Sequence of histologic alterations in porcine liver and ... · process,suchasproteolysis,intheaffectedcells. Berembom etal. ' stated that autolysis and necrosisresult from isolating

9 3

SEQUENCE OF HISTOLOGIC ALTERATIONS

IN PORCINE LIVER AND GALL BLADDER

UNDERGOING POSTMORTEM AUTOLYSIS

BY

MICHAEL ADEKUNLE SALAKOD.V.M., Ahmadu Bello University, Zaria, Nigeria, 1975

A THESIS

submitted in partial fulfillment of the

requirements for the degree

MASTER OF SCIENCE

Department of Pathology

KANSAS STATE UNIVERSITY

Manhattan, Kansas

1978

Approved by:

Major Professor Xj

LD „

• T4-|CJ7£ TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

INTRODUCTION 1

REVIEW OF LITERATURE 3

PA.PER I - SEQUENCE OF HISTOLOGIC ALTERATIONS INPORCINE LIVER AND GALL BLADDER UNDERGOINGPOSTMORTEM AUTOLYSIS AT 26°C.

Introduction 13

Materials and Methods 13

Results 15

Discussion 20

Summary 2 2

References 24

Tables 25

Figures 30

PAPER II - SEQUENCE OF HISTOLOGIC ALTERATIONS INPORCINE LIVER AND GALL BLADDER UNDERGOING . .

POSTMORTEM AUTOLYSIS AT 4°C.

Introduction 40


Results 42

Discussion 45

Summary 4 7

References 49

Tables 51

Figures 56

PAPER III - SEQUENCE OF HISTOLOGIC ALTERATIONS IN

PORCINE LIVER AND GALL BLADDER UNDERGOINGPOSTMORTEM AUTOLYSIS 2 4 AND 48 HOURS AFTERFREEZING AT -10°C. PaS e

Introduction 66


Results 67

Discussion 70

Summary '*

References 73

Tables

Figures

GENERAL DISCUSSION 74

APPENDIX

ABSTRACT •

ACKNOWLEDGEMENTS

The author wishes to express his gratitude to his major

Professor, Dr. S. M. Kruckenberg, for his advice, guidance

and tolerance throughout his stay in the Department of

Pathology at Kansas State University.

The advice and supervision of Dr. S. M. Dennis, Pro-

fessor and Head, Department of Pathology is also appreciated,

Sincere appreciation is extended to the Ahmadu Bello Univer-

sity, Zaria, Nigeria, and to the KSU/AID for the opportunity

for this training. Dr. J. E. Cook and Dr. H. W. Leipold are

complemented for their advice, and Dr. S. G. Lake for help-

ing in the necropsy procedures

.

Finally, the author wishes to thank his parents for

giving to their children the education they did not have the

opportunity to get, and to his wife for her endurance and

perseverance.

INTRODUCTION

Postmortem decomposition or autolysis, is defined as

enzymatic self -digestion of cells or tissues after death.

It is an important phenomenon that must be taken into con-

sideration for accurately interpreting cellular and tissue

changes. Because it follows death of an animal as a

whole, inflammatory cells are not expected to be present as

in tissue necrosis. Autolysis was first noted in 1871

by Hoppe-Seyler , and Jacoby introduced the term "autolysis"

21in 1900. The extent of postmortem autolysis is influenced

by how long an animal has been dead, environmental factors

prior to and following death, body weight, external insula-

tion, nutritional status, age and species of animal.22

'20

' 30 ' 36,1 '2

Some work has been done on the study of postmortem

decomposition, 4' 5 '

6 ' 10 ' 11 '19

'23

'24

' 27 but most investiga-

tions have been directed towards the later stages of the

1

8

process, such as proteolysis, in the affected cells.

Berembom et al. ' stated that autolysis and necrosis result

from isolating a tissue from its normal supply of nutrients.

This results in interruption of metabolism by toxic, physical

7 8and chemical agents. Bradley ' claimed that autolysis can

be prevented by good blood supply, and Wilhelmi e_t al.37

' 38

demonstrated that- glucose and good blood supply helped to

prevent autolytic changes with anoxia.

The role of lysosomes can not be overemphasized: These

inner cytoplasmic organelles contain hydrolytic enzymes,

including acid phosphatase.28 These enzymes digest the

tissues which produce them and hence the term "autolysis"

or self-digestion.

Splitter and McGavin33 noted that hepatocyte nuclei

in guinea pigs disappeared almost entirely by karyolysis,

and that nuclear fading accompanied by margination of nuclear

chromatin was present by 6 hours after death at 26 C. Al-

2 7though Munger and McGavin did not observe pyknosis m

chicken hepatocytes , they observed karyorrhexis at 6 and

12 days postmortem at 4°C.

The concept of postmortem autolysis, simple as it

appears, is important. Recent studies indicated that indi-

vidual and species variation may be more significant than

previously thought.26

'33 This experiment was designed to

study the process in pigs.

The objectives of this study in pigs were to:

1. determine the sequence of histological changes in

the liver and gall bladder resulting from post-

mortem autolysis;

2. estimate the time postmortem when tissues would

not be useful under light microscopy;

3. determine the time postmortem giving the most

readable sections;

4. study the sequential histological changes in

liver and gall bladder in the intact carcasses

maintained at -10 C, 4 C, and 26 C.

2a

REVIEW OF LITERATURE

The importance of postmortem decomposition has been

realized for over a century. For a complete and accurate

histopathologic interpretation of cellular and tissue re-

actions, a pathologist must be able to differentiate between

22 20 31antemortem necrosis and postmortem decomposition. ' '

Since Hoppe-Seyler reported dissolution of dead tissue

without previous putrefaction in 1871, several workers have

33investigated the concept of autolysis in man, guinea pigs,

? 7 X ft \X 428chicken, rat, ' and other laboratory animals. '

21Jacoby introduced the term "autolysis" in 1900, after Sol-

kowski reported that postmortem decomposition was due to

3 2 7 8digestion of tissue by the enzymes it produced. Bradley '

claimed that autolysis can be prevented by good blood supply

3 7 3 8and Wilhelmi ejt al_. ' demonstrated that glucose and ade-

quate blood supply helped to prevent autolytic changes from

anoxia. It can be rightly assumed that, so far, most inves-

tigations on autolysis have been directed towards the later

18stages of the process, such as proteolysis in affected cells.

