DEATH OF LIVER TISSUE

CHAPTER

20

DEATH OF LIVER TISSUE A Review of Cell Death, Necrosis, and Autolysis GUIDO MAJNO

Department of Pathology, Harvard Medical School, Boston, Massachusetts

I.

TERMINOLOGY: "CELLULAR DEATH," "NECROSIS," "AUTOLYSIS"

II.

268

METHODS FOR THE STUDY OF C E L L DEATH IN LIVER TIS-

270

SUE III.

STUDIES ON LIVER IMPLANTS

IV.

A. Gross Changes B. Histological Changes C. Chemical Changes D. Physical Changes: Evidence of Protein Denaturation E. The Significance of Cellular "Coagulation" F. The Competition between Autolysis and Coagulation G. Injurious Effects of Dead Liver Tissue H. Cellular Edema and the Significance of "Cloudy Swelling" STUDIES ON LIVER TISSUES in Vitro AND POST-MORTEM A. Metabolic Changes; The Failure of Oxidative Phosphorylation B. Chemical and Histochemical Changes C. Gross and Histological Changes D. Physical Changes E. Electron Microscopy of Liver Tissue Isolated at 25°C F. Electron Microscopy of the Perfused and Ischemic Liver G. Considerations on the Morphology of the Liver Obtained Post-Mortem

V.

270

272 272 274 282 289 292 292 294 296 296 299 301 302 302 303 306

DEATH OF A LIVER C E L L : A TENTATIVE SEQUENCE OF EVENTS

307

References

309 267

268

GUIDO MAJNO

Cellular death is a major component of liver disease, both in frequency and in extent. During acute carbon tetrachloride poisoning onethird of the liver may consist of dead cells (Stowell and Lee, 1950). The causal agents of liver "necrosis," its morphologic patterns, its local and general consequences are discussed in other chapters of this book (see Chapters 6, 7, 14, 21, 22, and 2 7 ) . Here we intend to deal with cellular death for itself; that is, to review the chemical, physical, and morphologic changes in liver parenchyma, when it is caused to die either in vivo, in vitro, or through somatic death. The information available on this topic, though still very incomplete, is far greater than for any other organ, because the uniform structure of the liver—which has caused it to be used as a model tissue for so many cytological and biochemical studies—has also made it the organ of choice for the study of cellular death. The results are therefore of broad interest in the field of cellular pathology. I.

Terminology: "Cellular Death/ 7 "Necrosis/ 7 "Autolysis 77

"Cellular death9' versus "necrosis." Let us assume that a liver lobe is deprived of its blood supply by a clamp applied to the vascular peduncle. Ultimately, the cells in this lobe will die; but in this process we should be able to distinguish several steps: ( 1 ) A period of reversible alterations. If the clamp is released at any time during this period, all or most of the liver cells should recover. ( 2 ) A point of no return, beyond which the cells will be irreversibly damaged. This is the critical step in the process, and we may agree (for want of a better definition) to take this point as the time of cellular death. ( 3 ) A period of irreversible changes, culminating in the total destruction of the cell. The "point of no return" is known, for rat liver at least: the bulk of liver parenchyma is not able to recover from a period of ischemia longer than 45 minutes (Baker, 1956). The experimental data (Table I ) show some scatter, so that we may assume a critical time range—rather than a "point"—varying between 30 and 60 minutes. 1 This allows us to say that most liver cells are dead, according to our definition, within the first hour of total ischemia. Now, as we will see below, the morphologic changes in tissue sections are almost nil at this time. With our present methods, the cells look amazingly normal; in fact they will continue to look normal for several We agree with Baker (1956) that the data reproduced in Table I show that rat liver can survive 20-30 minutes of ischemia without serious injury; the same appears to be true for dog liver (Baker, 1956). Baker also places "the limit of tolerance" ( ? ) between 30 and 45 minutes. In terms of cell survival it seems to us that the data point quite clearly to a time limit of 30 minutes to 1 hour. 1

20.

269

DEATH OF LIVER TISSUE

hours longer. It is generally agreed that about 8 hours must elapse before we can pronounce a cell dead (Himsworth, 1950). At this late time we call it necrotic, basing our diagnosis on the only reliable sign: the destruction of the nucleus. TABLE

I

ABILITY OF RAT LIVER TO RECOVER FROM TRANSIENT ISCHEMIA OF INCREASING B 1

DURATION* >

r no. ot rat in group 1 2 3 4 5 6 7 8 9 10 Mean

Percentage necrosis in sections after ischemia lastinj Ë!

!

2 Min.

10 Min.

20 Min.

30 Min.

45 Min.

0.04 0.23 0 0.005 0.03 0 0.1 0.07 0.93 0

0 0 0.04 0 0.09 0 0.014 0.16 0.08 1.8

0 0.5 0.07 0.24 36.0 0.25 0.07 0.1 0.063 0.008

0.5 0.54 0.62 0.60 0.28 2.6 28.0 0.18 0.9 95.0

94.0 7.6 82.0 2.7 75.0 1.2 85.0 90.0 89.0 7.5

0.14

0.2

3.7

12.7

53.5

α

From Baker (1956). b Recovery is judged by the percentage area of necrotic liver tissue found in histological sections 18 hours after the return of circulation.

It is obvious that if the terms "cell death" and "necrosis" are used interchangeably, as they usually are, much confusion will ensue. In a section of normal tissue, for instance, all cells are dead, but not necrotic. Because pathologists have long given up the hope of recognizing the early stages of cell death, they have become accustomed to ignore the issue and to identify—for practical purposes—cell death with the advanced "postmortem" changes of necrosis. In defining the latter, one authority has recently stated that a safe criterion is "absence of the cells." If applied to the diagnosis of somatic death, these standards of diagnosis would certainly lead a coroner into trouble. Autolysis. This term, introduced by Jacoby (1903), aptly describes the process whereby the cell digests itself by means of its own enzymes (see Bradley, 1922b). The work of de Duve (1959) (see also Novikoff 1959, 1961 ) has shown that these enzymes include a group of hydrolases, thus extending the original concept, which tended to equate autolysis with proteolysis alone. The complementary term, also proposed by Jacoby, heterolysis (digestion by enzymes from a source external to the cell) has not been widely used. Autolysis is usually studied on organs isolated in vitro or taken from a dead body; hence it is often implied that it is characteristic of post-

270

GUIDO MAJNO

mortem conditions; for the same reason a number of studies have failed to recognize the distinction between the aseptic phenomenon of autolysis, and putrefaction which implies bacterial growth. It should be quite obvious that the basic process of cellular self-digestion may occur in the living body as well as in vitro, even though it will be partly modified by an environment of live tissue. The very cell need not be dead for autolysis to occur. As we shall see in our study of ischemic tissue, the chemical events which lead to complete dissolution of the cell can be traced back to the first few minutes, when the injury is still reversible. Bradley ( 1922b ) maintained long ago that the phenomena of autolysis might be applied to the interpretation of atrophy (another reversible condition), and more recently de Duve ( 1959 ) has pointed out that the concept of turnover also implies a form of continuous "autolysis in vivo" In summary, we consider autolysis to be a process of (aseptic) selfdigestion of the cell, due to hydrolytic enzymas, initially reversible, and occurring both in vivo and in vitro. II.

Methods for the Study of Cell Death in Liver Tissue

In order to reconstruct the sequence of events of cell death, the study of liver parenchyma deprived of blood supply has long been a favorite method, because it can be applied to large samples of tissue; the time of onset can be accurately determined; all cells are simultaneously affected; and the condition can be simulated in vitro. Several variants have been used: ( 1 ) Isolation of liver tissue "in vivo Γ Large fragments of fresh liver from a donor animal (hereafter referred to as implants) are introduced into the peritoneal cavity of another animal of the same species. Such implants of liver and other organs have been used as models of ischemic infarcts for almost a century. ( 2 ) Isolation of liver tissue in vitro under various physical and chemical conditions. ( 3 ) Transient ischemia of liver lobes in vivo; and ( 4 ) Perfusion of the isolated liver with unphysiologic fluids. Biochemical studies of the liver after toxic or dietetic injury (Himsworth and Glynn, 1945; Popjâk, 1948; Gupta, 1956) are certainly useful, but not for a sequential study of cellular death; in these livers it is practically impossible to extricate the simultaneous phenomena of early injury, cell death, necrosis, repair, hypertrophy and regeneration. III.

Studies on Liver Implants

The situation of an implant, though more complex to analyze than that of a fragment kept in vitro, is a more faithful model of "necrosis" as it occurs in liver disease. It also provides some interesting data on

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DEATH OF LIVER TISSUE

271

the effect of dying and dead cells on the surrounding live tissues. Some changes which are induced by the host (e.g., calcification) have no equivalent in vitro. A more detailed survey of the literature and analysis of the problems under consideration may be found in a paper from this laboratory (Majno et al., 1960).

FIG. 1. Death of liver tissue in vivo: "implants" of rat liver left in the peritoneal cavity for 0 to 200 hours (as indicated). The characteristic whiteness of longstanding "necrosis" can be traced back to as early as 4 and even 2 hours, when histological changes are still minimal (compare with Fig. 10C). At 200 hours the fragment is surrounded by granulation tissue arising from the omentum. Scale in millimeters. From Majno et al. (1960).

