Intracytoplasmic Inclusion Bodies in Hepatocytes During Prolonged Extracorporeal Circulation

Path. Res. Pract. 172,349-362 (1981) Department of Pathology and Department of Surgery, Hiroshima Umverslty School of Medlcme, Japan

Intracytoplasmic Inclusion Bodies in Hepatocytes During Prolonged Extracorporeal Circulation H. KAJIHARA, M. YAMAMOTO, H. YAMADA, T. MOCHIZUKI, and K. TAGUCHI

Summary Hyalme mcluslOn bodies appeared m hepatocytes of acutely congested livers produced by prolonged extracorporeal circulatIOn. The mcluslOns were posltlve by PAS, PTAH and ZlehlNeelsen stammg and were colored light green to green by Masson's tnchrome stammg. In methylene blue-stamed sectIOns, they were dlVlded mto two groups, pale to light blue (Type I) and dark blue (Type II). By electron microscopy, small vacuolar structures which contamed small amounts of flocculent matenal appeared near the vascular pole of hepatocytes at early stages of the expenment. With lapse of time, they became larger and had compact amorphous matenal. These mcluslOns corresponded to the Type I mcluslOns seen by light microscopy. By electron microSCOpiC cytochemistry, these mcluslOns were usually posltlve to the aCid phosphatase reactIOn and negative to the DAB reactIOn. With an mcrease m serum free hemoglobm, electron-dense mcluslons correspondmg to the Type II mcluslOns appeared m the hepatocytes. They were strongly positive to the DAB reactIOn. Large ones were usually positive to the aCid phosphatase reactIOn In the penpheral narrow nm. Large mvagmatlOns of cytoplasmiC membranes and large hydropIc vacuoles were observed at the late stage of the expenment. They con tamed frequently ftbrm strands or a few erythrocytes. AutophagiC vacuoles and myelm figures were also mcreased m the hepatocytes at me late stage of the bypass circulatIOn.

Introduction Extracorporeal circulation is commonly used during open heart surgery. Although improvements in both instruments and management of metabolic disturbances have contnbuted much to the decrease in morbidIty and mortality, bypass circulation is non-physiologic and, if prolonged, is still often associated with pathophysiological disturbances in vanous organs, which eventually induce an irreversible state after the operation. It has been recognized by many investigators that following establishment of the cardIOpulmonary bypass circulation, liver functIOn is more or less impaIred (Andersen, et 24 Path Res Pract Vol 172

350 . H. KaJlhara et al.

aI., 1958; Mundth, et aI., 1967; Neutze, et aI., 1974; Lepert, et aI., 1977). Hemodilution is also widely used during bypass circulation to diminish hemolysis and disorders of coagulation during and after the operation. However, Lepert et ai. (1977) have pointed out that hemodilution increased vascular resistance in the liver, and decreased hepatic blood flow. Biernat et ai. (1974) also reported that the liver cell damage which follows extracorporeal circulation with hemodIlutIOn were more severe than those with whole blood. It could be supposed that the damage of the liver which results from extracorporeal CIrculation is related mainly to hypoxia. However, there is little information concerning morphological changes in the liver dunng bypass circulation. To investigate the morphological changes of the bver during extracorporeal circulation, dogs were subjected to the bypass circulation and the liver was examined by light and electron microscopy.

Materials and Methods Thirty SIX adult mongrel dogs, welghmg 10 to 16 kg, used m thiS study, were anesthetized by mtramuscular mJectlOn of 15 to 20 mg/kg Ketaral (2-o-methylammo-cyclohexanone-hydrochlonde). Five dogs, sacnflCed Immediately after anestheSia, served as controls. The chest of the remammg dogs was opened by a longltudmal sternal splIttmg mClSlon. The supenor and mfenor vena cavae were cannulated through the nght atnum. Both femoral artenes were also cannulated for cardiopulmonary bypass Circulation and for momtonng of blood pressure. As descnbed m our (mg/dll