The liver is the largest organ in the body and since it

receives, through the portal blood, most of the absorbed

products from the small intestine, it is clear that many of

its functions relate to the metabolism of the absorbed pro-

14ducts. Among other functions of the liver are secretion

of bile; metabolism of protein; carbohydrate and fat; detoxi-

fication of harmful substances; storage of vitamins; destruc-

tion of red blood cells; formation of blood proteins, i.e.,

synthesis of fibrinogen, albumin, globulin and prothrombin.

The liver is also responsible for phagocytosis of foreign

particulate matter, conjugation of toxic substances and

steroid hormones and finally, hemopoiesis in utero and

potentially, in adults.

In vitro and in vivo experiments have been performed

with variable successes and setbacks. Results of in vitro

experiments by Cruickshank were influenced by inability to

collect tissue samples aseptically, edema, rapid cellular

plasmolysis, and retardation of nuclear changes in organs

kept in physiological saline. Organs kept in moist chambers

did not give accurate results since the nuclei degenerated

rapidly, like necrotic tissue in situ would degenerate.

Nevertheless, various observations were made regardless of

whether the experiments were conducted in vivo or in vitro .

These include Cruickshank' s observation in 1912 that auto-

lysis altered the histologic appearance of tissues, and that

the process of autolysis was also modified by the presence

of pathogenic bacteria. Gallagher et al. noted that the

initial autolytic process in the liver of the rat resulted

1

8

from failure of definitive enzyme systems. The labile

oxidative phosphorylation system, and the loss of respiratory

co-factors were believed to trigger the series of reactions.

It was also found that the addition of glucose and L-glutamate

18protected and reversed these failures. Morrione et_ al .

observed that microscopic sections prepared from livers

fixed in Zenker's solution or formalin had little or no de-

crease in stainable glycogen, when fixation was delayed for

as long as 10 hours after death or surgical biopsy. They

concluded from necropsies performed as long as 10 hours post-

mortem, that the microscopic appearance of glycogen in liver

was a good index for stainable hepatic glucose content at

time of death. However, chemical analysis for glycogen

revealed marked decreases for corresponding periods.

Studies have also been done on the role of cellular

components including deoxyribonucleic acid (DNA) , mitochon-

dria, and cell membrane. DNA studies by Stowell suggested

35that DNA was probably lost rapidly following death. This

was indicated by the rapid loss of the Fuelgen positive

19reaction. Ito, in his investigations, concluded that

mitochondria, granular reticulum, the nuclear envelope and

cellular membranes were more resistant to autolytic changes

than plasma membranes, Golgi complex and smooth surface

19 20reticulum. ' However, Trump reported that mitochondria

was the first to be affected by autolytic changes, and that

various regions of the plasma membrane degenerated differ-

ently. ' He noted loss of microvilli, interruptions in

membrane continuity, bleb formation and enlargement of the

space of Disse in the sinusoids, but not in bile canaliculi.

Following cell death, an early cytoplasmic change is

increased eosinophilia due to increased binding of eosin by

cytoplasmic protein as a result of pH change and loss of

9 23cytoplasmic basophilia. ' Swelling of mitochondria can

be observed as eosinophilic globules, and granulation and

irregular spaces represent denatured and digested cytoplas-

mic organelles.

Splitter and McGavin noted in guinea pig hepatocytes

undergoing autolysis that nuclei disappeared almost entirely

by karyolysis, and that nuclear fading accompanied by mar-

gination of nuclear chromatin was present by 9 hours post-

mortem. They also observed that hepatocyte cytoplasmic

basophilia started to disappear centrilobularly by 0.75

hours and was completely lost by 3 hours. Pyknosis of Kup-

ffer cells was also observed to have begun as early as 1.5

hours. Perhaps the most significant observation was indivi-

dualization of hepatocytes and the presence and prominence

of the space of Disse. They also observed separation of

the bile duct epithelium from the basement membrane at 18

hours

.

27Hunger and McGavin found that Kupffer cells and

periportal lymph nodules became pyknotic and fragmented

earlier than fading and karyolysis of nuclei of epithelial-

type cells of the bile ducts and hepatocytes. They also

indicated that pyknosis did not occur in chicken hepatocytes;

and that karyorrhexis was observed only 6 and 12 days post-

mortem at 4 C. Because most periportal lymph node cells and

most Kupffer cells either fragmented or disappeared by 12

hours, they suggested that Kupffer cells recognizable after

long periods may actually have been different, perhaps "en-

dothelial" cells.

Discussions about autolysis would be incomplete without

mentioning hydrolytic enzymes, and in particular lysosomes,

which are part of the inner organelles and contain the enzymes.12

3 13The concept of lysosomes was first introduced in 1955. '

Lysosomes contain great amounts of acid phosphatase and

2 8other hydrolytic enzymes. Pekkanen, in studying the

passage of acid phosphatase from lysosomes in bovine liver

undergoing autolysis, found that the total acid phosphatase

activity decreased with time. Sawant e_t a_l. also noted that

the stability of lysosomal membranes decreased when the pH

31of their surrounding deviated towards acidity or alkalinity.

The activity of lysosomal enzymes seems to be closely asso-

ciated with events resulting in or preceding physiological

or pathological cell death, and hence are important in the

autolytic process.

REFERENCES

1. Anderson, W. A. D., Pathology, Vol. 1, 7th ed. , C. V.

Mosby Co., St. Louis, Mo., (1972), pp. 94-95.

2. Anderson, W. A. D., Pathology, Vol. 2, 7th Ed., C. V.

Mosby Co., St. Louis, Mo., (1972), pp. 1321-1324.

3. Appelmans, F., Wattiauz, R. , and DeDeuve C, Tissue

Fractionation Studies 5. The Association of Acid

Phosphatase with a Special Class of Cytoplasmic

Granules in Rat Liver. Biochem J. 59, (1955), pp.