272

GUIDO MAJNO A.

GROSS CHANGES

If a fragment of rat liver, roughly cubic in shape and 6-8 mm. on edge, is introduced into the peritoneal cavity of a normal rat, it will promptly gain weight and become somewhat paler; by 3-4 hours it will begin to show the characteristic whiteness of necrotic tissue ( Fig. 1 ). After 12-18 hours it is usually attached to the omentum or to some other peritoneal surface, and in subsequent days it is surrounded by a layer of granulation tissue. Slow reabsorption ensues; two or three months later all that remains is a small yellowish nodule, often calcified. If the fragment is smaller, it may remain free ( see under Section III, G ) ; weeks later it will then appear as a free body, consisting of dead liver tissue surrounded by a thin capsule of connective tissue, which presumably survives through exchange with the peritoneal fluid. B.

HISTOLOGICAL CHANGES

One hour after implantation, when we know that the cells are doomed, formol-fixed preparations could easily be mistaken for normal. Glycogen, however, has almost vanished: only traces remain in the center of the specimen, especially as granules spilled into the extracellular spaces. The few peripheral cells which retain their glycogen are in all likelihood the surviving cells described by Cameron and Oakley (1934). This rapid disappearance of glycogen emphasizes the difference between death of cells in vivo, at body temperature, and in vitro at room temperature (or post-mortem) (see Section IV, B ) . Still at 1 hour, and even earlier, a peculiar change is found in the cells at the periphery of the specimen (especially if Helly's fixative is used instead of formol): The cytoplasm is filled with semitransparent granules of uniform size, somewhat reminiscent of caviar ( Fig. 2 ). In the central part, for several hours it is impossible to recognize the cells as altered unless they are compared with a control, simultaneously fixed, embedded, cut, and stained; the cytoplasm stains more uniformly, without the large basophilic clumps characteristic of freshly fixed rat liver. The overall effect, however, is to give the cells a clearer, more "healthy" appearance (see Fig. 10, C ) . Nuclear changes begin to appear toward the 8th hour (pycnosis, lysis, peripheral condensation of the chromatin), and we can now say that the tissue shows signs of "early necrosis." In this sequence, two features are remarkable: ( 1 ) the naked eye fares better than the microscope, in that it allows recognition of the tissue as dead 3-4 hours earlier. The reason for this paradox will become apparent later: In essence, the optical properties which identify the gross tissue as necrotic (whiteness and opacity) depend on protein coagulation. These optical

20.

DEATH OF LIVER TISSUE

273

FIG. 2. Early cellular changes in a liver implant. Above: Normal liver (control). Peritoneal surface at the top. Below: Surface of a 15-minute implant. The arrows point to cells with a "foamy" aspect presumably due to swelling of intracellular organelles. A single cell-layer is involved at this early stage, but within the next 2 hours the foamy change has progressed to a depth of about 1 mm. Fixation: Helly; hematoxylin and eosin. Magnification: X 1600.

274

GUIDO MAJNO

properties are lost in tissue sections, owing to the combined effects of fixation, staining, and clearing. ( 2 ) The cells closest to the surface, which might be thought to have better chances of survival, actually show the earliest and severest changes. We will return to this point when we discuss the electron-microscopic findings (Sections IV, Ε and IV, F ) . C.

CHEMICAL CHANGES

1.

Changes in Wet and Dry Weight The implants continue to swell for about 6 hours ( Fig. 3 ). Obviously the cells are taking up water, for the dry weight drops; but once again, I40 r

-,

130-

0

6

12

<

18

24 2 HOURS DAYS —>

3

FIG. 3. Progressive changes in weight of liver implants. Above: Data from 60 nonsterile implants. Note steady increase in weight (uptake of water) for 6 hours. Below: Data concerning 4 sterile implants. Every implant was recovered and reweighed at each time point. Formation of adhesions was thus prevented. After the initial swelling, the tissue begins to decline in weight. From Majno et al. (1960).

the microscope is of little help: cellular swelling is not apparent in the tissue sections. While the implants are swelling, they also lose a measurable amount of solids, and after 6 hours they begin to lose weight (Fig. 3 ) . We therefore have a biphasic curve—swelling, followed by shrinkage—which is strongly reminiscent of the phenomena observed in

20.

DEATH OF LIVER TISSUE

275

injured, isolated cells of various types, e.g., sea-urchin eggs (Lucké and McCutcheon, 1926). The mechanism whereby the dying cells take up water has been studied extensively (see Leaf, 1956); the hypothesis now favored is that the cell, under normal conditions, continues to extrude sodium ions by means of an energy-requiring "sodium-pump"; when the energy supply fails, the pump fails, sodium ions enter the cell, and osmotic swelling ensues. However, we would like to point out that the phenomenon of cellular swelling is actually far more complex than mere osmotic hydration of the cytoplasm; within the cell membrane are contained several other membrane-limited compartments (nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes), and as we will see in the paragraph concerning electron microscopy, these compartments do not necessarily swell all together. Each cellular compartment may respond to different factors. For liver mitochondria, for instance, it has been suggested that there may be a dynamic balance between swelling (induced by several agents, including reduced glutathione) and an "active" contraction (Lehninger and Schneider, 1959). 2.

Changes

in pH

Measurements of pH within the microscopic environment of the cell encounter serious technical and theoretical difficulties. If these are overcome by making the pH determination on a large population of cells (assumed to be homogeneous) ground to a pulp, other objections arise (see Caldwell, 1956). It is a reassuring fact, however, that determinations made in the two ways—on single cells and on brei—are in substantial agreement (Caldwell, 1956). Thus the determination of pH on tissue pulp, though relatively gross, represents a useful and reproducible value. For normal rat liver we found it to be pH 7.2; in implants, it dropped to 6.4 within 1 hour (Fig. 4 ) . This was certainly to be expected, for it is a well-known fact that the intracellular pH drops as a result of injury of various kinds, and more particularly of ischemia (see Bradley, 1922b; Caldwell, 1956; Majno et al, 1960, for literature). Phosphoric acid is principally responsible for the change; lactic acid also accumulates (Sevringhaus, 1923a,b). Less expected was the reverse trend which occurred after an hour (Fig. 4 ) : the curve rose, until at about 8 hours it reached 7.6, the same value as found in the blood of normal, ether-anesthetized rats. The sequence of events suggests that acid accumulates intracellularly; then, as the cellular membranes break down, this acid diffuses out, and the internal medium of the implant becomes a single "open space" which tends to equilibrate with the extracellular body fluids. By contrast, if the fragment of liver

GUIDO MAJNO

276

is prevented from equilibrating with an outer environment by being isolated in vitro, in air saturated with moisture, the pH still drops but the secondary rise is absent (Fig. 4 ) . VARIATIONS

IN

pH

8.0 7.2

6.5 pH

4~

5.5 4.5 0 «

IMPLANTS, STERILE IMPLANTS, NON STERILE INCUBATED, 37° C. INCUBATED, 4°C. _l

4

HOURS

8

I L Γ2 I 3 5 ~ ~ l 2 < DAYS

I

I

I 3 5 7 —MONTHS —

FIG. 4. Changes in pH in liver tissue, isolated under various conditions. Top curve (#, o ) : peritoneal implants. Note return to slightly alkaline value, indicating of equilibration with tissue fluids. Lower curves: in vitro under sterile conditions, at 3 7 ° C (x) and 4°C. ( A ) . These samples remain acid. All determinations were made on tissue brei with a Beckman pH-meter. From Majno et al. (1960).

3.

Ability to

Respire

We do not know the actual oxygen uptake by the implant in situ; it is probably very small. However, we can measure the tissue's capacity to respire, by taking slices at various time intervals and supplying them with oxygen and glucose in vitro. This gives us an idea of the rate at which the potential respiratory function is destroyed. Figure 5 shows that it survives somewhat longer than the cell itself; by 5 hours it is reduced to less than 1 0 % of normal. More searching metabolic studies will be discussed under Section IV, A. 4.

Changes

in Nitrogen-

and Phosphorus-Containing

Compounds

Berenbom et al. ( 1955b ) have studied implants of mouse liver, with regard to total, acid-soluble, and protein N, free amino acids, total, acidsoluble, and protein P, RNA, DNA, and other variables. Table II summarizes their results, and Figs. 6 and 7 compare these data with similar ones obtained in vitro, with fragments kept at 37°C. in an atmosphere saturated with vapor ( Berenbom et al., 1955a ). The mechanisms of these changes will be discussed in the section (IV, B ) dealing with autolysis in vitro. A major difference between in vivo and in vitro conditions is illustrated in Fig. 6 (right): several breakdown products accumulate in

0

347 82 34.8 2.2 26 8.6 3.6 0.86 0.20 8.6 2.3

± 34 ± 26 ± 1.4 ± 0.3 ± 3 ± 2.4 ± 0.2 ± 0.04 ± 0.02 ± 1.2 ± 0.3

Initial value (mg./gm. wet wt. )

From Berenbom et al. (1955b).