200 180 160 140 120 100

, p--d

80 60

,{

I

I

I

I

I

I

I

P

,

, ,,

P'"",,,.,,-..o

~_.h-----l~-

40 20 0

2

3

4

5

6

(hI

Fig. 1. Sequential changes m serum free hemoglobm concentrations of three dogs (0---0, !:::'---!:::', and x --- x) durmg extracorporeal circulatIOn. After 11/2 h, hemoglobm concentratIOns were elevated slgmflCantly (21.0, 32.2 and 43.6 mg/dl). Thereafter, they were progressively mcreased (after 2h; 35.2, 55.3 and 61.6 mg/dl: after 3 h; 59.9, 65.1 and 81.2 mg/dl: after 6 h; 81.0, 107.1 and 202.3 mg/dl).

IntracytoplasmIc InclusIOn BodIes m Hepatocytes . 351 prevIous study (Ka)lhara, et aI., 1977), bypass CIrculatIon was mstltuted by means of a roller pump and bubble oxygenator primed wIth a hemodtluted mIxture, conslstmg of 75% heparlmzed blood and 25% lactIC rmger solution. Blood was oxygenated wIth a gas mIxture of 98% O 2 and 2% CO 2 , Bypass cIrculatIOn was contmued for 1,2,3, and 6 h, and 7 dogs were sacrifIced at the end of each period. Ftve dogs m each period were for electron mIcroscoPIc exammatlOn and 2 were for electron mIcroscopIc cytochemIstry. Serum free hemoglobm concentratIons were measured at mtervals m an addItIonal 3 cases of the 6 h bypass cIrculatIOn (FIg. 1). For electron mIcroscopIC exammatlon, hvers were removed ImmedIately after sacrifIce. Small pIeces of hver were fIxed overmght m cold 3% glutaraldehyde buffered wIth phosphate at pH 7.4 and postflxed for 1 h m 2% buffered OS04 solutIon at pH 7.4. The materials were then dehydrated through graded alcohol and embedded m Epon. Thm sectIOns were stamed wIth uranyl acetate and lead, and then exammed wIth a lEOL 100 B electron mIcroscope. For electron mIcroscopIC cytochemIstry, the hvers of 2 dogs m each period were perfused through the portal vem for about 15 mm wIth appropriate fIxatIve. Small pIeces of the hver were fIxed for an additional 1 h m the same ftxatlve. They were then cut mto 40 to 50 !l thICk sections wIth a Sorvall TC-2 tIssue sect10ner and mcubated m the followmg medIa: (1) Barka medIUm for aCId phosphatase (Barka and Anderson, 1962) and (2) DAB medIUm for peroxIdase reaction (Graham and Karnovsky, 1966). After embeddmg m Epon, thm sections were exammed wIthout stammg. Additional samples of hver were fIxed m 10% formalm and embedded m paraffm. The sections were stamed WIth the followmg: hematoxtlmeosm, Masson's trichrome, PAS, Zlehl-Neelsen, PTAH, methylene blue, OIlred 0, Sudan Black B, methylgreen-pyronm, Ralph method for hemoglobm, and DAB method for hemoglobm. Unstamed sectIOns were exammed for autofluorescence WIth a fluorescence mIcroscope.

Results 1. Serum free hemoglobin Figure 1 shows sequential changes in serum free hemoglobin concentration of 3 dogs. Before the experiment, the serum free hemoglobin concentrations were 9.1, 10.5 and 20.1 mg/dl respectively. After 1 Yz h bypass circulation, the serum hemoglobin concentratIOns were significantly elevated (21.0, 32.2 and 43.6 mg/dl). Thereafter, they rose progreSSIvely (after 2 h; 35.2, 55.3 and 61.6: and after 3 h; 59.9, 65.1 and 81.2) and reached 8 to 20 times at the end of the experiment (81.0, 107.1 and 202.3 mg/dl). 2. Macroscopic findings Liver congestion appeared as early as 1 after the initiation of bypass circulation and became progressively more severe. After 6 h, the liver was reddish to purplIsh black. Congestion and hemorrhage were conspicuous in the parenchyma of the lIver at this stage and it was very difficult to perfuse with Ringer solution immediately after stopping the bypass circulatIOn. The formation of ascites, retroperitoneal edema including pancreatic edema, and hemorrhage in vanous organs were frequently observed, as shown in Table 1.

