolar septa. After 30 and 90 minutes, the
Pulmonary Changes After Temporary Ischemia'
.
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,
••
""I
• •
•
•
•
. •,
'
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·I
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~·
. I!
Fig. 4. Necrosis of the right upper lobe. Intact subpleural zone with leukocytic infiltration. 360 minutes of ischemia, 3rd day after operation. Hematoxylin and eosin; X 160.
epithelium of the bronchi is necrotic; after 300 minutes there are a few necrotic vessels; and after 360 minutes, all vessels and smooth muscles of the bronchi are disaggregated to an amorphous material. However, a narrow subpleural zone always remains intact, even in those cases in which an occluding thrombosis of a large branch of the pulmonary artery has caused a permanent ischemia followed by total necrosis (Fig. 4). Controls: During the first days, the remaining lobes of the right side not subjected to ischemia show a slight perivascular extravasate around the hilum, dilated lymphatic vessels and a few dilated alveoli, especially in those cases in which the upper (or middle) lobe had shown long-lasting atelectasis. Occasionally a few atelectatic alveoli are seen. Except for a few collapsed alveoli, the left lung appears normal. In the lungs of the sham-operated animals, one finds only a few collapsed alveoli on the 1 st day; there are no pathologic findings at all on the znd day.
276 . M. AMTROR
Discussion Postischemic extravasate: In our opinion the postischemic extravasate is due to an insufficiency of the terminal vascular system (ROTTER, 1959). Its protein content increases with the duration of ischemia. Accordingly, we found different kinds of extravasates varying from an edema, poor in protein, to protein-rich exsudates. In all cases we found an interstitial and intraalveolar extravasate in the upper (or middle) right lobe on the 1 st day. We consider the alveolar casts, which appeared no earlier than after a 10-minute period of ischemia, as proof for the increasing protein content after longerlasting ischemic periods. The severity of the postischemic lesion of the vessel walls can also be demonstrated by the erythrocytic diapedesis (ROTTER, 1959). From the 2nd day on, after a temporary ischemia of 10 minutes and more, we found crystals which are most likely oxyhemoglobin or related compounds (FUCHS, 1966) and which we consider a sign of a massive erythrocytic extravasation. It seems noteworthy that extensive extravasation in the rat can already be seen after 5-minute ischemia, while in dogs (EISENBACH, 1968) and in monkeys (DAVIS, 1952) an extravasate was not observed under equal or comparable conditions not before 2 or 3 hours. These differences may be due to, among other factors, a higher vulnerability of the rat to ischemia. It is well known that the basal metabolic rate (KLEIBER, 1947) and with it the vulnerability to ischemia (OETLIKER, 1961) decreases in proportion to the increasing weight of the animal. Atelectasis: After resorption of the intraalveolar extravasation an atelectasis develops from the 3 rd day on, and regresses after a 5-minute period of ischemia until the 7th day. After an ischemia of ro to 30 minutes, reinflation was incomplete on the loth day; after an ischemia of 90 minutes and more, no reinflation could be seen up to the roth day. The tendency of the atelectatic lobe to reinflate thus shows a conspicuous relation to the duration of the preceding ischemic period. A spontaneous atelectasis, which is not uncommon in the albino rat, and an atelectasis caused by the operative technique, namely by a pneumothorax, can be excluded with great probability. Opposing the assumption of a spontaneous atelectasis is the localization to only one, the right side; opposing both assumptions is the tendency of the lungs to reinflate, indicating a distinct relationship to the duration of the preceding ischemia. Oxygen-poisoning due to artificial respiration for 35 minutes can also be excluded, since ultrastructural changes have been demonstrated at the earliest after 24 hours of breathing pure oxygen (KISTLER et aI., 1967). We find it more likely that the intraalveolar extravasation causes the atelectasis by destroying the intraalveolar lining (surfactant, antiatelecta-