Dry weight Lipid Total nitrogen Acid-soluble nitrogen Protein nitrogen Free amino acids Total phosphorus Acid-soluble phosphorus Protein phosphorus PNA DNA

Analysis

T A B L E II

92 86 92 58 92 49 78 57 74 80 97

± ± ± ± ± ± ± ± ± ± ±

Va

6 14 2 10 4 8 4 3 2 1 2

86 85 74 26 79 20 23 21 42 4 68

DAYS 0

± ± ± ± ± ± ± ± ± ± ±

2 4 12 1 7 7 2 4 2 5 1 2 ± ± ± ± ± ± ± ± ± ± ±

—

26 35 4 41

± 10 ± 4 ± 1 ± 11

—

68 ± 2 —

—

103 ± 6 —

7 64 137 66 21 67 24 20 46 23 3 33

5 2 12 4 5 2 5 3 8 4 1 9

4 ± 1 15 ± 1

—

42 ± 11

—

20 ± 4 48 ± 7 15 ± 3

—

126 ± 7

—

14

Per cent of initial value ± standard deviation: Days of incubation in peritoneal cavity

CHEMICAL CHANGES IN IMPLANTS OF MOUSE LIVER BETWEEN 6 HOURS AND 28

59 84 65 22 52 19 23 12 22 4 10

± ± ± ± ± ± ± ± ± ± ±

28 5 7 3 4 8 2 3 1 1 1 2

20. DEATH OF LIVER TISSUE 277

278

GUIDO MAJNO

100 < oc

ο

ο hζ

ÜJ

o\

40

-

Vo

G ν O

u oc uj CL

0

1 1 2

1 1 3 4 HOURS

1 5

FIG. 5. Oxygen uptake of liver slices taken from implants at stages of 20 minutes to 5 hours. The capacity to respire in the presence of glucose persists for several hours after the cells have been irreversibly damaged. Medium: KrebsRinger-phosphate with glucose. Results were calculated in microliters of oxygen per 100 mg. fresh weight and expressed as a percentage of the value for normal liver slices. From Majno et al. (1960).

FIG. 6. Chemical changes in fragments of mouse liver isolated either in vitro (o) or in vivo, in the peritoneal cavity ( · ) . From Berenbom et al. (1955b).

DEATH OF LIVER TISSUE

20.

279

vitro, but seemingly decrease in vivo where outward diffusion is possible. Figure 7 shows that several enzymes still retain some activity even after 2 or 3 days, i.e., long after the death of the cells; the rates of decay are not markedly different in vivo and in vitro.

ALKALINE PHOSPHATASE

·= in vivo. 0= in vitro

72 HOURS

I 6 OF

INCUBATION

FIG. 7. Changes in enzymatic activity in fragments of mouse liver isolated either in vitro (o) or in vivo, in the peritoneal cavity ( · ) . From Berenbom et al. (1955b).

5.

Accumulation

of

Calcium

There is now evidence from several sources that when liver tissue dies in vivo, it accumulates calcium at a very rapid rate. This phenomenon is particularly interesting, because in some respects it challenges the traditional concept of pathologic or "dystrophic" calcification. The latter, as it is commonly observed in necrotic tissue, consists of irregular deposits of calcium salts which are obvious only in long-standing foci. On account of these characteristics, it is generally held to be a late and somewhat unpredictable phenomenon. If we follow the calcium content of liver implants, we obtain the curve shown in Fig. 8. It is obvious that calcium accumules quite constantly, and well before the cells are histologically "necrotic" By

280

GUIDO MAJNO

the end of the second hour it has doubled, and thereafter it continues to increase for at least 10 days. In the same study we compared liver with other tissues, also implanted in the peritoneal cavity of the rat. After 1 week, the calcium in dog tendon was unchanged; in rat liver, lung, and muscle it had increased tenfold; in rat kidney, one hundredfold ( Majno and La Gattuta, 1962, unpublished results ). 1500

γ-

ι cd

LU

Bï >-

(Τ Ο 1000

8 3, 500-

DAY

10

FIG. 8. Accumulation of calcium in liver tissue dying in vivo: rat liver implants, wet ashed and analyzed by the method of Wallach et al. (1959). The outer rim, to a depth of about 1 mm., was discarded.

The same tendency was found in other types of liver injury. Stowell and Lee ( 1950 ) observed calcified material in the center of liver lobules, 6-18 days after CC1 4 intoxication. Chemical analysis confirmed this observation (Reynolds, 1963) and added a further refinement: the calcium content rises and falls twice, suggesting that two different phenomena are taking place (Fig. 9A). The smaller, transient rise which appears immediately after the administration of CC1 4 (Fig. 9 B ) may indicate that liver cells were mildly injured, began to admit some calcium, then recovered and expelled it. The second and larger peak corresponds in all likelihood to the calcification and subsequent reabsorption of dead

TIME A F T E R THE ADMINISTRATION OF C C I ^ , HOURS

TIME AFTER THE ADMINISTRATION OF C C I 4 , HOURS

DEATH OF LIVER TISSUE

FIG. 9. Calcium content of liver in the course of experimental CC1 4 intoxication (rat). (A) (left) Note the small early peak presumably corresponding to a reversible lesion. The secondary peak probably represents calcification of necrotic cells, followed by their reabsorption. ( Β ) (right) Detail of the early peak. From Reynolds (1963) (courtesy of Dr. E. S, Reynolds).

20. 281

282

GUIDO MAJNO

cells. It should be noticed that at the 2-hour stage the curve traced by Reynolds is swinging downward to a level which is barely above normal (Fig. 9 B ) , whereas our own curve at this point shows a very significant increase (Fig. 8 ) . It may be that at this very early stage, in CC1 4 poisoning, the calcification of dead cells is "diluted out" by the greater bulk of surviving tissue; or that cells poisoned in this fashion do not die as fast as those of an implant. An increased calcium content was also found in dietetic injury (Wachstein et al., 1962); in thioacetamide poisoning, the amount of calcium chemically determined correlated well with the extent of his2 tologically recognizable "necrosis" (Gupta, 1956). The mechanism whereby calcium accumulates in dead cells is not known. It is tempting to speculate, however, that the calcium ions may be fixed in the dead tissue by denatured proteins, because of valences made available through the unfolding of the polypeptide chain which is associated with denaturation. If this were the case, the necrotic tissue would act in a manner comparable to that of an ion exchange resin. The denaturation of cellular proteins in relation to cell death will be discussed in the following paragraph. D.

PHYSICAL CHANGES: EVIDENCE OF PROTEIN DENATURATION

A striking gross characteristic of dead tissue, in the liver as well as in other parenchymatous organs, is that it often becomes white ( Fig. 1 ) ; hence the term "white infarct." The significance of this change has received very little attention; it is generally assumed that the protoplasm is in some way coagulated, as suggested by the traditional term of "coagulation necrosis." Weigert, who introduced this term in 1880, meant "transformation of the tissue into a mass similar to coagulated fibrin." The comparison with fibrin may no longer be warranted, but the term was well chosen, for the naked-eye appearance is strongly suggestive of protein coagulation such as it occurs, for instance, in egg white through 2

We have taken the liberty of misquoting this paper. Gupta's data show that the calcium content of the liver after 0, 4, 6, 12, 24, and 48 hours (in micrograms calcium per gram wet weight of tissue) was 29, 29, 29, 260 ( + ), 1275 ( + + + ) , and 280 ( + + ) , respectively ( + signs refer to degree of necrosis). Histological sections showed clear-cut signs of cellular injury at 6 hours, and full-fledged necrosis at 12 hours. By correlating the chemical and the morphologic data, it is difficult to escape the conclusion that calcium is accumulating in dead cells. The author's original interpretation, however, is that calcium plays a specific role in the pathogenesis of thioacetamide intoxication, by poisoning enzyme systems and thus "killing" the cells. We feel that a misunderstanding may have arisen by considering "necrosis" as equivalent to "cell death."

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283

heating. W e attempted to go one step further, and to seek evidence of protein coagulation. This is not a simple task, because we are dealing with an extremely "impure" system, the cellular protoplasm. It is also necessary to define what is meant by "coagulation": this term has been applied to the cellular protoplasm with different meanings, i.e., a wholly reversible change (Gaidukov, 1910; Heilbrunn, 1956; Ungar et al, 1957; Fischer et al, 1961), or one which is partially (Lepeschkin, 1924) or wholly irreversible (Weigert, 1880). We have used the term to mean irreversible denaturation with formation of large insoluble aggregates, but will further qualify this statement under Section III, E . We must therefore seek experimental evidence that dying or dead cells contain aggregates of denatured protein. This we have done, by four methods: ( 1 ) dark field microscopy, ( 2 ) ultraviolet microscopy, ( 3 ) measurement of optical density, and ( 4 ) measurement of soluble protein. 1.