352 . H. KajIhara et al. Table 1. MacroscopIC fIndIngs of varIOUS organs durIng extracorporeal CIrculatIon Hours after

No

Subendocardial hemorrhage

ECC

dog.

r-Vent

I

+ + +

I

2

at

2 3 4 5 6 7 8

6

+

..... +

-

9

.....

10

+

II

3

-

12 13 14 15 16 17 18 19 20

I-Vent.

-

Liver congestion

.... + + + +

'*+

.....

+ +

'* .....

-

+ +

Infestinal hemorrhage

Adrenal hemorrhage

it

+

+ + -

+ +

-

+ + + + + + + +

-

-

+

'*-

+ +

it

+

+

....

.....

it

'*+

'* '*

..... '*

....

+

'*it

it

.... it

.....

.... .... it

-

.....

it

-

+

+ +

+ + -

'* ** *i t

....

*

-

-

-

....

'*

+ -

-

.....

it

Lung hemorrhage

Pancreas edema

.....

+ +

+

+ + + + +

'*+

-

-

+ it

-

-

it

+ +

'*+ *+ +

3. Light microscop'c findings After 1 h of bypass circulation, congestion of centrolobular areas was observed. The central vein and the vascular sinusoids of the centrolobular areas were distended with blood, in which neutrophiles were increased in number. Small vesicular structures were occasionally recognized in the hepatocytes of the centrolobular areas, some of which were slightly eosinophilic in H-E sections, and slightly positive by PAS and Ziehl-Neelsen staining. In methylene blue-stamed epon sections, they were pale or light blue and were designated as Type I inclusion bodies. After 2 h, centrolobular congestion became more conspicuous and the hepatocytes of the central areas were slightly atrophic. Eosinophilic spherical inclusions, ranging mostly from 3 to 6 I.t in diameter, were frequently recognized in the hepatocytes of central areas. These inclusions were slightly to moderately positive by PAS, PT AH and Ziehl-Neels ell staining, and were light green to green after Masson's

Intracytoplasmic InclUSion Bodies

III

Hepatocytes . 353

Fig. 2. At 2 h after bypass CtrculatlOn. Two types of mclusions, dark (thick arrows) and hght (thm arrows), are observed 10 the hepatocytes. Epon section, methylene-blue stammg.

trichrome staining. After staining with methylene blue, two populations of inclusions were identified in the hepatocytes. One was pale or light blue and the other was dark blue (Fig. 2). The inclUSIOns whlCh were strongly positive for methylene blue first appeared at thts stage and were named Type II inclusions. They were usually smaller than Type I inclusions. In Epon sections of tissue incubated in DAB medmm and paraffm secttons reacted with DAB, the Type II inclusions were positive. After 3 h, extravasation was usually observed around the central veins and vascular sinusoids of the central area. The inclusion bodies were increased 10 number and stze and were found not only in centrolobular hepatocytes but also 10 penpheral hepatocytes. They were usually sphencal, but sometimes polymorphous. In the methylene blue-stained sections, both Type I and II inclusions could be tdenttfied. However, fusion of both types or intermediate forms between Type I and II were frequently observed at this stage. After 6 h, damage to the sinusoidal walls and extravasation were conspicuous. The inclusions, mostly intermedtate forms, were increased in number and size, up to 25 !.I. in diameter. They contamed no lipid and showed no autofluorescence. The staining characteristics of both types of inclusion body are described in Table 2. Large hydropic vacuoles frequently appeared in the hepatocytes of the centrolobular areas at this stage. These vacuoles occasionally contained one or a few erythrocytes.