Pulmonary Changes After Temporary Ischemia' 277
tic factor; GIESE KING, 1971; WEIBEL, 1968) and thus inducing the alveolar collapse after resorption of the extravasate. The regeneration of the surfactant, the production of which is supposed to take place in the pneumocytes II (GIESE KING, 1971; HATASA and NAKAMURA, 1965, KIKKAWA et aI., 1965), obviously starts with a different delay depending on the duration of the preceding temporary ischemia. This could be, as in the transitory tubular insufficiency of the kidney, caused by a variable postischemic insufficiency of the pneumocytes II. In dogs (EISENBACH, 1968) and in monkeys (DAVIS, 1952) atelectases were also observed after temporary ischemia. However, EISENBACH attributed them to a postoperative insufficient respiration, while DAVIS did not give an explanation. Our hypothesis is supported by the observation of atelectasis (HORINE and WARNER, 1934; HUBER and EDMUNDS jr., 1967) and of a decrease of surfactant activity and an increase of surface tension (GIAMMONA et aI., 1966) after ligature of the pulmonary artery. It is also supported by the observation of changes of the osmiophilic lamellated bodies within the pneumocytes II after ligature of the pulmonary artery (HUBER and EDMUNDS jr., 1967) and after breathing CO 2 (SCHAEFER et al., 1964). The latter are related to the acidosis. A metabolic acidosis, as well as the hypoxidosis which is closely connected to it, is one of the main sequences of ischemia. Necrosis: After temporary ischemia necroses of the interalveolar septa, the bronchial epithelium and the smooth muscles of the bronchi and the vessels are seen. As in dogs (EISENBACH, 1968), the septa perish first. This observation was striking in two respects. First, the conspicuously short duration of temporary ischemia after which a necrosis of the septa can be found, the vascular and connective tissue usually has a low vulnerability to ischemia (WALTHER, 1963); secondly the septa become totally necrotic in spite of the structural differences of the constituting cells. Both observations are probably related to hemodynamic factors prolonging the ischemia. We have not found hyaline thrombi; also, vasoconstriction in the sense of the reflex of v. EULER and LILJESTRAND (v. EULER and LILJESTRAND, 1946) does not seem very probable to us to be the cause of necrosis. Further experiments in progress rather suggest that after ischemia the blood circulation is restricted locally almost to the point of stasis because the viscosity of the blood is increased by extravasation of the plasma. Emplrysema: After reinflation of the lobe, a conspicuous dilatation of the alveolar air spaces can be noticed in areas with several necrotic interalveolar septa. Whether this condition is considered a true emphysema, as it seems, in the sense of the definition of the Report of a Committee of the WHO (abnormal increase in the size of air spaces distal to the terminal bronchiole,
with destructive changes in their walls, WHO 1961), or just an extreme 19 Beitr. Path. Bd. 149
278 . M. AMTHOR
overinflation, or an emphysema due to overinflation (HARTUNG, 1964) depends on the significance one attaches to the necrotic interalveolar septa. In all cases a survey of more than 10 days will be needed in order to decide on the chronicity of the process which is necessary in an emphysema in the sense of the WHO. An emphysema induced by ischemia has been reported by EISENBACH (1968) and STRAWBRIDGE (1960); and in the naturally ocurring pulmonary emphysema of humans and rabbits, the ischemia seems to be of some importance as a causal factor (MARTIN and BOATMAN, 1965).