Evidence

from Dark Field

Microscopy

With the use of the dark field it is possible to visualize submicroscopic particles down to a limit ( according to Lepeschkin, 1924 ) of 0.004 μ. We applied this method in the hope that it might help in detecting very fine aggregates of "coagulated protein" within the cells. To this end, we prepared sections of fresh, unfixed liver implants, cut in the cryostat together with a block of fresh normal liver to serve as a control, and covered with a mixture of glycerin and phosphate buffer (Majno et al, 1960). Figure 10A shows the aspect of a 4-hour implant, compared with a control: the dead cells have acquired a striking luminosity, indicating that they have become filled with submicroscopic particles. Viewed in this manner, the difference between the normal and the pathologic tissue is far greater than in ordinary histological sections (Fig. 10C). The appearance of submicroscopic particles could also explain the "whiteness" observed by naked eye: in either case the optical effect is due to light diffraction. Thus the different optical properties of live and dead tissue are not unlike those of water and snow. By comparing normal liver with implants at various stages, we found that at 15 minutes the implant is not yet noticeably different from the control, but at 30 minutes a difference is clearly distinguishable. Thus if the bright granules really represent protein aggregates, we are drawn to conclude that some protein coagulation occurs when the cell is still alive, i.e., still reversibly injured (for we have established above that the injury become irreversible at about 1 hour).

284

GUIDO MAJNO

20.

DEATH OF LIVER TISSUE

285

Having demonstrated that "granules" appear, we will now provide some evidence that they represent denatured protein. 2.

Evidence

from

Ultraviolet

Microscopy

It has been shown by Teale and Weber (1957, 1959) that proteins, which do not exihibit the phenomenon of autofluorescence when examined by ultraviolet light, may become autofluorescent upon denaturation. This phenomenon is interpreted as follows: the three aromatic amino acids, tyrosine, tryptophan, and phenylalanine, exhibit a characteristic fluorescence in water solution; however, when the same amino acids are incorporated into a protein molecule, a solution of this protein does not necessarily show any fluorescence. This may be explained by a specific internal "quenching" within the protein molecule. Accordingly, the fluorescence which appears with denaturation may be dependent upon an unfolding of the polypeptide chains associated with denaturation. Fresh, unfixed sections of liver implants, placed in saline and examined in ultraviolet light, showed a greenish autofluorescence which increased as the stage of the implant advanced. At the 4-hour stage the difference with the control is quite marked (Fig. 1 0 B ) . This in itself is not a new finding. Though the mechanism had not yet been explored, it was known decades ago that several tissues which were not normally fluorescent could be altered chemically or physically in such a way as to become fluorescent. Hamperl (1934) described the phenomenon in tissues which had been fixed, Fahr ( 1943a, b ) in tissues which had been boiled, or which were simply necrotic. However, one new fact did emerge from our study of implants by ultraviolet light: the autofluorescence becomes apparent at the same time as the darkFIG. 10. Illustrating the problem of recognizing cell death before the onset of "necrosis": Comparison of three different techniques (left: control livers; right: 4-hour implants). (A) Dark field microscopy. "Snowwhite" aspect of a slice of unfixed, unstained liver tissue examined in saline (right). Note contrast with the juxtaposed normal liver at left. The submicroscopic particles causing the brightness are probably aggregates of denatured protein. ( B ) Ultraviolet microscopy of the same field: The dead tissue is autofluorescent, in contrast with the control (left). This fluorescence is consistent with the presence of denatured protein. ( C ) Conventional histology ( formol-fixed tissue stained with hematoxylin and eosin). In the normal liver (left) the cytological structure is obscured by basophilic clumps; in the absence of these, the 4-hour implant (right) has a distinctly more "pleasing" aspect, with no indication that the cells are dead. Conclusion: For the early detection of cell death, dark-field and ultraviolet microscopy (applied to fresh tissues) are clearly of value; ordinary fixed and stained tissue preparations are unreliable.

286

GUIDO MAJNO

FIG. 11. Changes in optical density of rat liver tissue, during and after cell death (in liver implants). Unfixed, unstained slices 0.2 mm. in thickness, mounted

20.

287

DEATH OF LIVER TISSUE

field effect, between the 15- and 30-minute stages. This too, then, suggests that some protein denaturation occurs while the cell is still alive. 3.

Evidence

from Changes

in Optical

Density

If a fragment of liver is "denatured" by boiling and a slice 0.2 mm. in thickness is placed in saline and examined under the microscope by transmitted light, it will appear extremely opaque; by contrast, a slice of fresh tissue cut to the same thickness will be light brown in color and quite translucent. The difference is undoubtedly due to a coagulation of the cellular proteins, which represent the bulk of the liver solids. Now, if it is true that proteins coagulate also in the course of cellular death, we may also anticipate a change in optical density. This is the case (Fig. 1 1 ) : in late stages of cell death, the difference is quite striking (Majno et al, 1960). We also measured the optical density of the tissue by photometry, using slices of standard thickness gently compressed in a blood-counting chamber; control livers were compared with implants at various stages. When the values of optical density were plotted against time, there resulted a curve (Fig. 12) which shows that there is an initial drop, followed by the expected rise. The reason for this early "clearing" was not immediately apparent, because it occurred at a time when dark field and ultraviolet microscopy indicated that the cells already contained granules of coagulated protein. We reconciled these facts in the following interpretation: during the first 2 hours, the mitochondria, which are normally somewhat denser than the cytoplasm, tend to swell, thus approaching the background density of the cytoplasm. This should decrease the optical density of the tissue. At the same time, protein coagulation begins to occur, but its optical effect (an increase in optical density) is overwhelmed by the simultaneous hydrating process just described. After 4 - 6 hours, however, water uptake comes to an end while coagulation continues, so that the tissue now becomes opaque. 4.

Evidence

from Changes

in Extractable

Liver

Proteins

Protein denaturation, as defined above, implies a decreased solubility. We therefore measured the amount of soluble protein extractable from the liver, under normal conditions, and in implants of liver tissue which in a mixture of glycerol and phosphate buffer, and viewed by transmitted light. Top: control. Center: 30-minute stage. The tissue has become clearer, probably because hydration plays the main role at this stage. Bottom: 24-hour stage. The tissue has become almost completely opaque, as would be expected from protein coagulation. Photographs were taken and processed under standard conditions. Magnification: χ 150. Compare with measurements of optical density (Fig. 1 2 ) . From Majno et al. (1960).

288

GUIDO MAJNO

had been left in the peritoneal cavity for varying lengths of time (actually, the quantitative determination was done with a biuret method, hence "extractable peptide bond" would be more accurate than "extractable protein"). The result is shown in Fig. 13: during the first few hours, the amount of extractable peptide bond rises, then falls to onefourth of normal (referred to dry weight). Several factors are at play, hence we cannot state with certainty that the rise and fall in this _i < ο

ζ

üJ Ο ce Id CL

< ο CL ο

8 12 16 — HOURS —

8 10 12 14 90 92 DAYS •

FIG. 12. Changes in optical density of liver tissue, isolated under various conditions ( photometric measurements on unfixed slices of standard thickness ) : peritoneal implants; xxxxxx in vitro at 3 7 ° C ; in vitro at 4°C. There is always an initial drop (hydration?) followed by a rise which occurs more rapidly in vivo (consistent with protein coagulation). Compare with Fig. 11. From Majno et al. (1960).

FIG. 13. Changes in the amount of "peptide bond" extractable from liver implants at various stages. From Majno et al. (1960).

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nitrogen fraction represents a rise and fall in proteolysis. However, the shape of the curve fits very well with the concept that two phenomena are at play, with opposing effects: autolysis predominating in the first period, and coagulation during the second ( see Section III, Ε ) . Ε.

T H E SIGNIFICANCE OF CELLULAR "COAGULATION"

The evidence presented so far places on firmer ground the traditional notion that dying and dead liver cells contain coagulated protein. We can rely on six indications: the tissue becomes white, firm, opaque, fluorescent in ultraviolet light and bright in the dark field, and yields less soluble protein. None of these criteria, of course, is definitive from the physical chemist's point of view, but the cumulative evidence could not be reasonably questioned. W e have also indicated that this process of denaturation—like the concomitant process of autolysis—cannot be considered typical of cell death, because it occurs, to some extent, in cells which are still alive. Hence it is quite conceivable that it may also take part in processes other than ischemic cell death; it is interesting to note, for instance, that the hyaline bodies described by Mallory in alcoholic cirrhosis have long been considered "coagulated protein" and correlated with coagulation necrosis (see e.g., Szanto and Popper, 1951). There is also considerable evidence that the changes found by us in liver tissue have a counterpart in many other types of cells. The "brightness" of dead cells was known long ago in the broader field of biology (Russo, 1910). In cultures of chick embryo tissues, examined with the dark field, Lewis noticed in 1923 that cell death is accompanied by the appearance of "very small white granules" which he called deathgranules or d-granules. In 1932, Guillermond published beautiful drawings of plant cells, mushroom filaments, and protozoa viewed in the dark field to demonstrate the difference between viable protoplasm, optically empty or nearly so, and dead protoplasm which appears "snowwhite" (Figs. 14 and 1 5 ) . It is a cellular change of this kind which contributes to the dull color of injured banana peel (von Loeschke, 1950). Also the fluorescence phenomenon appears to have an equivalent in plant tissues: in the cross section of a cotton stem affected by wilt, the area which shows "vascular browning" is also fluorescent by ultraviolet light (Subba-Rao, 1959). It would be particularly interesting to understand the relationship between the denaturation as described by us and other phenomena also described as intracellular protein denaturation under wholly different conditions. Heilbrunn (1956) gathered a large body of data to prove that reversible coagulation is a general property of the cytoplasm, oc-

290

GUIDO MAJNO

curring under normal and pathologic conditions, and dependent upon calcium ions. "A 4 reversible denaturation" was also described as a result of stimulation in nervous tissue ( Ungar et al., 1957; Fischer et al., 1961 ) and as a physiological event of importance in determining the course of early morphogenesis (Ranzi, 1957).