354 . H. KaJlhara et al. Table 2. StaInIng reactIons of IntracytoplasmIc InclusIOn bodIes appearIng In hepatocytes Results

Methods

Type I

Type ]I EosinophIlic

Hematoxylin-eosin

Eosinophi he

Masson trichrome

Green

PAS

(+)

(+)

Zlehl- Neelsen

(+)

(+)

PTAH

(+)

(+)

Methylene bl ue

(±)

H+)

011 red 0

(-)

(-)

Sudan black B

(-)

(-)

Methyl green-pyronin

(-)

(-)

Hemoglobin (Ralph)

(-)

(±)

DAB

(-)

(++-)

Autofluorescence

(-)

(-)

Green

4. Electron microscopic fmdings After 1 h bypass circulation, the inclusion bodies, rangmg mostly from 0.3 to 3 !l in diameter, were frequently observed m the hepatocytes of the centrolobular regions (Fig. 3). They were membrane-bound and contained small amounts of flocculent material and a few small vesicular structures. Electrondense cores were usually recognized in these inclusions. Relatively large incluSiOns were usually located near the biliary pole, while smaller ones were predominantly distributed near the vascular pole. These inclusions corresponded to the Type I inclusiOns seen by light microscopy. In addition to these inclusions, a small number of heterogeneous dense bodies were observed, which were located near the biliary pole and were conSidered to be lipofuscin pigments. Lipid droplets were absent m the hepatocytes at this stage. After 2 h, the inclusions containing flocculent material had become larger and the contents were usually more compact in comparison to those present after 1 h of bypass circulation. In addition, electron-dense inclusions, rangmg from 0.5 to 1.5 !l in diameter, appeared m the cytoplasm of hepatocytes (Fig. 4). They

FIg. 3. At 1 h after bypass CIrculatIon. A few InclusIOns (I) are observed III the hepatocytes. They contaIn a small amounts of flocculent materIal and eccentrIC dense cores. N: nucleus. X 9200. FIg. 4. At 2 h after bypass CIrculatIOn. A few dense InclusIOns (II) appear at thIs stage. The contents of these InclusIOns are homogIneous and have resemblance to those of erythrocytes. I: Type 1 InclusIon. V: hydropIC vacuoles. X 10 000.

IntracytoplasmIC InclusIOn Bodies

III

Hepatocytes . 355

356 . H. KaJlhara et al.

were also membrane-bound and usually homogeneous. On greater magnification, however, the matnx of these mclusions was finely granular, resembling that of erythrocytes. In larger ones, electron dense materials were frequently marginated with narrow lysosomal matnces. These electron dense inclusions were Identical with the Type II mclusions observed by light mIcroscopy. In cytochemical studIes, Type I inclusions were usually positIve to the acid phosphatase reactIOn (Fig. 5 a). On the other hand, Type II inclusions were usually negative, while m large ones reaction products for acid phosphatase were frequently observed in penpheral narrow spaces (FIg. 5 a and 5 b). The DAB reaction was strongly positIve in the matnx of the Type II inclusions (Fig. 6), while Type I inclusIOns were negatIve. Dense matenals in the penbIhary bodIes also exhIbited a strongly POSItIve DAB reaction. FUSIOn of these Type I and II mclusions to the penbIhary bodies was occasionally observed at this stage. After 3 h, both Type I and II inclusions were mcreased in number and size. The matrix of the Type I inclusIOns was usually condensed and was increased in electron density (Fig. 7). AutophagIc vacuoles and large hydropIc vacuoles were frequently seen in the hepatocytes of the centrolobular areas at this stage. After 6 h, both types of inclusion bodies became larger and irregular in shape. Fibrin strands were frequently recogmzed in large Type I inclusIOns containing relatively small amounts of flocculent matenal and in hydropIc vacuoles (Fig. 7). Large invagination of plasma membrane and hydropIc vacuoles were increased in number and contamed frequently one or a few erythrocytes (Fig. 8). A considerable number of myehn figures and autophagic vacuoles were also recognized m these hepatocytes.