348-445.

4. Baker, H. D. C, Ischaemic Necrosis of Mouse Liver

Tissue in the Peritoneal Cavity. Lab Invest. 10,

(1961): pp. 11-128.

5. Berenbom, M. , Chang, P. I., Betz, H. E. , and Stowell,

R. E. : Chemical and Enzymatic Changes Associated

with Mouse Liver Necrosis in vivo . Cancer Research,

15, (1955): pp. 1-5.

6. Berenbom, M. , Chang, P. I., and Stowell, R. E. , Changes

in Mouse Liver Undergoing Necrosis in_ vivo . Lab

Invest. 4, (1955): pp. 315-323.

7. Bradley, H. C: Autolysis and Atrophy. Physiol.

Review, 18, (1938): pp. 73-196.

8. Bradley, H. C, Studies of Autolysis, VII. The Nature

of Autolytic Enzymes. J. Biol. Chem. , 52, (1922):

pp. 467-484.

9. Cheville, N. F. , Cell Pathology, 1st Ed., Iowa State

University Press, Ames, Iowa, (1976), p. 50.

10. Cruickshank, J. The Histological Appearances Occuring

in Organs Undergoing Autolysis, J. Path., 16 (1912),

pp. 167-183.

11. Dawkins, M. J. R. , Judah, J. D., and Rees, K. R.

:

Factors Influencing the Survival of Liver Cells

During Autolysis. J. Path. Bact., 77 (1959), pp.

257-275.

12. DeDuve, C: The Lysosome Concept. In Lysosome, Ciba

Foundation Symposium. Ed. V. S. de Reuck and M. P.

Cameron. J. A. Churchill, Ltd., London, (1963),

pp. 1-28.

13. DeDuve, C, Presman, B. C, Gianetto, R. , Wattiaux,

R. , and Appelmans, F. : Tissue Fractionation Studies

6. Intracellular Distribution Patterns of Enzymes

in Rat Liver Tissue: Biochem. J., 60 (1955), pp.

604-607.

14. Dellman, H. , Brown, E. M.: Textbook of Veterinary

Histology, 1st Ed. Lea and Febiger, Philadelphia,

PA, (1976), pp. 253-260.

15. Dorland's Illustrated Medical Dictionary, 25th Ed.,

Saunders. Philadelphia, PA.. .

16. Duke's Physiology of Domestic Animals, 8th Ed., Cornell

University Press; Ithaca, NY, (1970), pp. 369-397.

17. Florey, H. : General Pathology, 4th Ed., W. B. Saunders

Co., Philadelphia, PA, (1970), pp. 444-448.

10

18. Gallagher, C. H. , Judah, J. C, and Rees , K. R. , Enzyme

Changes During Liver Autolysis: J. Path. Bact.,

72, (1956): pp. 247-256.

19. Ito, S. : Light and Electron Microscopic Study of Mem-

branous Cytoplasmic Organelles. In the Interpretation

of Ultrastructural, Ed. by Harns , R. J. C, (1962):

pp. 129-148.

20. Ito, S. : Postmortem Changes of the Plasma Membrane.

In Electron Microscopy. Fifth International Con-

ference for Electron Microscopy, Philadelphia, PA,

August 29-September 5, 1962. Ed. 'by Breese, S. S.,

Vol. 2, pp. 1-5, New York, Academic Press, Inc. 1962.

21. Jacoby, M. , cited by Florey, H. , General Pathology,

4th Ed., W. B. Saunders Co., Philadelphia, PA, (1970),

pp. 444-448.

22. Jubb, K. V. F., and Kennedy, P. C, Pathology of Domestic

Animals, Vol. 2, Academic Press, NY, (1963), p. 247.

23. Lavia, M. F., and Hill, R. B., Principles of Pathobio-

logy, 2nd Ed., Oxford University Press. New York,

(1975), p. 13.

24. Majno, G. , LaGattuta, M. L., and Thompson, T. E.:

Cellular Death and Necrosis: Chemical, Physical

and Morphologic Changes in Rat Liver. Virch. Arch.

Path. Anat., 333, (1960), pp. 421-465.

25. Minjer, A., Histological Examination of the Breakdown of

Hepatic Glycogen by Postmortem Glycogenolysis and

by Action of Saliva, J. Path. Bact., 73, (1957), p. 11.

11

26. Morrione, T. G. , and Mamelok, H. C, Observations on

the Persistence of Hepatic Glycogen After Death,

Am. J. Path., 28, (1952), pp. 497-502.

27. Munger, L. L., and McGavin, M. D., Sequential Postmortem

Changes in Chicken Liver at 4, 20, or 37°C. Avian

Diseases, 16, No. 3, (1972), pp. 587-605.

28. Pekkanen, T. J., Distribution of Acid Phosphatase

During Autolysis in Bovine Liver, Acta., Vet. Scand.

,

11, (1970), pp. 283-294.

29. Runnels, R. A., Monlux, W. S., and Monlux, A. W.

,

Principles of Veterinary Pathology, 7th Ed., The

Iowa State University Press, Ames, Iowa, (1965), pp.

236-239.

30. Sawant, P. L., Desal, I. D., and Tappel, A. L. , Factors

affecting the lysosomal membranes and availability

of enzymes. Arch. Biochem. , 105, (1964), pp. 247-253.

31. Smith, H. A., Jones, T. C, and Hunt, R. D. , Veterinary

Pathology, 4th Ed., Lea and Febiger, Philadelphia,

PA, (1974) , pp. 21-23.

32. Solkowski, E. , cited by Florey, H. : General Pathology,

4th Ed., W. B. Saunders Co., Philadelphia, PA, (1970),

pp. 444-448.

33. Splitter, G. A., and McGavin, M. D. , Sequence and Rate

of Postmortem Autolysis in Guinea Pig Liver. Am.