FIG. 14. Cell death as demonstrated with the dark field in epidermal cells from leaves of Iris germanica. Left: live cell; nucleus and nucleolus barely visible (Gl and F: lipid granules). Right: dead cell. Nucleus and cytoplasm "snowwhite." These changes are very similar to those that occur in dying liver cells. From Guillermond (1932).

W e find the above-quoted studies of Ungar and collaborators ( 1957 ) uniquely interesting. These authors found that the stimulation of nervous tissue produces reversible changes in protein configuration, which are closely related to the reversible denaturation induced by urea. The criterion used for the study of this protein change, based on spectrophotometric measurements of ultraviolet absorption, indicated a rupture of hydrogen bonds, which "loosens the protein molecule, unmasks aminoacid side-groups," and "perhaps changes its affinity for different ions." The authors point out that denaturation is probably a reversible phenom-

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enon, and that native and denatured proteins may be present together in equilibrium, as physiological states of the same protein: "It is questionable whether the term 'denatured,' with its original connotation of an unphysiological state, is the proper designation for a modified but still functional form of the protein molecule." These far-reaching statements induce us to revise our own views on the "denaturation" which occurs in cell death. W e had originally envisioned this physicochemical change as an irreversible one, though not

FIG. 15. Cell death as demonstrated with the dark field in filaments of Saprolegnia. Left and center: living filaments (Gl, granules of lipid material; C, chondriosomes ). Right: dead filament, in which the entire protoplasm has become bright and granular. This and Fig. 14 illustrate the general phenomenon of protoplasmic coagulation in cell death, as seen also in liver cells (compare with Fig. 1 0 ) . From Guillermond (1932).

necessarily fatal to the cell as a whole. It seems more likely that there is a whole range of possible changes in the structure of cellular proteins; the process of intracellular "denaturation" should thus be conceived as a continuum, from an early and reversible stage (possibly related to physiological states) to a final irreversible one. 3 3 The mechanism of this denaturation remains to be established. An acid pH may be involved in initiating the change, but coagulation continues in vivo even after the pH has returned to slightly alkaline values (Fig. 1 2 ) . The possibility of enzymatic denaturation has been suggested (Cain, 1943; Ungar et al., 1957), but final proof is lacking.

292

GUIDO MAJNO F.

T H E COMPETITION BETWEEN AUTOLYSIS AND COAGULATION

The numerous chemical studies of "dying liver tissue" which have followed that of Jacoby have focused on the phenomenon of autolysis, and more particularly on the breakdown of the liver proteins by proteolytic enzymes. Our own studies have focused on a phenomenon which is, in a broad sense, opposite: coagulation. We will now conclude that in fragments of liver tissue, caused to die by lack of circulation, two processes compete for the destruction of the cellular proteins: autolysis, and coagulation: and that this competition is probably a general phenomenon, valid for a wide variety of cells. Coagulation tends to "freeze" the tissue into a solid mass, and thus to bring autolysis to a halt; autolysis tends to overcome coagulation and to dissolve the tissue. It is likely that this balance can be altered by environmental factors, such as the availability of fluid. Dead brain tissue, for instance, usually tends to be dissolved; but under appropriate circumstances the "protein" of brain infarcts may persist (Dixon, 1 9 5 6 ) . In "zonal necrosis" of liver pathology (as opposed to the artificial situation of the implant), the cells presumably die bathed in blood, because the scaffolding of the sinusoids persists. Whether they "coagulate" under these conditions we do not know for sure, but it appears likely in view of the calcium accumulation ( see Section III, C ). Within days or weeks they then disappear—but the part played by autolysis, heterolysis, and phagocytosis, remains to be seen. The outcome of the balance between autolysis and coagulation is probably not indifferent to the surrounding tissue, as we will briefly discuss below. G.

1.

Local

INJURIOUS E F F E C T S OF DEAD LIVER TISSUE

Effects

It is an old observation that a fragment of boiled liver tissue, placed in the peritoneal cavity, can persist for weeks almost unchanged, as a free body (see Wells, 1 9 2 5 ) . The fact seems rather surprising (Fig. 1 6 ) , but after all, if the tissue is reduced to a mass of coagulated, insoluble material with a relatively smooth surface, it stands to reason that the surrounding tissues should be almost unaware of its existence, except for a mild mechanical irritation. A large fragment of liver in a stage of advanced necrosis, transferred aseptically to a new animal, is equally inert (Majno et al., 1 9 6 0 ) . On the other hand, a similar fragment of fresh tissue behaves quite consistently as an irritant; it induces adhesions and the formation of granulation tissue.

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These observations suggest that the coagulation which takes place in dying tissue may serve a useful function. Among the products of autolysis are polypeptides, which are powerful mediators of inflammation (Spector, 1951). By bringing autolysis to a halt, coagulation may significantly minimize the local effects of tissue death.

FIG. 16. Photograph illustrating the point that, once coagulated, a fragment of liver tissue fails to elicit inflammation in the peritoneal cavity of a rat. This fragment is shown 1 week after it had been coagulated by boiling and then implanted under sterile conditions. No adhesions have formed. A similar result is obtained if the implant is a fragment of liver which has previously undergone coagulation necrosis in another animal (under sterile conditions). From Majno et al. (1960). 2.

General

Effects

The notion that necrotic liver parenchyma may pour abnormal and injurious materials into the blood stream is already found in Jacoby's early papers (see Bradley, 1922b). Experimental proof met with unforeseen complications, and the problem seems to have been dropped unanswered. The salient established facts are the following: ( a ) Enzymes may certainly escape from injured liver parenchyma, and appear in the blood in sufficient amounts to be useful for diagnostic purposes Chapter 1 8 ) . This has been shown in clinical (see Page and Culver, 1960) as well as in experimental situations (Rees et al., 1961). No ill

294

GUIDO MAJNO

effects have been correlated with the presence of these enzymes in the blood, ( b ) The toxic syndrome accompanying extensive hepatic necrosis is not necessarily an autointoxication by products of necrotic tissue. The symptoms may be explained by insufficiency of the remaining parenchyma, ( c ) The so-called autolytic peritonitis described by a number of authors, a fatal (experimental) condition caused by fragments of liver or other organs introduced aseptically into the peritoneum, is in reality a fulminating infection with Clostridium welchii (syn. C. perfringens). These experiments were performed on dogs, which have since become notorious for harboring C. welchii in their tissues under normal conditions. The experimental protocols, describing bloody, foamy exudates and crepitating tissues, are self-explanatory (see Wangensteen and Waldron, 1928; Andrews and Hrdina, 1931). ( d ) The method of choice for assaying the toxicity of autolyzing tissues has been to place large fragments of various organs into the peritoneal cavity. This approach is open to two major criticisms. First, autolyzing tissues liberate materials which have local inflammatory effects; the animals will therefore develop a local disease, peritonitis, which is bound to complicate the experiment with its own set of symptoms. Furthermore, once this aseptic peritonitis is established, bacteria of intestinal origin may easily come to infect it. ( e ) The decisive experiment would be to extract various fractions of soluble material from organs undergoing autolysis in the demonstrated absence of bacteria; and to inject these fractions intravenously (see Boy ce and McFetridge, 1937). Controlled experiments of this kind, to our knowledge, have not been done, (f) There remains the possibility, in theory at least, that the dead liver tissue might liberate into the blood stream materials sufficiently "foreign" to induce an antibody reaction; and that these antibodies, in turn, might induce an autoimmune liver disease. Enzymes, and possibly other materials of hepatic origin, do circulate in the blood stream in relation to acute and chronic liver injury ( see Espinosa and Insunza, 1962 ). However, in reviewing the evidence, Steiner et al. ( 1961 ) concluded that the concept of "autoclasia, as a mechanism of initiation or self-perpetuation of liver injury," had yet to be substantiated. H.