Zusammenfassung Bei 84 weifien, weiblichen Wistar-Ratten wurde der Ober(oder Mittel-)lappen der rechten Lunge einer temporaren Ischamie von 5, 10, 15, 30, 90, 300 und 360 Minuten ausgesetzt. In Paraplast® eingebettete Praparate des rechten Ober(bzw. Mittel-)lappens, der tibrigen rechten Lunge sowie der linken Lunge wurden nach I, 2, 3, 5, 7 und 10 Tagen untersucht. Wir fanden ein tiber 3 Tage andauerndes intraalveohires Extravasat im rechten Ober(bzw. Mittel-)lappen, welches in seinem Gehalt an Eiweifi und Erythrocyten eine Abhangigkeit von der Lange der Ischamie erkennen lien. Dieses Extravasat war offenbar Folge einer postischamischen Insuffizienz der terminalen Strombahn, verbunden mit einem von der Dauer der temporaren Ischamie abhangigen Anstieg der Gefafidurchlassigkeit. Dem Extravasat folgte eine Atelektase, deren Dauer his zur Wiederentfaltung eine auffallende Beziehung zu der vorausgegangenen Ischamie zeigte. Diese Beobachtung wird von uns so gedeutet, dafi es nach einer Zerstorung des alveolaren Oberflachenfilms durch das intraalveolare Extravasat zu einem verzogerten Ersatz des Films infolge einer postischamischen Insuffizienz der den Film produzierenden Pneumozyten II kommt. Nach tempo rarer Ischamie fanden sich Nekrosen der Alveolarsepten (10-15 Min.), der Bronchusepithelien (30-90 Min.) und der glatten Muskulatur (300-360 Min.). Die Wiederbelebungszeit der Septen war hierbei nicht nur sehr kurz, sondern auch fur aIle am Septumaufbau beteiligten Zelltypen einheitlich. Es wird deshalb angenommen, dafi es infolge einer Viskositatserhohung nach einer durch Plasmaextravasation bedingten Hamokonzentration zu einer Verminderung oder einem Stillstand der Durchblutung gekommen war, wodurch die Ischamie verlangert wurde. Nach der Wiederentfaltung des Lungenlappens wurde eine Erweiterung der Alveolen-tragenden Luftraume beobachtet, die Folge von Nekrosen einzelner Alveolarsepten zu sein scheint. Acknowledgments The author is grateful to Mrs. R. L. CASEY, lecturer at Ventura College, for her kind linguistic assistance, and to Mrs. W. ZELLER for her excellent technical work.
References AMTHOR, M.: Beitr. Path. I47, 401 (1972) BLADES, B., PIERPONT, H. c., SAMADI, A., and HILL, R. P. Surg. Forum 4, 255 (1953) BORRIE, ]. and LICHTER, I.: Thorax I9, 383 (1964)
Pulmonary Changes After Temporary Ischemia' 279 BURCK, H.-C.: Histologische Technik, 2nd ed. Thieme Verlag, Stuttgart (1969) CONNAUGHTON, P. ]., BAHUTH,]. ]., and LEWIS, F.].: Dis. Chest 41, 404 (1962) DAVIS, H. A., GORDON, W. B., HAYES, E. W., and WASLEY, M. T.: Arch. Surg. 64,464 (195 2) EISENBACH, J.: Untersuchungen tiber die temporare Ischamie von Leber und Lunge bei Normothermie in Hinblick auf chirurgische Fragestellungen. Habilitationsschrift, Frankfurt/Main (1968) FUCHS, U.: Path. Microbiol. 29, 8 (1966) GIAMMONA, S. T., MANDELBAUM, ]., Foy, ]., and BONDURANT, S.: Circul. Res. 18, 683 (1966) Supp. I GIESE KING, R.: Verh.Dtsch. Ges. Path. ff, 22 (1971) HARTUNG, W.: Lungenemphysem, Pathologie und Klinik in Einzeldarstellungen. Vol. XIV. Springer-Verlag, Berlin - Gottingen - Heidelberg (1964) HATASA, K. and NAKAMURA, T.: Z. Zellforsch. 68,266 (1965) HORINE, C. and WARNER, Arch. Surg. 28,137 (1934) HUBER, G. L. and EDMUNDS JR., H.: ]. appl. Phys. 22, 1002 (1967) KIKKAWA, Y., MOTOYAMA, E. K., and COOK, C. D.: Amer. J. Path. 47, 887 (1965) KISTLER, C. S., CALDWELL, P. R. B., and WEIBEL, E. R.:]. Cell. BioI. 33, 605 (1967) KLEIBER, M.: Physiol. Rev. 27,5 I I (1947) LADEWIG, P.: Z. mikroskop. Technik f f, 21 5 (193 8) MARTIN, H. B. and BOATMAN, E. S.: Med. thorac. 22, 171 (1965) McLAUGHLIN, R. F., TYLER, W. S., and CANADA, R. 0.: Amer. Rev. resp. Dis. 94, 380 (1966) OETLIKER, 0.: Virchows Arch. path. Anat. 334, 56 (1961) PREECE, A.: A Manual for histologic Technicians. Little, Brown and Comp., BostonToronto (1959) ROMEIS, B.: Mikroskopische Technik, 16th ed. Oldenbourg-Verlag, Mtinchen-Wien (1968) ROTTER, WG.: in Angiologica. Editor: M. RATSCHOW. Thieme Verlag, Stuttgart (1959) SCHAEFER, K. E., AVERY, M. E., and BENSCH, K.:]. din. Invest. 43, 2080 (1964) STRAWBRIDGE, H. T. G.: Amer.]. Path. 37, 391 (1960) V. EULER, U. S. and LILJESTRAND, G.: Acta phys. Scand. 12, 302 (1946) WALTHER, D.: Verh. Dtsch. Ges. Path. 47,214 (1963) WEIBEL, E. R. and GIL,].: Resp. Physiol. 4,42 (1968) World Health Organization, Chronic Cor Pulmonale, Technical Report Series No. 213, Report of an Expert Committee, Geneva (1961) ZIMMERMANN, H.: Frankf. Z. Path. 69,477 (1959)