J. Vet. Res. Vol. 35, No. 12, (1974)-, pp. 1591-1596.

12

34. Stowell, R. E. , Chang, J. P., and Berenbom, M.

,

Histochemical Studies o£ Necrosis of Mouse Liver

Tissue in Peritoneal Cavity. Lab. Invest. 10,

(1961), pp. 111-128.

35. Trump, B. F. , Goldblatt, P. J., and Stowell, R. E.

,

An Electron Microscopic Study of Early Cytoplasmic

Alterations in Hepatic Parenchymal Cells of Mouse

Liver During Necrosis in vitro (Autolysis). Lab.

Invest. 11, (1962), pp. 986-1015.

36. Trump, B. F., Goldblatt, P. J., and Stowell, R. E.,

Studies on Necrosis of Mouse Liver in Vitro,

Ultrastructural Alterations in Mitochondria of

Hepatic Parenchymal Cells. Lab. Invest., 14, (1965),

pp. 343-371.

37. Wilhelmi, A. E. , Engel, M. G., and Long, C. N. H.

,

Influence of Feeding on the Effects of Hepatic

Anoxia on the Respiration of Liver Slices in Vitro .

Am. J. Phys., Vol. 147, (1946), p. 181.

38. Wilhelmi, A. E. , Russell, J. A., Engel, F. L. , and

Long, C. H. H. The Effects of Hepatic Anoxia on

the Respiration of Liver Slices in Vitro . Am. J.

Phys., Vol. 144, (1947), p. 669.

12a

PAPER I


IN PORCINE LIVER AND GALL BLADDER UNDERGOING


13

INTRODUCTION

Autolysis is the enzymatic self-digestion of tissues

after death. The rate of autolysis is influenced by how

long an animal has been dead, environmental factors prior

to and following death, body weight, external insulation,

4 7 8nutritional status, age and species of animal. ' '

The first changes observed are loss of cytoplasmic

basophilia and granularity. Cytoplasmic eosinophilia be-

comes evident due to increased affinity for eosin by the

cytoplasmic proteins. In the nucleus, typical changes are

7 8pyknosis, karyolysis, and karyorrhexis .

' Occasionally,

margination of nuclear chromatin are observed, particularly

.9in guinea pigs.

Karyolysis is secondary to pyknosis in Kupffer cells.

Swelling and pyknosis are the most constant autolytic changes

gin Kupffer cells. Gall bladder epithelial sloughing is

rapid.

MATERIALS AND METHODS

Sixteen pigs of both sexes weighing about 17 kg and 6

to 8 weeks old were electrocuted by connecting one electrode

behind an ear and another on the tail and a current of 110

volts was passed for 10 seconds between the 2 electrodes.

Two pigs were necropsied as quickly as possible following

electrocution, and 1 x 6 x 3 cm sections of liver were re-

moved from the lateral aspects of the left and right lateral

14

lobes and immediately placed in 10% buffered neutral formalin

(BNF) to serve as controls. A piece of gall bladder was

also taken. The other pigs were kept in a chamber at a con-

stant temperature of 26°C and relative humidity of 50%.

Two pigs were necropsied at 3, 6, 12, 24, 48 and 96 hours

postmortem, respectively, and sections of liver and gall

bladder were taken and placed in 101 BNF.

Another set of control tissues was collected by anes-

thetizing 2 pigs by Rompuna intramuscularly. Blood was

allowed to flow by cutting the femoral vein and 101 BNF

was perfused under pressure by connecting a tube containing

the solution to the left ventricle. Perfusion was stopped

when the clear color of 10% BNF drained from the femoral

vein instead of blood. The pigs were necropsied and similar

liver and gall bladder sections were taken and fixed in 10%

BNF.

The tissues were dehydrated through graded ethanols,

cleared in xylene, and cut at 6 microns. They were then

stained with Harris' hematoxylin and eosine Y (H§E) . Some

sections were stained with periodic acid-Schiff (PAS) for

glycogen and control slides digested by diatase (PAS-diatase)

were also prepared to rule out glycoproteins which also

accept this stain. Sections were also stained with Spicer's

stain for cytoplasmic RNA and nuclear DNA.

9.

5 ,6-Dihydro-2- (2 ,6-xylidino) -H-l ,3-thiazine ; Haver-Lockhart

,

BAYVET Division, Cutter Laboratories, Inc., Shawnee, Kansas66201.

15

Microscopic observations were under magnifications

of 40 and 400 and scored as "present" or "absent", negative

(-) or positive (+) , or adequately described. The degree

of positivity ranged from plus one ( + 1) to plus three ( +++J ,

as indicated below.

Grade Remarks

+ Slightly present

++ Moderately present

+++ Distinct

RESULTS

Gross Findings

The perfused liver appeared lighter than non-perfused

which was mahogany red, the difference being attributed to

the effect of formalin flushing erythrocytes from the per-

fused liver. The color was retained for about 3 hours post-

mortem after which it became increasingly lighter and ir-

regular. Texture and firmness decreased from 6 hours post-

mortem and by 96 hours, the liver sections were virtually

"poured" into the 101 BNF. Bile imbibition started before

3 hours postmortem and increased with time depending on the

amount of bile present in the gall bladder.

16

Microscopic Findings

General Architecture

Persistence of the lobular pattern throughout the en-

tire experiment was remarkable. Even at 96 hours postmortem,

the interlobular fibrous connective tissue was still dis-

cernible. Both sets of control (zero hour perfused and zero

hour non-perfused) had few erythrocytes within the sinusoids

and blood vessels, though the sinusoids were dilated and

interlobular connective tissue was widened due to formalin

in the perfused sections (Fig. 1) . The portal triads were

prominent and all vessels were dilated in the controls (Fig. 3)

As time between death and necropsy increased, the amount

of blood pooling in the sinusoids increased until between 12

and 24 hours when hemolysis was advanced enough to mask rea-

sonable observations. Glycogen depletion also occurred

with time, but there appeared to be lobular selectivity re-

garding the extent of depletion. Glycogen depletion was most

marked in the midzonal region of the affected lobules.

Individualization of hepatocytes started at about 6

hours and increased progressively until 48 hours (Table 1).