CELLULAR E D E M A AND THE SIGNIFICANCE OF "CLOUDY SWELLING"

The liver has shared with the kidney the privilege of being the organ of choice in the search for a mythical entity, which goes under the name of "cloudy swelling." This name, which has accumulated more than a century of confusion, is still used as if it represented a definable entity in cellular pathology. In the past, it may have served a useful function as a catch-all, to include practically any cytoplasmic change

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at the limit of visibility; this was, however, a pseudosynthetic function, which is no longer compatible with our present knowledge of the cell. We would not indulge in another stab at this venerable relic, were it not that it is threatening a revival under the more subtle, and thus more misleading, disguise of biochemistry and electron microscopy. The gist of the argument against "cloudy swelling" is set forth below (for a more complete discussion the reader is once again referred to our paper on cell death). Around 1846, Rudolf Virchow, then at the beginning of a career which was to last 56 more years, noticed that certain tubular cells of the kidney in "Bright's disease" contained opaque granules; some cells were also swollen. This cellular condition he called "Cloudy Swelling." The granules, which conferred the "cloudiness," were probably droplets of reabsorbed protein, concentrated in what we would now call "phagosomes" (see Novikoff and Essner, 1960). From this simple observation, "cloudy swelling" was later extended—and not only by Virchow—to include practically every form of abnormal cell, which was either enlarged, or contained granules, or enjoyed both conditions. "Cloudy swelling" was reproduced, in the liver and in the kidney, literally by all means. These included postmortem autolysis, compensatory hypertrophy, starvation, fever, poisoning with bacterial toxins or heavy metals, and immersion of the tissue in distilled water— just to mention a few (Bell, 1913). There is little need to belabor the point that a cytological entity corresponding to the name of "cloudy swelling" never existed, including Virchow's original observation, which we should now consider under the heading of phagocytosis. With the advent of electron microscopy and of cellular fractionation, it was found that mitochondria were able to swell under a variety of circumstances (see Rouiller, 1960). There has been an unfortunate tendency to identify this phenomenon with the traditional "cloudy swelling." This merely adds to the confusion, because there is no cytological change we can safely call by this name in light microscopy. Even the reference to "cloudiness" is misleading: when the mitochondria swell, the optical density drops, both in vivo (in the tissue as a whole, see Fig. 11) and in vitro. Many have now taken to use of the term at the level of gross pathology, thus precluding any reference to a precise cellular change. A descriptive term is certainly in order when it is necessary to describe a cell which is swollen, or contains swollen mitochondria; but then cellular edema and mitochondrial swelling are the only terms which can be safely used; self-explanatory terms which merely describe morphologic symptoms of cellular disease, without suggesting that they correspond to a disease entity.

296 IV.

GUIDO MAJNO Studies on Liver Tissue in Vitro

and Post-Mortem

When liver tissue dies either in vitro or with the body as a whole, the process of cell death is not affected by exchanges with a living environment. Thus the task of recognizing the sequence of chemical events which are characteristic of cellular breakdown is simplified. Most studies of liver autolysis fall into this category. We will include under the same heading a method for the study of autolysis in vitro which shares some features of an experiment in vivo: perfusion of the isolated liver with unphysiologic fluids. A survey of the data is complicated by the great variety of conditions used by different authors for producing autolysis: fragments or homogenates of fresh liver have been kept in air, nitrogen, fluids of various temperatures, with or without aseptic precautions; sometimes the liver is simply left in situ after death. The following generalizations should be kept in mind: ( 1 ) if liver tissue is deprived of circulation and kept at room temperature, it can be taken as a model for the "autopsy liver'; and ( 2 ) if kept at 3 7 ° C , it will duplicate the conditions prevailing in the center of a recent infarct, before significant exchanges with the surrounding tissues have occurred. A.

METABOLIC CHANGES; THE FAILURE OF OXIDATIVE PHOSPHORYLATION

When the liver is deprived of its blood supply, and the cells start to slide along the slope of chemical and morphologic breakdown, a very complex interplay of events occurs. However, the initiating factors should be one, or more, of the following three: lack of oxygen; lack of substrate; and lack of perfusion, possibly leading to local accumulation of injurious products. Thanks to the pioneer work of Gallagher et al. (1956b), we now begin to have some insight into the role of these factors (Table I I I ) . The first step in this work was to study the ability of rat liver to oxidize various substrates after 1 hour of autolysis in Ringer at 3 8 ° C . In homogenates of these livers, the oxidation of succinate was unaffected (in fact it rose, possibly because the enzymes had become more easily accessible to the substrate). Pyruvate, however, was oxidized to the extent of about 7 5 % of normal. The oxidation of L-malate was similarly affected. It seemed unlikely that this could depend on destruction of enzyme protein, because malic dehydrogenase activity could still be demonstrated. It was then found that, for some of the substrates at least, these deficiencies could be reversed by the addition of cofactors such as dephosphopyridine nucleotide ( D P N ) . The loss of cofactors (either by enzymatic destruction, or by diffusion through increased

20. DEATH OF LIVER TISSUE

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permeability) thus appeared to be one of the early events in the breakdown of the respiratory process. The addition of cofactors, however, did not induce complete reversal, a result indicating that failure of other mechanisms was at play. The mechanism likeliest to fail seemed to be that of oxidative phosphorylation, i.e., the mechanism whereby the cell converts the energy liberated in the oxidation of tricarboxylic acid intermediates for the synthesis of TABLE

III

THE DECAY OF OXIDATIVE PHOSPHORYLATION" IN AUTOLYSIS: T H E RELATIVE ROLE OF THREE FACTORS—LACK OF OXYGEN, LACK OF SUBSTRATE, LACK OF PERFUSING FLUID

0

/hr.)

Ρ esterified ( μπιο^ /hr.)

P:0

13.1 16.6 15.0 15.8 16.0

0 4.1 1.8 22.6 22.0

0 0.25 0.12 1.43 1.38

Oxygen utilized ( μαίοηΐ8

Conditions of liver tissue 6

No perfusion, ' no substrate, no oxygen Perfusion,** no substrate, no oxygen 6 Perfusion, no substrate, oxygen Perfusion, substrate/ no oxygen 9 Perfusion, substrate,- oxygen a

System for oxidative phosphorylation: standard medium, 30 μ M L-glutamate, 100 μΜ glucose, 0.5 ml. hexokinase solution, and NaF at a final concentration of 0.014 M. Enzyme preparation was 0.5 ml. homogenate equivalent to 50 mg. fresh tissue. 0 Rearranged from Gallagher et al. ( 1956b ) . c Fragments immersed in Krebs-Ringer-phosphate medium at 38°C. for 1 hour. d With Krebs-Ringer-phosphate medium at 38°C. for 1 hour at the rate of 60 ml. per hour. e Bubbled into the perfusing medium to which approximately 7% of human hemoglobin was added. / Glucose, 0.1%. ο L-Glutamate, 10 μΜ/ml.

ATP. In effect, it was found that within 30 minutes of incubation at 38°C, oxidative phosphoryhtion failed completely, despite the fact that added substrates were oxidized (see Fig. 1 7 ) . Having thus found that oxidative phosphorylation could be used as a very sensitive tool for assaying liver damage, the next step was to isolate and perfuse rat livers with fluids of various composition, in order to separate the injurious effects of the three factors mentioned above: lack of oxygen, of substrates, and of perfusion. The results, summarized in Table III, show that the key factor is lack of substrate. A liver which has been simply immersed in Ringer for an hour shows no oxidative phosphorylation at all; similarly, a liver which has been

298

GUIDO MAJNO

perfused with Ringer (but without oxygen and without substrate) carries out only minimal oxidative phosphorylation, a result implying that the often postulated accumulation of toxic substances plays a negligible role. The addition of oxygen (supplied by hemoglobin in the Glucose •ADP ATP

Acetyl CoA^

J Citrate

ADP ATP Oxaloacetate

L-Malate

/ Fumarate

FIG. 17. Effect of anoxia and other agents on ATP synthesis during the breakdown of glucose by glycolysis and the tricarboxylic acid cycle. Symbols: O, in the presence of oxygen; = anoxia; • • • • calcium or DNP; CC1 4. From Gallagher et al (1956b).

perfusing medium) does not improve the situation. On the other hand, if glucose is added even under anaerobic conditions, oxidative phosphorylation is preserved; it can even be restored if it had previously been made to fail by lack of substrate. In summary, oxidative phosphorylation is shown to be one of the most labile systems in cells deprived of blood supply; the failure is

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primarily due to shortage of substrate (glucose, or some tricarboxylic acid cycle intermediate) normally supplied by the blood. It should be added that exhaustion of the supply of ATP does not yet mean that the cell is irreversibly injured; however, numerous other reactions of the normal cell's economy will be necessarily affected. W e will return to this point in discussing the sequence of events in cell death (Section V). B.

1.

CHEMICAL AND HISTOCHEMICAL CHANGES

Drop in pH

This effect, which probably plays a key role in initiating the phenomena of hydrolysis, has already been mentioned ( see Section I I I , C ) . It occurs also, but with long fluctuations, in sterile homogenates (see Bradley, 1922b). 2.

Hydrolysis

of Cellular

Constituents

The hydrolytic breakdown of the liver cell has served as a model for the study of autolysis ever since this concept was introduced by Jacoby. For about half a century, the hydrolysis of cellular proteins received by far the greatest share of attention (see, e.g., Bradley's review, 1922b). In recent years, de Duve's discovery of the lysosomes, intracellular bodies packed with hydrolytic enzymes, has provided an entirely new basis for the study of autolysis, and the reader is referred to the excellent review by Novikoff ( 1961 ) for further details of this fundamental work. The trigger mechanism of hydrolytic breakdown is not yet entirely clear. In vitro, in a suspension of liver tissue, the pH drops, and it has long been known that this is an essential step in activating the proteolytic enzymes. The pH drops also in vivo, when the circulation is interrupted (Section III, C ) ; it is possible that this same change may somehow bring about the rupture of the lysosomes and the liberation of their contents. About a dozen acid hydrolases have been found in association with the lysosomal fraction of liver homogenates; a critical discussion of this topic will be found in the review by Novikoff (1961) which was just mentioned. We will merely add that the enzymes which take part in the breakdown of proteins are a variety of endo- and exopeptidases. Of the former, three have been identified (though not crystallized): cathepsins A, B , and C , which are the counterparts—with respect to their specificity—of pepsin, trypsin, and chymotrypsin, respectively. However, their pH optimum is close to 6; and cathepsins Β and C are maximally active in the presence of sulfhydryl compounds, e.g., cysteine and glutathione (see Fruton and Simmonds, 1958).