c.:
Doz. Dr. M. AMTHOR, Senckenbergisches Zentrum der Pathologie, D-6 Frankfurt am Main, Theodor-Stern-Kai 7, Federal Republic of Germany
Senckenbergisches Zentrum der Pathologie der Universitat Frankfurt am Main, Abteilung I (Leiter: Prof. Dr. W. ROTTER)
Pulmonary Changes After Temporary Ischemia of Varying Duration Lungenveranderungen nach abgestufter temporarer Ischamie M.
AMTHOR
With 4 Figures' Received January 23, 1973 . Accepted April 5, 1973
Summary In 84 white female Wistar rats, the upper (or middle) lobe of the right lung was subjected to temporary ischemia of 5, 10, 15, 30, 90, 300 and 360 minutes. Paraplast®embedded sections were examined I, 2, 3, 5, 7, and 10 days after the procedure. We found an intraalveolar extravasate lasting for 3 days. The protein and erythrocyte content of the extravasate depends upon the duration of the preceding temporary ischemia. The appearance of the extravasate is considered to be due to postischemic insufficiency of the terminal vascular system with an increase in permeability according to the duration of the ischemia. The extravasate phase is followed by an atelectatic phase, the duration of which correlates well with the duration of the preceding temporary ischemia. In our opinion this is due to a destruction of the surfactant by the intraalveolar extravasate followed by delayed replacement of the surfactant because of a postischemic insufficiency of the surfactant-producing pneumocytes II. Following temporary ischemia, necroses can be observed as follows: interalveolar septa (10 - I 5 min.), bronchial epithelium (30 -90 min.), smooth muscles (300 - 360 min.). The time of temporary ischemia after which necroses of the septa occurred was not only extremely short but also equal in length for all component cell types. Consequently, the assumption is made that a viscosity increase following a hemoconcentration caused by an extravasation of plasma led to a circulatory restriction, or stasis, which in turn prolonged the state of ischemia. After reinflation, enlargement of the alveolar air spaces was observed. This appears to be due to necrosis in a number of alveolar walls.
Experiments dealing with temporary ischemia can provide essential information about tissue susceptibility to lack of oxygen and energy supply (for references, see ROTTER, 1959; ZIMMERMANN, 1959)' One finds, however,
Pulmonary Changes After Temporary Ischemia' 27 1
only sporadic work related to temporary ischemia of the lung, although this would appear to be especially important in operations involving ischemia and transplantations. These previous investigations have dealt with monkeys, dogs and sheep (BLADES et aI., 1953; BORRIE and LICHTER, 1964; CONNAUGHTON et aI., 1962; DAVIS et aI., 1952; EISENBACH, 1968; HUBER et aI., 1967). Smaller animals, however, in spite of a few pecularities of the pulmonary anatomy (McLAUGHLIN et aI., 1966) would be time saving and offer technical advantages, thus allowing larger series which can compensate for the considerable individual differences in reaction to be expected. However, a special device for insufflation anesthesia of small animals is necessary (AMTHOR, 1972). The following work was carried out to investigate the specific reversible and irreversible pulmonary changes following temporary ischemia of varying duration.