At 96 hours postmortem, many lobules contained clear air or

gas spaces. Aggregates of extramedullary hematopoietic

centers were observed along some interlobular septa. These

cells were more hyperchromatic than hepatocytes in the con-

trols, but slowly lost their staining ability with time.

They were still observable as dark basophilic, round cells

even at 96 hours postmortem.

17

Erythrocytes

Erythrocytes were scanty in both sets of controls in

the sinusoids and vascular tissue (Fig. 2). This was ex-

pected in perfused pigs, and in recently killed pigs blood

flowed freely from the small sample collected in 101 BNF

.

Erythrocytes tended to be present in greater numbers from 3

till 12 hours (Table 11). Clumping of erythrocytes within

sinusoids and vessels, as well as loss of acidophilic stain-

ing, was observed at 6 hours. At 12 hours, most erythro-

cytes had disappeared as result of hemolysis and some dark-

brown pigments of acid hematin was visible in some sinusoids.

Ghost cells of erythrocytes were observed at 48 hours.

Sinusoids

Sinusoids were narrow in the zero hour non-perfused

sections but dilated in perfused tissues though blood cells

were not visible. At 3 hours postmortem, the sinusoids

were observed to contain blood and the width or extent of

dilation depended on the amount of blood present.

Portal Areas

The bile duct epithelium was preserved for as long as

12 hours postmortem. However, it contained pyknotic

nuclei and the cytoplasm was slightly eosinophilic. At 24

hours, sloughing and separation of the bile duct epithelium

was observed. The endothelial cells lining the veins were

still intact, though swollen, and with pyknotic nuclei at

24 hours (Fig. 4). At 48 hours, the portal triads were more

18

discernible than at 96 hours postmortem, and some pyknotic

nuclei were observed in the bile duct epithelium, though

most cells had sloughed and separated from the lamina propria.

Hepatocyte Cytoplasm

The cytoplasm was faintly basophilic and finely granu-

lar in both sets of control at zero hour. Large clear

spaces or vacuoles were observed in most hepatocytes that

were PAS positive, and negative for PAS-diatase, indicating

glycogen. Cytoplasmic basophilia started to disappear dif-

fusely at about 3 hours. At 6 hours, the cytoplasm was

more eosinophilic, with aggregation of basophilic material

in "clumps" in most hepatocyte cytoplasm. These basophilic

clumps persisted until 12 hours when the cytoplasm became

mainly eosinophilic and the granularity was completely lost.

At 96 hours, part of the cytoplasm was replaced by vacuoles

(Fig. 6). The fate of glycogen, determined by PAS and PAS-

diatase, indicated independent lobular activity. The dis-

appearance was greatest in most cases in the midzonal region,

and less so in the centrolobular and periportal areas. In

some lobules, glycogen disappearance was mainly centrolobular,

while in some, the pattern was periportal. Changes in

glycogen concentration was first noted at 3 hours and became

prominent by 6 hours. Total glycogen loss was not achieved

throughout the entire experiment, since PAS positive mater-

ials were present in hepatocyte cytoplasm even at 96 hours

postmortem.

19

Hepatocyte Nucleus

In both sets of control, the hepatocyte nuclei were

centrally placed and relatively large. Typically, one

nucleus was present per cell, but many hepatocytes had two

nuclei. The chromatin granules were mostly one or two, but

few cells had more than two. At 3 and 6 hours, the number

of chromatin granules per nucleus increased to an average

of 5 per nucleus. They were mostly scattered within the

nuclei, though many hepatocyte nuclei had marginally ar-

ranged chromatin. At 12 hours postmortem, some nuclei were

pyknotic and some karyorrhexic, but most appeared swollen

and fading. With increased cytoplasmic eosinophilia, nuclear

fading was more prominent at 24 hours (Table III). Some

nuclei appeared fragmented. At 96 hours, only ghost-like

appearance of nuclei was visible, as most had disappeared

(Fig. 6).

Kupffer Cells

The Kupffer cells appeared mostly flattened with hyper-

chromatic nuclei. The zero hour perfused controls, however,

had swollen Kupffer cells. Swelling and pyknosis of nuclei

of the Kupffer cells were observed to increase with time

and at 12 hours, they were oblong shaped (Table IV). At,

96 hours, nuclear fading was noted, though the nuclei were

still more hyperchromatic and discernible than hepatocyte

nuclei. Swelling of the entire cell was so marked in some,

that they resembled cells of the extramedullary hematopoie-

tic centers.

20

Gall Bladder

The amount of sloughing of gall bladder epithelium

was remarkable by 3 hours postmortem (Table V). Most of

the epithelial cells were separated from the lamina propria

and only spared in places where epithelial mucus was abun-

dant on the surface (Fig. 9). In such areas, the nuclei

were pyknotic and the cytoplasm eosinophilic. Exposed

lamina propria displayed vacuoles resembling pox vesicles

in areas where the epithelium had sloughed. The cells of

the lamina propria immediately below the epithelium were

vacuolated, empty and devoid of nuclei (Fig. 10). The

changes were less severe progressively towards the muscular

layer, the nuclei being present and pyknotic. The serosal

surface appears swollen and edematous.

DISCUSSION

The perfused liver sections were included in the experi-

ment to eliminate the possibility of autolysis between death

and collection of specimens in the zero hour non-perfused

control. Except for dilation of sinusoids and widening of

the interlobular spaces in the perfused pigs , no other

microscopic changes were detectable with the light microscope

Gross findings were in accordance with the basic prin-

7 8ciples of pathology ' namely, progressive loss of color

and strength, increased odor of putrefaction, and bile

21

imbibition. Double nucleated hepatocytes are frequently

2encountered in young, growing animals. Extramedullary

hematopoietic centers are also commonly encountered in

2 3young and sometimes in adult animals. ' The persistence

of fibrous connective tissue reflects the fact that they

are probably the last to undergo postmortem autolysis.

Glycogen was not completely lost, since stainable

glycogen was present even at 96 hours. Morrione and Mamelok

qand later, Splitter and McGavin made similar observations.