300

GUIDO MAJNO

The fate of the lipids was greatly clarified by Sperry et al. (1942). In slices and fragments of rat liver, incubated for 1-2 days in Tyrode with a preservative, they found that the total amount of cholesterol remained unchanged; however, as the proteins broke down into soluble products which diffused away, the cholesterol—being insoluble— remained in the tissue, leading to an apparent increase. As for the phospholipids, they did show a 2 6 % decrease due to decomposition; however, this decrease was proportionately less than that of the dry 10 r

Hours after death FIG. 18. Decreasing glycogen content, chemically determined in ten rat livers kept at room temperature in moist airtight containers; no asepsis. From Morrione and Mamelok (1952).

weight, with the result that there was an apparent concentration, as there was for cholesterol. This mechanism probably plays a role in determining the oft-quoted "phanerosis" of lipid in dead cells. Glycogen tends to disappear, but not nearly as fast at room temperature as in vivo: chemical analysis of 10 rat livers kept at room temperature (Morrione and Mamelok, 1952) showed a rapid decrease during the first 6 hours, apparently independent of the initial concentration; thereafter the decrease was slower (Fig. 1 8 ) . Histologically a difference could be found between 0 and 4 hours, but not thereafter; livers with 5.4 and 2.9% glycogen were not appreciably different. Biopsy specimens kept at room temperature showed histologically little or no decrease

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during the first 6 hours. The over-all results indicated that the glycogen content observed histologically up to 10 hours after death is a good indication of the amount of glycogen present at the time of death. These data shed light on those of Kent (1957), who used histological grading alone: On dog livers kept (septically) at 37°C. and 4 ° C , glycogen disappeared quite gradually from the liver in the course of 6 days. The composite picture of cellular breakdown, as it occurs in liver fragments when their hydrolytic enzymes are set free both in vivo and in vitro, is best summarized by the work of Stowell and collaborators (see Berenbom et al., 1955a, b; Chang et al., 1958): Figures 6 and 7 show that the rates of breakdown are not greatly different in vivo and in vitro. The loss of enzymatic activities shown in Fig. 7 does not necessarily mean, of course, that the enzyme proteins are being hydrolyzed; other forms of inactivation may also occur. If the liver is maintained at 25°C. the loss of enzymatic activity is somewhat slower (Taft, 1960). Of five hydrolytic enzymes tested, three were unchanged and two actually showed an increase ( 5'-nucleotidase and glucose-6-phosphatase ). RNA remained constant for 6 hours; but in half an hour there was a 4 0 % increase in inorganic phosphates (when the cell is to be considered still viable, especially at 25°C.) (see also Flock, 1936). Dog liver maintained (septically) at 37°C. and 4°C. was studied histochemically by Kent (1957). Within the limitations of histologic grading, the results indicate that alkaline phosphatase remained unchanged for as long as 15 hours at 3 7 ° C ; succinic dehydrogenase (at 37°C.) started to drop only at about 4 hours and remained unchanged for 6 days at 4°C. (though there was some diffusion); DNA at 37°C. showed a sharp drop between 6 and 12 hours. C.

GROSS AND HISTOLOGICAL CHANGES

Rat liver tissue kept aseptically at 37°C. in a humid atmosphere does not fluidify. Contrary to the traditional belief that autolysis occurs characteristically in vitro, and coagulation only in vivo, the tissue become stiff and firm with a dull brownish tinge (resembling a white infarct discolored by blood pigments). At 4°C. it will become softer and begin to show some "whiteness" only after 2-3 months, a phenomenon indicating that some coagulation eventually takes place even at this temperature. Histologically, it has been noticed that in vitro at 37°C. changes occur more slowly than in vivo (Cherry, 1950). After 1 hour, at 3 7 ° C , fragments of mouse liver fixed in Stieve's fluid (Chang et al., 1958) showed a slight dilatation of the sinusoids and of Disse's spaces; early pycnotic changes in liver cell nuclei began at 6 hours, and full

302

GUIDO MAJNO

"necrosis" was established within 24 hours. A loss in cytoplasmic and nucleolar RNA was noticeable at 6 hours and pronounced at 24 hours (at which time the chemical determinations indicated losses of 24 and 6 8 % , respectively). Loss of nuclear DNA was slower; with methyl green it appeared to begin at 24 hours and was almost complete by 48; the Feulgen reaction showed greater loss at both stages. Some caution should be exercised, however, in equating basophilia with nucleoprotein content of the cell. In the first place, DNA and RNA may leak out from the nucleus and cytoplasm, causing a diffuse stain not readily appreciated. Furthermore, studies on the liver of CCl 4-poisoned rats indicate that RNA may be chemically present in normal amounts without demonstrable basophilia. In human liver obtained by biopsy, basophilic staining is not striking and digestion with RNase does not occur. These data suggest that RNA may exist in different physicochemical states (see Himes et al., 1954). And finally, as pointed out by Craig (1960), different methods of fixation and staining introduce another variable. Formol fixation and hematoxylin staining may not demonstrate "basophilia" as intensely as fixation in chromate-containing fluids followed by toluidine staining. D.

PHYSICAL CHANGES

The curve of optical density drops, then rises in vitro as well as in vivo, though not as rapidly (Fig. 1 2 ) . This indicates that coagulation is accelerated, but not conditioned, by exchange with liver tissues. E.

ELECTRON MICROSCOPY OF LIVER TISSUE ISOLATED AT 25°C.

Fear of postmortem autolysis has always been an incentive to rapid fixation, but with the advent of electron microscopy immediate histological fixation became a ritual, culminating in the practice of fixation within the live animal. In some cases this urgency has proved to be justified; but recent studies by Ito (1962) show that good structural detail can be demonstrated also in tissues presumably undergoing autolysis. Ito has examined, with the electron microscope, fragments of rat, bat, and monkey liver after they had been left at 25°C. for periods up to 36 hours. The results appeared unorthodox: after 2 hours, the autolyzing tissue was almost undistinguishable from the control; and even after 6 hours it was possible to find cells in an excellent state of preservation (Fig. 1 9 ) . Of the various membranous systems of the cell, the plasma membrane appeared to be the most fragile; the components of the Golgi apparatus also showed early swelling; but the nucleus, the endoplasmic reticulum, and also the ribosomes were remarkably resistant.

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These statements however, need to be qualified. In the first place, it must be emphasized that the images of good cellular preservation, such as those shown in Fig. 19, represent selected areas; elsewhere the cells show greater damage. At any given stage, it is possible to find a spectrum of cellular alterations. Hence, we agree that the current pessimistic attitude (e.g., toward the usefulness of autopsy material) is not fully justified; but rapid fixation is still indicated. The merit of Ito's work is to point out that morphologic changes due to autolysis—up to a point —are not as inexorable as previously thought. It should also be understood that these findings concern autolysis outside the body and at room temperature. Both factors weigh heavily. For instance, protein coagulation is demonstrable by optical density measurements after 6 hours if the liver fragment is kept within the living body; after 16 hours if it is incubated at 37 ° C ; and after 3 months if the tissue is kept at 4°C. (Fig. 1 2 ) . When the tissue is kept in a moist petri dish, very little water is available for cellular swelling. No materials can be removed by convection or diffusion, and none (such as calcium) are added. It is quite conceivable, under these circumstances, that some of the chemical reactions of autolysis should be slowed down very significantly. Of this we have evidence also from the histology of the liver implants: the central portion of the fragment, which is the farthest removed from circulation, shows better cellular preservation at all stages; along the outer rim, cellular injury (especially cellular and mitochondrial swelling) is much more prominent. This leads one to speculate that for the survival of cellular structure (and possibly function) a period of partial ischemia, during which the circulation is reduced to a trickle, might possibly be more harmful than an equivalent period with no circulation at all. Finally, there is one aspect of Ito's findings which deserves special attention: when comparable samples of autolyzing tissue were embedded in methacrylate rather than in epoxy resin, the degree of cellular damage—at a given stage—was far greater. This suggests that the cellular structures of autolyzing tissue, even though morphologically preserved, are altered in some subtle manner, so that one embedding procedure is able to "hold them together," whereas the other is not. In other words, it may well be that much of the celluhr damage which we have traditionally attributed to autolysis is actually prepared by autolysis, but developed by fixation and embedding. F.

ELECTRON MICROSCOPY OF THE PERFUSED AND ISCHEMIC LIVER

Ashford and Porter (1962) have succeeded in maintaining rat liver for several hours in vitro at 37°C. by means of a perfusion apparatus.