Fig. 5 a. Hepatocytes Incubated In Barka medIUm for aCid phosphatase reaction after 2 h bypass circulatIOn. Type I InclUSIOn (arrow) IS pOSitive by aCid phosphatase reactIOn, while Type II (II) IS negative. x 12 000. Fig. 5 b. Same material as that of Fig. 5 a. Large Type II InclUSIOn (II) has reactIOn products for aCid phosphatase In peripheral narrow rim X 8 000. Fig. 6. Hepatocytes Incubated In DAB medIUm after 2 h bypass CIrculatIOn. Type II InclUSIOns (II) are strongly pOSitive for DAB reaction and have resemblance to the reaction of erythrocytes. Arrows: peroxisomes. x 12100.

IntracytoplasmIc IncIuSlon BodIes

10

H eP atocytes . 357

358 . H. KaJlhara et al.

Intracytoplasmic InclUSIOn Bodies m Hepatocytes . 359

Discussion Two types of intracytoplasmic inclusion bodies, Type I and II, were observed in hepatocytes of dogs subJected to extracorporeal circulatIOn. Type I inclUSIOns were slightly electron-dense and contamed variable amounts of flocculent matenal. In electron microscopIC cytochemistry, they were usually positive for the acid phosphatase reaction, but negative for the DAB reaction. On the other hand, Type II inclusions were electron-dense with a matrix which was homogeneous and finely granular under higher magnification, resembling that of erythrocytes. Electron-dense material of these inclusions was strongly positive for the DAB reactIOn, and the aCid phosphatase reactIOn was frequently positive in the penpheral narrow nm. These two types of inclusion bodies were membrane-bound and usually round. By hght microscopy, they were indistinguishable from one another in sections prepared by PAS, PTAH, Masson's trichrome and Ziehl-Neels en stammg. However, Type I inclusions were negative or slightly posltlve for methylene blue, while Type II mclusions were strongly positive. It is well known that hyaline droplets or inclusions appear in the hepatocytes m hypoxic conditions (Pichotka, 1942; Altmann, 1949, 1955; Anderson, et al., 1961; Oueda, 1963; David, et al., 1965) as well as in regenerating liver following partial hepatectomy (Domach and Weinbren, 1952; Pfeifer and Bannasch, 1968) and in certain other pathologic conditions (Trowell, 1942; Fisher and Fisher, 1954; Boler and Bibighaus, 1967). With respect to morphogenesis of hyaline inclusions appearing in hypoxic state, Altmann (1949, 1955) intensively investigated and stressed that these hyaline inclusions might onginate from an mcrease m membrane permiabihty caused by hypoxia and from an exceSSIve ImbibItion of serum protein. In the present study, small watery inclusions appeared near the vascular pole of the hepatocytes at an early stage of the experiment. They became larger and developed into typical Type I inclUSIOns with compact amorphous contents. As suggested by Altmann (1949, 1955), it seems likely that fusion and dehydratIOn play an important role in the development of large Type I mcluslOns containing compact amorphous matenal. Boler and Biblghaus (1967) observed morphologically simillar inclusions m hepatocytes of dogs dunng endotoxin shock and consid-

fig. 7. Hepatocytes after 3 h bypass circulation. Type I mcluslOns (I) represent van able denSity, shght to moderate. One of them contams flbrm strands (arrow). M: myelm figure. X 5200. Fig. 8. Hepatocytes after 6 h bypass CIrculatIOn. Large hydropIC vacuoles appear m the hepatocyte. One of them contams an erythrocyte (E). M: myelm figure. X 6900.