Material and Methods Eighty-four conventional white, female Wi star rats with an average weight of 2II g (167 g - 250 g) were divided into 7 groups, each consisting of 12 animals in which the upper lobe (in a few cases the middle lobe) of the right lung was subjected to temporary ischemia of 5,10,15,30,90,300, and 360 minutes. All animals were given food (pellets) and water ad libitum. The operations were performed under insufflation anesthesia with a mixture of ether and oxygen (barbiturates and chloral hydrate had proved unsuitable for our purposes). After the thorax was opened at the 4 th or 5 th intercostal space, the upper lobe of the right lung was pinched off at the hilum with a small clamp. In animals with a very small upper lobe, the middle lobe was used. (Animals with spontaneous atelectasis or pneumonia were not used.) During the temporary ischemia, the open thorax was covered with a dressing moistened with a warm solution of isotonic sodium chloride. After completion of ischemia, the thorax was closed with one Z-type suture with metal pins adapting the skin. A draining tube was not needed. Periods of ischemia of 90, 300, and 360 minutes were achieved by operating in two sessions. For this purpose, we used a small household rubber band which had been pulled through a 3 mm long, very narrow glass tube, thus forming a loop on each end. One loop was laid around the lobe, then the rubber band was stretched by pulling on the free loop and fixed in this position by a small metal pin. Complete circulation arrest to the operated lobe was demonstrated in two operated animals by intracardial injections of lissamin-green (10% solution, I ml/ 100 g body weight) and in one animal by intracardial injection of acridine-orange (0,5 mg /100 g body weight). After installation of the tourniquet, the thorax and skin were temporarily closed. Shortly before ischemia was discontinued, a rethoracotomy was performed and the tourniquet removed by cutting the rubber band below the pin. Then the thorax and skin were closed. All animals received chloramphenicol (Catilan®-Hoechst 10 mg/lOo g body weight) on the day of the operation and on the 2nd, 3 rd, 5 th, 7th, and 9th postoperative day to prevent pneumonia which had been observed in preliminary experiments.
27 2 .
M. AM THOR
Two animals out of each group were sacrificed on the 1St, 2 nd, 3rd, 5 th, 7 th and loth day after the operation by decapitation under anesthesia while preserving the trachea. The right upper lobe (or middle lobe if used), the remaining right lung, and the left lung were removed separately. Fixation was accomplished by 4% formalin. The tissues were passed through graded alcohol, methylbenzoate and benzene and embedded in Paraplast®. Staining: hematoxylin and eosin (ROMEIS, 1968); connective tissue according to Domagk (ROMErs, 1968); elastic fibers according to Hart (BURCK, 1969); fibrin stain according to Ladewig (LADEWIG, 1938) and Mallory (PREECE, 1959); acid mucopolysaccharides (Chesa-Stain, BURCK, 1969), and PAS-reaction (ROMEIS, 1968). Controls:
The remaining lobes of the right lung and the left lung of the experimental animals were examined regularly. In addition, six animals were subjected to a sham operation with thoracotomy and artificial respiration of 35 minutes duration. Three animals were sacrificed on the first postoperative day, and three on the second day. Embedding and staining procedures were the same as in the other animals.
Results After temporary ischemia of varying duration quite uniform changes are found within the first days. The perivascular connective tissue of the great hilar vessels is interspersed with an extravasate enclosing several sometimes extremely dilated lymphatic vessels. Also, the interalveolar septa are broadened by an extravasate. The alveolar epithelial cells exhibit clear and finely vacuolated cytoplasm. The alveoli, like the bronchioles and the bronchi, are expanded containing a proteinaceous and hemorrhagic extravasate. The interstitial extravasate extending into the interalveolar septa and around the great vessels and the dilatation of the lymphatic vessels start to recede slowly after the 2 nd day; on the 5th day they can hardly be demonstrated, while they have disappeared completely on the 7th day. The intraalveolar extravasate vanishes fairly rapidly after the 2nd day. On the 3rd day it is found only in a few alveoli, while it has disappeared completely on the 5th day. However, while it disappeares completely without leaving any residues after a 5-minute period of ischemia, after interruption of the circulation for 10 minutes and longer great brownish red bar- or needle-type crystals as well as alveolar casts rich in fibrin and consisting of amorphous eosinophilic material sometimes including a few alveolar cells are seen after the 1st day. The alveoli not containing any extravasate are collapsed; the intraalveolar extravasate is immediately followed by atelectasis (Fig. I). After the 3rd to 5th day, the parenchyma expands increasingly, apparently depending upon the duration of the preceding temporary ischemia. After a 5-minute ischemia the whole alveolar parenchyma and the bronchi are com-
Puimonary Changes After Temporary Ischemia' 273 Slt1 day
'0
intraalveolar exlravasate
0
::'" .E c'" ~ u
E .~
emphysema
intt'rstltial extravasate dllatallon of Iymphalic vessels
I
inlraalveolar ext ravasate
m
sema
ateleclasls :
interstitial extrayasate dilatation of lymphatic vessels
intraalveolar extravasate
atelectasis
interstillel extravasate dilatation of lymphatic vessels
Fig.