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When transient "ischemia" was produced in this system by clamping the inflow tube for 45 minutes, the changes in fine structure were not as dramatic as had been anticipated. There was consistently a segregation of dense chromatin masses against the nuclear membrane (see also our Fig. 1 0 C ) . The mitochondria lost the dark granules which are characteristically present (see also Fig. 1 9 ) ; they acquired abnormal shapes, and there were also abnormalities in thickness and orientation of the cristae. The endoplasmic reticulum showed vesiculation, and in the Golgi apparatus the peripheral vesicles were enlarged and gorged with dense granules. Through swelling of the hepatic cells, the sinusoids and the bile canaliculi were constricted. Most of these changes were reversed 1 hour after the perfusion had been resumed (Ashford et al., 1962). These authors point out, however, that changes which are morphologically "mild" may not be mild for the cell; and that cellular disruption may possibly occur as an abrupt phenomenon. Furthermore, though they did not directly observe cells in the act of breaking up, they noted that the sinusoids contained cellular debris, a fact indicating that disruption must have occurred in unseen parts of the preparation. The findings on ischemic livers are thus consistent, in their "mildness," with those on autolyzing livers. Anoxic livers have also been studied with the electron microscope, but the results are conflicting. Mölbert and Guerritore ( 1957 ) examined the liver of guinea pigs which had been maintained at an atmospheric pressure of 170-180 mm. Hg for 45-60 minutes. The liver cells showed extensive swelling of mitochondria and endoplasmic reticulum, as well as large clear vacuoles of uncertain origin; the nuclear chromatin showed marginal condensation. These changes were partially reversible (it should be noted that the control sections do not appear free of artifacts). Bassi et al. (1960) found no such changes in hypoxic rats; the main features were smooth, round vesicles unrelated to mitochondria and with contents of varying electron density. The authors suggest that FIG. 19. Electron microscopy of liver autolysis. Surprisingly good preservation of cellular detail (top) in rat liver allowed to stand at 25°C. for 6 hours. Below: control. The mitochondria (Ai) of the autolyzing tissue are slightly swollen and have lost their granules (arrows). Note the excellent preservation of the endoplasmic reticulum (E) and of the other cellular membranes. N, Nucleus; G, glycogen. The field shown above was selected among those showing optimal preservation; elsewhere in the section the damage was greater (see text). Osmiumfixed tissue embedded in Epon, and stained with lead (Karnovsky, 1961). Magnification: χ 16,500. From Ito (1962) (courtesy of Dr. S. Ito).

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the striking vacuolation of hypoxic livers, as seen by light microscopy in conventional preparations, may not be a true mirror of the cellular lesion. G.

CONSIDERATIONS ON THE MORPHOLOGY OF THE LIVER OBTAINED POST-MORTEM

It is now well recognized that the histological image of the liver obtained at autopsy may be significantly different from the image prevailing a few hours earlier in life. To summarize the information

FIG. 2 0 . Effect of agonal changes on the histological appearance of the liver. Left: liver from patient who died as a result of head injury, following a short agonal period. Right: Liver in instantaneous death caused by airplane crash. Hematoxylin and eosin. Magnification: X 2 2 0 . From Popper and Schaffner ( 1 9 5 7 ) .

available, it seems that three sets of phenomena are at play in distorting the original cytological picture. 1. Agonal changes are almost inevitable unless death occurs instantaneously, as in a plane crash; they are probably more obvious in the liver than in any other organ. According to Popper (1948, 1954) these changes are recognizable if the agonal period is longer than 10 minutes. This would imply that from tissue obtained at autopsy we should practically never be able to visualize the liver exactly as it was in the patient, a view which is certainly supported by comparing biopsy and autopsy material. The characteristic and most obvious changes are three (Fig. 2 0 ) : disappearance of the glycogen (most likely a reflection of anoxia and prolonged fasting); a darker, uniform staining of the cytoplasm; and a widening of the perisinusoidal spaces (Popper, 1954).

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2. Postmortem changes, for a few hours at least, may be of less significance (morphologically) than those that occur during the agonal period. This we can assume for several reasons: ( a ) The temperature has dropped and circulation has ceased; both factors are of major importance in determining the rate of morphologic changes. ( b ) Though glycogen disappears (as shown by chemical determinations), even a 5 0 % decrease would be scarcely appreciable in tissue sections, ( c ) W e have direct evidence from Ito's work that with appropriate methods the tissue may be shown to be relatively intact for several hours (especially by light microscopy ). 3. Technical artifacts. We have become accustomed to assume that our ordinary histological sections faithfully reflect the morphology of the tissue as it was at the time of fixation. The higher standards of fixation and embedding required by electron microscopy have made us aware of the fact that the routine methods for light microscopy may actually be quite "rough," especially when the object is to recognize fine detail rather than architectural change. That the degree of distortion observed in "autolyzing" material may vary with the fixation and embedding procedures is brought out by the observations of Chang et al. (1958) and by those of Ito (see Section IV, E ) . The implication here, as already noted, is that if liver is kept for several hours at room temperature, morphologic detail can be preserved to a remarkable degree even at the level of the electron microscope, but that at the same time the tissue has become more susceptible to damage by certain embedding procedures (perhaps on account of beginning proteolytic digestion). Thus it is not unlikely that with the use of more delicate procedures, such as those employed for electron microscopy, it may actually be possible to circumvent some of the damage which so far has been loosely labeled "postmortem change." V.

Death of a Liver Cell: A Tentative Sequence of Events

We will now attempt to piece together some of the facts presented in this chapter and to reconstruct as best we can the chemical and morphologic drama of cellular death. This scheme, we hasten to add, will be a very tentative one, referring primarily to liver tissue as it dies in vivo when suddenly deprived of circulation. As the blood flow ceases, the cell does not face immediate disaster. Many of its key metabolic reactions depend on ATP; now the lack of oxygen and of blood-borne oxidizable substrates deprive it of the major source of energy for the synthesis of ATP, but a store of glycogen is available in the cytoplasm, and for a while (perhaps 15-30 minutes, not

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longer) the less efficient process of glycolysis will act as an alternative source. Glycolysis, however, entails the production of acids which will accumulate locally due to the lack of blood flow. Within about half an hour, phosphoric and lactic acid are present in such amounts that the cell and its organelles find themselves soaked in a fluid with a pH of about 6. In the meantime, some of the cellular membranes begin to malfunction, though little morphologic change is yet apparent. Cofactors leak out of the mitochondria; as the energy supply begins to fail, the "sodium pump" runs down and potassium leaks out of the cell, while sodium and calcium seep in. Calcium is a powerful inhibitor of oxidative phosphorylation, even in concentrations one-tenth that of the serum (Potter, 1947). This may well be the coup de grâce to the synthesis of ATP. The Golgi apparatus, parts of the endoplasmic reticulum, and the mitochondria begin to swell; a critical turn occurs when the lysosomes break up (perhaps as an effect of the low pH) and set their hydrolases free into the cytoplasm. Favored by the acid environment, these enzymes begin to dissolve the cell's contents. With breakdown processes prevailing, the number of osmotically active molecules increases rapidly, and despite the leaky state of the membranes, swelling of the whole cell ensues, limited by the relatively scarce availability of fluid. Not all the swelling, however, is due to osmotic forces. Among the substances which accumulate is reduced glutathione, which in addition to being a cathepsin activator is also capable of inducing mitochondrial swelling (Lehninger and Schneider, 1959). Now while some of the cellular protein is being destroyed by hydrolysis, some also begins to coagulate. Even this does not mean that the cell must necessarily die. However, by the end of the first hour, the combined structural and functional damage is such that recovery is no longer possible. If enough fluid is available, the cell—now doomed—continues to swell, until the membranes give way and the intra- and extracellular compartments merge. Many enzymes still retain their ability to function; but even if they are still active, this activity is no longer coordinated. Diffusion occurs; some enzymes leak out of the cell and may be washed away into the blood stream. As the acids also diffuse out and equilibrate with the body fluids, the pH shifts toward slight alkalinity, and eventually the proteolytic and other hydrolytic enzymes (if they and their substrates had not yet been removed by diffusion or by coagulation) will cease to operate. Within 4 or 5 hours, protein coagulation is advanced enough to be recognized (in an infarct) by a whitish aspect and a firmer consistency. Toward the eighth hour the nucleus begins to break down; remains of nucleoprotein spill into the extracellular spaces, where they join cellular debris and remains of glycogen which had outlived the cell's

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last-minute demands. Though the outline of the cell and of the nucleus may persist, the cell body has now become an inert extracellular object which continues to absorb calcium. To the surrounding tissues it is but a mild physical irritant, ready to be destroyed by "heterolysis" or by phagocytosis. We have reasons to believe that parts of this scheme are valid for many other types of cells and tissues besides the liver. For example, a close parallel can be drawn with the death of a very different tissue: the crystalline lens, which undergoes—in a developing cataract—practically all the physical, chemical, and morphologic changes we have described in our liver implants (see Majno et al., 1960). Partial comparisons could be drawn with cells of plants, algae, and fungi. However—despite the fact that we usually kill the cells in order to study them—we must admit that the surface of this topic, cell death, has been barely scratched. REFERENCES

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