Although centrolobular and periportal glycogen depletion

were observed, most lobules had midzonal depletion, the

picture being similar to necrosis presented by pigs fed

aflatoxin and horses fed moldy hay. Splitter and McGavin

9also observed similar glycogen loss in guinea pigs.

Hepatocyte individualization was observed at 6 hours,

9the time reported by Splitter and McGavin. They observed

hepatocyte individualization as early as 6 hours postmortem

at 26°C. It is possible that the thick fibrous connective

tissue, responsible for the unique lobulation in porcine

liver, is also responsible for holding the cells together

and hence delaying individualization. It was also observed

that while hepatocyte nuclei either faded or became pyknotic,

the Kupffer cell nuclei became primarily pyknotic. Fading

was secondary.

Loss of cytoplasmic basophilia and hence an increase

in acidophilic staining is due to loss of nucleoproteins

ribonucleic acid and deoxyribonucleic acid from the

22

o

cytoplasm and nucleus. However, the persistence of baso-

philia in the form of cytoplasmic clumps in one area of the

cell while the rest of the cells gradually took to acido-

philic staining indicated that the cytoplasmic RNA was lost

gradually within 12 hours postmortem. The clumps stained

positive for Spicer's stain, indicating that they contained

RNA.

The rate of gall bladder epithelial sloughing is worth

mentioning. Total sloughing was achieved within 3 hours.

2Since the porcine gall bladder does not have a submucosa,

the lamina propria was always exposed following epithelial

desquamation. The cells of the lamina propria were devoid

of nuclei and cytoplasm and appeared like pox vesicles.

This was the most consistent feature of this study as varying

reactions were displayed by different cells. For example,

only about 70% of the nuclei examined revealed fading, the

rest pyknosis and fragmentation; 50% of the lobules presented

midzonal glycogen depletion, 25% centrilobular , 15% peri-

portal and about 10% diffuse glycogen depletion. It is,

however, significant that mucus appears to play a preser-

vative role, as gall bladder changes were less severe where

epithelial mucus was abundant.

SUMMARY

Sixteen pigs were electrocuted to study the sequence

of histologic changes in the liver and gall bladder.

23

Twelve pigs were maintained at 26° and 50% relative humid-

ity, and four served as controls and were necropsied imme-

diately. Liver and gall bladder sections were taken from

2 pigs at 3, 6, 12, 24, 48, and 96 hours postmortem, res-

pectively, and fixed in 10% BNF

.

In hepatocytes, loss of cytoplasmic basophilia was

first observed at 3 hours and cytoplasmic clumping was pro-

minent by 6 hours. Both loss of cytoplasmic granularity

and individualization of hepatocytes were observed by 6

hours postmortem. Karyorrhexis of hepatocyte nuclei was

not observed throughout the test period of 96 hours, but

fragmentation of nuclear chromatin started as early as 3

hours postmortem. Hemolysis of erythrocytes began at 6

hours and was advanced at 12 hours.

Unlike hepatocytes, Kupffer cells nuclei became pyknotic

rather than karyolytic. These changes were observable by

3 hours postmortem and became progressively more prominent

with time. Loss of cytoplasmic glycogen was not of any par-

ticular pattern, though midzonal depletion was most prominent

Sloughing of gall bladder epithelium was complete by 3

hours postmortem. Exposure of the underlying lamina pro-

pria was observed and cells were vacuolated. The epithe-

lium was preserved only where epithelial mucus was abundant.

The conventional porcine hepatic lobulation was maintained

throughout the entire experiment.

24

REFERENCES

1. Cheville, N. F. , Cell Pathology, 1st Ed., Iowa State

University Press, Ames, Iowa, (1976), p. 50.

2. Dellman, H. , Brown, E. M. : Textbook of Veterinary

Histology, 1st Ed., Lea and Febiger, Philadelphia,

PA, (1976), pp. 253-260.

3. Duke's Physiology of Domestic Animals, 8th Ed., Cornell

University Press, Ithaca, NY, (1970), pp. 369-397.

4. Jubb, K. V. F., and Kennedy, P. C: Pathology of

Domestic Animals, Vol. 2, Academic Press, New York,

NY, (1963) , p. 1247.

5. McGavin, M. D. and Kanke , R. : Hepatic Midzonal Necrosis

in a Pig Fed Aflatoxin and a Horse Fed Moldy Hay,

Vet. Path., 14: 182-187, (1977).

6. Morrione, T. G., and Mamelok, H. C: Observations on

the Persistence of Hepatic Glycogen After Death, Am.

J. Path., 28, (1952), pp. 497-502.

7. Runnels, R. A., Monlux, W. S. , and Monlux, A. W. , Prin-

ciples of Veterinary Pathology, 7th Ed. , The Iowa

State University Press, Ames, Iowa, (1965), pp. 236-239

8. Smith, H. A., Jones, T. C, and Hunt, R. D.: Veterinary


PA, (1974) , pp. 1-23.

9. Splitter, G. A., and McGavin, M. D.: Sequence and Rate

of Postmortem Autolysis in Guinea Pig Liver, Am. J.

Vet. Res., Vol. 35, No. 12, (1974), pp. 1591-1596.

25

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Fig. 1. Zero hour perfused liver. Note the effect of 10%BNF perfusion on Kupffer cells. H§E stain; x 400.

Fig. 2. Zero hour non-perfused control liver. H§E stain;x 400.

31

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32

Fig. 3. Zero hour perfused liver. Note dilation of vesselsof the portal triad and the interlobular septa. H§E stain;x 40.

Fig. 4. 12 hours liver, 26°C. Note loss of stainingability, nuclear fading and early separation of bile ductepithelium. H§E stain; x 400.

33

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34

Fig. 5. 24 hours liver, 26°C. Note hepatocyte individual-ization, acid hematin from Hemolyzed blood and the presenceof glycogen in hepatocyte. PAS stain; x 250.

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35

Fig. 5

36

Fig. 7. Zero hour control gall bladder, non-perfusedH$E stain; x 40.

Fig. 8. Zero hour control gall bladder, perfused H§Estain; x 100.