360 . H. KaJlhara et al.

ered that the contents of these inclusions might be mainly fibrinogen which was newly synthesized. As a result of our present study, however, it seems unlikely that so much fibrinogen could be synthesized within 2 or 3 h under such hypoxic condition. The same mclusions appear in the regenerating hepatocytes after partial hepatectomy. As to the morphogenesis of these hyaline inclusions, Pfeifer and Bannasch (1968) emphasized that an increase in sinusoidal pressure plays an important role m the appearance of the inclusion body, Mori and Novikoff (1977), thereafter, demonstrated that inclusions developed initially by addition to smaller pmocytotic structures. Electrondense cores were frequently recognized in the Type I mclustons. The nature of these cores was not clear in the present study. However, they might occur as a result of coacervation of imbibed matenal (Altmann, 1949, 1955). Of paticular interest is the appearance of the Type II mclustons. They were membrane-bound and contamed a homogeneous dense matrix, resembling that of erythrocytes. They were strongly posItive to the DAB reaction. Small Type II inclusions first appeared near the vascular pole of hepatocytes, when serum free hemoglobin concentrations mcreased significantly. These fmdmgs indicate that dense contents of Type II inclusions are hemoglobin molecules taken up by hepatocytes. They enlarged gradually and usually had lysosomal enzyme in the peripheral rim. By means of a cytophotometric method, Beneke and Scomazzoni (1962) previously demonstrated the presence of hemoglobm in the hyaline droplets of the hepatocytes after injection of CCl 4 in the spleen of rats and considered that serum protein containing hemoglobin molecules entered into the hepatocytes as a result of mcreased membrane permiability. Moreover, Goldfischer and his co-workers (1970) reported that circulating hemoglobin molecules were engulfed in the hepatocytes by pinocytosis after hemoglobin injectIOn or after mductIOn of hemoglobmemia by injection of dIstilled water. With low doses, hemoglobin uptake was detectable only in the Kupffer cells. They also recognized the appearance of aCId hydrolase activity in the hemoglobin containing vacuoles. As a result of our present study, It might be concluded that, (1) hepatocytes have the capacity to take up circulating hemoglobin molecules, (2) degradatIOn of hemoglobin molecules occurs in the membrane-bound pertIcles and, (3) uptake of hemoglobin molecules is different from that of other serum components in the hepatocytes. Large hydropic vacuoles, frequently containing fibrin strands or a few erythrocytes, were observed in the late stage of bypass CIrculation. These vacuoles were formed by a large invagination of the cytoplasmic membrane, which might be related to the severe hYPOXIa and increase m sinusoidal pressure. Acknowledgement. The authors wish to thank Mr. T. Noml for excellent technical assistance, Prof. K. H. Chfton for readmg and MISS Y. Saiki for typmg the manUSCrIpt.

Intracytoplasmic InclusIOn Bodies m Hepatocytes . 361

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(1967) Neutze, J. M., Drakeley, M. J., Barratt-Boyes, B. G., and Hubbert, K.: Serum enzymes after cardiac surgery using cardiopulmonary bypass. Amer. Heart J. 88,425-442 (1974) Oueda, P. R.: AnOXIC changes of liver cells: electron mICroscopIc study after IllJection of collOidal mercury. Laborat. Invest. 12,386-394 (1963) Pfeifer, U., and Bannasch, P.: Zum Problem der "hyalillen Eiwed~tropfen" 1m Cytoplasma der Leberparenchymzellen: Llcht- und elektronenmlkroskoplsche Untersuchungen nach 3/4Hepatektomle. Vlrchows Arch. B Zellpath. 1,365-388 (1968) Plchotka, }.: Tlerexpenmentelle Untersuchungen zur pathologlschen Hlstologle des akuten Hohentodes. Beltr. Path. Anat. 107, 117-155 (1942)

362 . H. Kajlhara et al. Trowell, O. A.: The expenmental productIOn of watery vacuolatIOn of the hver. J. PhyslOl. 105, 268-297 (1946)

Received m revised form November 21, 1980 . Accepted December 18, 1980

Key words: Intracytoplasmic mclusion body - Hepatocyte - Extracorporeal ctrculation - Ultrastructure - Cytochemtstry Assoc Prof. Hirokl Ka)lhara, Department of Pathology, Hiroshima Umverslty School of Medlcme, 734 Hlroshlma-shl, Mmaml-ku, Kasuml 1-2-3, Japan