1.
Sequence of the postischemic lesions.
number of animals
24
septa mloralveolallg bronchial epllhellum
12
smooth muscles
D
8
t isch 10' ,15' n= 2L
tisch. 30',90' n=2L
number of cases With thrombosIs
I iseh
300',360' n=24
Fig, 2. Number of cases with wide-range necrosis of the septa and of bronchial epithelium and smooth muscles. Bottom: cases with total necrosis following occlusive thrombosis of a large branch of the pulmonary artery.
274 . M. AMTHOR
Fig. 3. Extreme enlargement of the alveolar air spaces in the upper right lobe (right). The left lung of the same animal shows an inconspicuous parenchyma (left). 5 minutes of ischemia, 5 th day after the operation. Hematoxylin and eosin; X 85.
pletely expanded at this time. After 10, 15, and 30 minutes of ischemia, however, the lobe expands only partially; the findings on the loth day are comparable with those on the 5th day after 5 minutes of ischemia (Fig. 1). Finally, after an ischemia of at least 90 minutes, the whole lobe remains atelectatic up to the loth day. During the reexpansion of the lobe, a considerable enlargement of the alveolar parenchyma is frequently noticed (Fig. 3). The cause probably lies in several shortened and necrotic interalveolar septa. Wide-range necrosis of the lung tissues is not found, however, until after an ischemia of more than 5 minutes. The degree of necrosis increases with the duration of ischemia. Accordingly, if considerable individual differences in ischemic tolerance are taken into account, a conspicuous grading of the time of ischemia after which necrosis occured can be seen between interalveolar septa, the epithelium of the bronchi, and the smooth muscles of the vessels and the bronchi (Fig. z.). Thus, after a temporary ischemia of 10 and 15 minutes, the necroses are limited to the interalveolar septa. After 30 and 90 minutes, the
Pulmonary Changes After Temporary Ischemia'
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Fig. 4. Necrosis of the right upper lobe. Intact subpleural zone with leukocytic infiltration. 360 minutes of ischemia, 3rd day after operation. Hematoxylin and eosin; X 160.
epithelium of the bronchi is necrotic; after 300 minutes there are a few necrotic vessels; and after 360 minutes, all vessels and smooth muscles of the bronchi are disaggregated to an amorphous material. However, a narrow subpleural zone always remains intact, even in those cases in which an occluding thrombosis of a large branch of the pulmonary artery has caused a permanent ischemia followed by total necrosis (Fig. 4). Controls: During the first days, the remaining lobes of the right side not subjected to ischemia show a slight perivascular extravasate around the hilum, dilated lymphatic vessels and a few dilated alveoli, especially in those cases in which the upper (or middle) lobe had shown long-lasting atelectasis. Occasionally a few atelectatic alveoli are seen. Except for a few collapsed alveoli, the left lung appears normal. In the lungs of the sham-operated animals, one finds only a few collapsed alveoli on the 1 st day; there are no pathologic findings at all on the znd day.