37

Fig. 8

38

Fig. 9. 3 hours gall bladder, 26°C. Note total absenceof epithelium and vacuolization of lamina propria. H$Estain; x 100.

Fig. 10. 12 hours gall bladder, 26°C. Note also autolysisin the musclaris. H§E stain; x 100.

39

Fig. 9

Fig. 10

39a

PAPER II




40

INTRODUCTION

Though numerous factors such as, how long an animal has

been dead, environmental factors prior to and following

death, body weight, external insulation, nutritional status,

age and species of animal are known to affect postmortem

autolysis,4

'8 not all factors affecting this phenomenon are

known.2 External insulation, such as fleece and subcutaneous

fat are known to affect the rate of postmortem autolysis.

These tend to keep the body temperature high and so acce-

lerate autolysis.

In studies on guinea pigs by Splitter and McGavin, it

was found that postmortem autolysis occurred twice as rapidly

at 37°C than at 20°C.10 Such morphologically altered find-

ings in the liver included bile duct epithelial sloughing,

hepatocyte individualization, loss of cytoplasmic granular-

ity and basophilia, pyknosis of Kupffer cells and sloughing

of gall bladder epithelial cells. This experiment was de-

signed to study the morphological histopathologic findings

in porcine liver and gall bladder undergoing postmortem

autolysis at 4 C.


A total of 16 pigs, weighing about 17kg and between 6

and 8 weeks of age, were used. They were electrocuted by

41

passing a current of 110 volts for 10 seconds between elec-

trodes placed behind an ear and the tail. They were then

kept at 4°C and two were necropsied at 3, 6, 12, 24, 48 and

96 hours postmortem, respectively. Liver sections taken

from both the right and the left lateral lobes were fixed

in 10% buffered neutral formalin (BNF) immediately after

removal.

For controls, 2 pigs were necropsied immediately and

fixed in 101 BNF. A second set of controls was obtained by

anesthetizing 2 pigs by Rompun intramuscularly. Blood

was allowed to flow by cutting the femoral vein and 10%

BNF was perfused under pressure by connecting a tube con-

taining the solution to the left ventricle. Perfusion was

stopped when the clear color of 10% BNF drained from the

femoral vein instead of blood. The pigs were necropsied

and similar liver and gall bladder sections were taken and

fixed in 10% BNF.

The tissues were dehydrated through graded ethanol,

cleaned in xylene, and cut at 6 microns. They were then

stained with Harris 1 hematoxylin and eosine Y (H$E) . Some

sections were stained with periodic-acid Schiff (PAS) and

PAS-diatase. Spicer's stain was also used, to detect

changes in cytoplasmic RNA and nuclear DNA.

-5,6-Dihydro-2- (2 ,6-xylidino) -H- 1 , 3- thiazine ; Haver -Lockhart,BAYVET Division, Cutter Laboratories Inc. , Shawnee Mission,66201.

42

RESULTS

Gross Findings

There was progressive loss of color and strength and

progressive increase in odor and bile imbibition. These

changes were not prominent until after 24 hours. The

livers were more friable than those of the control but were

not liquified as a result of autolysis by 96 hours post-

mortem.



There appeared to be little difference in the changes

observed in the interlobular connective tissue between the con-

trols and 96 hour livers at 4°C. The lobular pattern re-

mained relatively unaltered. The presence of erythrocytes

in the sinusoids was remarkable and hemolysis at both 12

hours and 24 hours postmortem was not pronounced at 4 C.

Glycogen was observed to decrease in amount as time post-

mortem increased. However, from the extent of reaction

with PAS and PAS-diatase, it decreased only slightly with

time postmortem.

Hepatocyte individualization was not prominent until

12 hours postmortem (Table I) , the number observed at 24

and 48 hours was small (Fig. 4). Staining ability of the

extramedullary hematopoietic centers were also less than ob-

served for the controls with time postmortem.

45

Erythrocytes

The number of sinusoidal erythrocytes was higher than

the controls from 3 hours onwards. At 12 hours postmortem,

hemolysis was not significant as erythrocyte clumping was

marked (Table IV). Loss of acidophilic staining was ob-

served, but only slightly. Little acid hematin was seen

since hemolysis was slight. These changes were more pro-

nounced at 24 hours postmortem.

Sinusoids

Sinusoidal dilation was marked as a result of conges-

tion. Except for the non-perfused control, sinusoidal

collapse was not observed throughout the study.

Portal Areas

Preservation of bile duct epithelium was observed for

as long as 24 hours. The nuclei were pyknotic and the cyto-

plasm slightly eosinophilic. Slight epithelial sloughing

was observed. The nuclei of endothelial cells lining the

veins were swollen at 24 hours, but the endothelial cells

were still intact. The cytoplasm, however, was slightly

eosinophilic. Sloughing, pyknosis and loss of cytoplasmic

basophilia were less severe at 96 hours postmortem than

would be expected at higher temperatures.


The significant observations included delay in loss

of cytoplasmic basophilia. Traces of eosinphilia in the

cytoplasm was not observed until 6 hours postmortem (Table I)

44

Condensation of cytoplasmic RNA was pronounced and persisted

until about 24 hours postmortem. At this period, cytoplasmic

granularity was almost completely lost. Glycogen depletion

also presented independent lobular activity. Centrolobular

loss of glycogen appeared to be predominant over midzonal

and periportal depletion. It was also observed that the

rate of depletion first detected at 3 hours postmortem by

PAS and PAS-diatase, was slow, since concentration of gly-

cogen granules within the cytoplasm only slightly altered

with time postmortem.

Hepatocyte Nuclei

The major observation was the intactness of nuclear

chromatin. Starting from 3 hours to 96 hours postmortem,

it was observed that most hepatocytes contained 1 or 2 ,or

occasionally 3 nuclear chromatin as in the controls (Fig. 2).