276 . M. AMTROR
Discussion Postischemic extravasate: In our opinion the postischemic extravasate is due to an insufficiency of the terminal vascular system (ROTTER, 1959). Its protein content increases with the duration of ischemia. Accordingly, we found different kinds of extravasates varying from an edema, poor in protein, to protein-rich exsudates. In all cases we found an interstitial and intraalveolar extravasate in the upper (or middle) right lobe on the 1 st day. We consider the alveolar casts, which appeared no earlier than after a 10-minute period of ischemia, as proof for the increasing protein content after longerlasting ischemic periods. The severity of the postischemic lesion of the vessel walls can also be demonstrated by the erythrocytic diapedesis (ROTTER, 1959). From the 2nd day on, after a temporary ischemia of 10 minutes and more, we found crystals which are most likely oxyhemoglobin or related compounds (FUCHS, 1966) and which we consider a sign of a massive erythrocytic extravasation. It seems noteworthy that extensive extravasation in the rat can already be seen after 5-minute ischemia, while in dogs (EISENBACH, 1968) and in monkeys (DAVIS, 1952) an extravasate was not observed under equal or comparable conditions not before 2 or 3 hours. These differences may be due to, among other factors, a higher vulnerability of the rat to ischemia. It is well known that the basal metabolic rate (KLEIBER, 1947) and with it the vulnerability to ischemia (OETLIKER, 1961) decreases in proportion to the increasing weight of the animal. Atelectasis: After resorption of the intraalveolar extravasation an atelectasis develops from the 3 rd day on, and regresses after a 5-minute period of ischemia until the 7th day. After an ischemia of ro to 30 minutes, reinflation was incomplete on the loth day; after an ischemia of 90 minutes and more, no reinflation could be seen up to the roth day. The tendency of the atelectatic lobe to reinflate thus shows a conspicuous relation to the duration of the preceding ischemic period. A spontaneous atelectasis, which is not uncommon in the albino rat, and an atelectasis caused by the operative technique, namely by a pneumothorax, can be excluded with great probability. Opposing the assumption of a spontaneous atelectasis is the localization to only one, the right side; opposing both assumptions is the tendency of the lungs to reinflate, indicating a distinct relationship to the duration of the preceding ischemia. Oxygen-poisoning due to artificial respiration for 35 minutes can also be excluded, since ultrastructural changes have been demonstrated at the earliest after 24 hours of breathing pure oxygen (KISTLER et aI., 1967). We find it more likely that the intraalveolar extravasation causes the atelectasis by destroying the intraalveolar lining (surfactant, antiatelecta-
Pulmonary Changes After Temporary Ischemia' 277
tic factor; GIESE KING, 1971; WEIBEL, 1968) and thus inducing the alveolar collapse after resorption of the extravasate. The regeneration of the surfactant, the production of which is supposed to take place in the pneumocytes II (GIESE KING, 1971; HATASA and NAKAMURA, 1965, KIKKAWA et aI., 1965), obviously starts with a different delay depending on the duration of the preceding temporary ischemia. This could be, as in the transitory tubular insufficiency of the kidney, caused by a variable postischemic insufficiency of the pneumocytes II. In dogs (EISENBACH, 1968) and in monkeys (DAVIS, 1952) atelectases were also observed after temporary ischemia. However, EISENBACH attributed them to a postoperative insufficient respiration, while DAVIS did not give an explanation. Our hypothesis is supported by the observation of atelectasis (HORINE and WARNER, 1934; HUBER and EDMUNDS jr., 1967) and of a decrease of surfactant activity and an increase of surface tension (GIAMMONA et aI., 1966) after ligature of the pulmonary artery. It is also supported by the observation of changes of the osmiophilic lamellated bodies within the pneumocytes II after ligature of the pulmonary artery (HUBER and EDMUNDS jr., 1967) and after breathing CO 2 (SCHAEFER et al., 1964). The latter are related to the acidosis. A metabolic acidosis, as well as the hypoxidosis which is closely connected to it, is one of the main sequences of ischemia. Necrosis: After temporary ischemia necroses of the interalveolar septa, the bronchial epithelium and the smooth muscles of the bronchi and the vessels are seen. As in dogs (EISENBACH, 1968), the septa perish first. This observation was striking in two respects. First, the conspicuously short duration of temporary ischemia after which a necrosis of the septa can be found, the vascular and connective tissue usually has a low vulnerability to ischemia (WALTHER, 1963); secondly the septa become totally necrotic in spite of the structural differences of the constituting cells. Both observations are probably related to hemodynamic factors prolonging the ischemia. We have not found hyaline thrombi; also, vasoconstriction in the sense of the reflex of v. EULER and LILJESTRAND (v. EULER and LILJESTRAND, 1946) does not seem very probable to us to be the cause of necrosis. Further experiments in progress rather suggest that after ischemia the blood circulation is restricted locally almost to the point of stasis because the viscosity of the blood is increased by extravasation of the plasma. Emplrysema: After reinflation of the lobe, a conspicuous dilatation of the alveolar air spaces can be noticed in areas with several necrotic interalveolar septa. Whether this condition is considered a true emphysema, as it seems, in the sense of the definition of the Report of a Committee of the WHO (abnormal increase in the size of air spaces distal to the terminal bronchiole,
with destructive changes in their walls, WHO 1961), or just an extreme 19 Beitr. Path. Bd. 149
278 . M. AMTHOR
overinflation, or an emphysema due to overinflation (HARTUNG, 1964) depends on the significance one attaches to the necrotic interalveolar septa. In all cases a survey of more than 10 days will be needed in order to decide on the chronicity of the process which is necessary in an emphysema in the sense of the WHO. An emphysema induced by ischemia has been reported by EISENBACH (1968) and STRAWBRIDGE (1960); and in the naturally ocurring pulmonary emphysema of humans and rabbits, the ischemia seems to be of some importance as a causal factor (MARTIN and BOATMAN, 1965).
Zusammenfassung Bei 84 weifien, weiblichen Wistar-Ratten wurde der Ober(oder Mittel-)lappen der rechten Lunge einer temporaren Ischamie von 5, 10, 15, 30, 90, 300 und 360 Minuten ausgesetzt. In Paraplast® eingebettete Praparate des rechten Ober(bzw. Mittel-)lappens, der tibrigen rechten Lunge sowie der linken Lunge wurden nach I, 2, 3, 5, 7 und 10 Tagen untersucht. Wir fanden ein tiber 3 Tage andauerndes intraalveohires Extravasat im rechten Ober(bzw. Mittel-)lappen, welches in seinem Gehalt an Eiweifi und Erythrocyten eine Abhangigkeit von der Lange der Ischamie erkennen lien. Dieses Extravasat war offenbar Folge einer postischamischen Insuffizienz der terminalen Strombahn, verbunden mit einem von der Dauer der temporaren Ischamie abhangigen Anstieg der Gefafidurchlassigkeit. Dem Extravasat folgte eine Atelektase, deren Dauer his zur Wiederentfaltung eine auffallende Beziehung zu der vorausgegangenen Ischamie zeigte. Diese Beobachtung wird von uns so gedeutet, dafi es nach einer Zerstorung des alveolaren Oberflachenfilms durch das intraalveolare Extravasat zu einem verzogerten Ersatz des Films infolge einer postischamischen Insuffizienz der den Film produzierenden Pneumozyten II kommt. Nach tempo rarer Ischamie fanden sich Nekrosen der Alveolarsepten (10-15 Min.), der Bronchusepithelien (30-90 Min.) und der glatten Muskulatur (300-360 Min.). Die Wiederbelebungszeit der Septen war hierbei nicht nur sehr kurz, sondern auch fur aIle am Septumaufbau beteiligten Zelltypen einheitlich. Es wird deshalb angenommen, dafi es infolge einer Viskositatserhohung nach einer durch Plasmaextravasation bedingten Hamokonzentration zu einer Verminderung oder einem Stillstand der Durchblutung gekommen war, wodurch die Ischamie verlangert wurde. Nach der Wiederentfaltung des Lungenlappens wurde eine Erweiterung der Alveolen-tragenden Luftraume beobachtet, die Folge von Nekrosen einzelner Alveolarsepten zu sein scheint. Acknowledgments The author is grateful to Mrs. R. L. CASEY, lecturer at Ventura College, for her kind linguistic assistance, and to Mrs. W. ZELLER for her excellent technical work.
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c.:
Doz. Dr. M. AMTHOR, Senckenbergisches Zentrum der Pathologie, D-6 Frankfurt am Main, Theodor-Stern-Kai 7, Federal Republic of Germany