Fragmentation was not severe, and only observed in a few

hepatocyte nuclei. The few nuclei with fragmentation of

chromatin did not present marginally arranged chromatin,

but diffuse scattering within the nuclei.-

Kupffer Cells

Nuclei of the Kupffer cells had swelling and pyknosis

early (Fig. 5). From 6 hours postmortem, rapid swelling

was observed. Many cells were rounded at 12 hours, and by

24 hours postmortem, pyknosis was a major change. Fading

of Kupffer cells was observed at 48 hours and 96 hours

postmortem, but this appeared to be more important in cells

with swollen rather than pyknotic nuclei (Table III).

45

Gall Bladder

Sloughing of gall bladder epithelium was not prominent

until 6 hours postmortem (Fig. 10) (Table V). At 3 hours,

pyknosis of nuclei, swelling of epithelial cells and increased

cytoplasmic eosinophilia of these cells were observed (Fig. 9),

At 6 hours postmortem, the epithelium was preserved only

where overlying mucus was abundant. The exposed lamina

propria was also vacuolated, simulating pox vesicles. These

lamina proprial cells were devoid of nuclei immediately be-

neath the epithelium. These changes were less severe pro-

gressively towards the muscular layers. The serosal surface

appeared swollen and edematous at 12 hours postmortem.

DISCUSSION

In comparing autolysis in the liver at higher tempera-

tures with that of 4°C, the effect of temperature on post-

mortem autolysis is well elucidated. Since the rate of auto-

5 8lytic changes increase with temperature, ' the reverse

also may be true. Hepatocyte individualization was signi-

ficantly delayed since it was observed 12 hours postmortem

at 4°C. Hemolysis of erythrocytes, prolongation of cyto-

plasmic basophilia, sloughing of the bile duct epithelium,

pyknosis of the Kupffer cells and sloughing of the gall

bladder epithelium were changes also delayed. Glycogen

depletion preferred a centrolobular pattern. This observa-

tion is worth mentioning, though hepatocytes did not react

46

uniformly in the amount of glycogen contained initially,

and the rate or pattern of glycogen depletion.

Of significance is the condensation of RNA of the nu-

cleoproteins in the cytoplasm. The reactions with Spicer's

stain were intense. Liver cell death will be accompanied

by inhibition of RNA synthesis which is accompanied by frag-

f\ 7mentation of the nucleolus, ' and inhibition of protein

synthesis by some fat accumulation, and the appearance of

large aggregates containing ribosomes between the stacks of

rough endoplasmic reticulum. ' The condensation of these

ribosomes probably increases at lower temperatures and hence

is responsible for the increased cytoplasmic "clumping"

observed at 4°C. This may also explain the fragmentation

of nuclear chromatin observed to be more prominent at 26 C

than at 4°C.

Although all conditions determining the rate of post-

2mortem autolysis are not known, temperature plays an im-

portant part. Morphological differences were also observed

in this study and these changes were visible by light micro-

scopy and the naked eye. Retarding the rate of autolysis

by lowering the temperature may be explained by assuming

that temperature drop reduces the rate or extent of deviation

of pH from neutral towards acidity and this in turn affects

the stability of lysosomal membranes which are responsible

for releasing lysosomal enzymes. Since the activity of

lysosomal enzymes are closely related with the external and

3internal environment of the cell, a slight deviation in pH

47

and the preservation of RNA at lower temperatures will af-

fect their activity less than at higher temperatures.

Similarly, the effect of temperature on nuclear chromatin

may be attributed to the preservative effect of lower tem-

peratures on DNA, since DNA is believed to be rapidly lost

9following death.

SUMMARY

Sixteen pigs were electrocuted to study the sequence

of histologic changes in the liver and gall bladder at 4 C.

Two of them were perfused with 10% BNF prior to death and

necropsied immediately after death with two others that

were not perfused to serve as controls. The rest were

maintained at 4°C and two each were necropsied 3, 6, 12,

24, 48 and 96 hours after electrocution, respectively.

Their livers and gall bladder were removed and fixed in 10%

BNF.

Significant microscopic findings included delay of

hepatocyte individualization till 12 hours postmortem.

Hemolysis was not severe and erythrocyte clumping and cre-

nation were more pronounced. Anisocytosis was also observed,

Loss of hepatocyte cytoplasmic granularity and basophilia

were significantly delayed, and centrilobular glycogen

loss appeared predominant over midzonal and periportal

loss. Fragmentation of nuclear chromatin was not observed.

48

Swelling and pyknosis of Kupffer cells was observed.

Gall bladder epithelial desquamation was noted at 6 hours.

Lower temperatures probably reduced the activity of lyso-

somal enzymes by preventing rapid pH changes from neutral

towards acidity. The characteristic porcine hepatic lobu-

lation was preserved throughout the time period of this

study.

49

REFERENCES

1. Faber, J. L. and El-mofty, S. K. ,The Biochemical

Pathology o£ Liver Cell Necrosis, American J. of

Path., Vol. 81, No. 1, (1975), pp. 273-250.

2. More, H. R. , and Crowson, C. N., Glomerulotublar

Nephrosis Correlated with Hepatic Lesions, Arch.

Path, 60, (1950): pp. 63-84.

3. Pekkanen, T. J., Distribution o£ Acid Phosphatase

During Autolysis in Bovine Liver, Acta Vet. Scand.

,

11, (1970), pp. 283-294.

4. Runnels, R. A., Monlux, W. S., and Monlux, A. W.

,

Principles o£ Veterinary Pathology, 7th Ed., The

Iowa State University Press, Ames, Iowa, (1965),

pp. 236-239.

5. Sewant, P. L. , Desal, I. D. , and Trappel, A. L.,

Fac-

tors affecting the lysosomal membranes and availability

of enzymes. Arch. Biochem. 105, (1964), pp. 247-253.

6. Shinozuka, I. I.. Farber, J. L., Kornish, Y., Ana-

karahanonta, J.: D-galactosamine and Acute Liver

Injury. Fed. Proc. 32: (1973), pp. 1516-1526.

7. Shinozuka, I. I.. Martin, J. T. ,Farber, J. L.

,

The

induction of fibrillar nucleoli in rat liver cells

by D-galactosamine and their subsequent re-formation

into normal nucleoli. 'J. Ultrastruc. Res. 44,

(1973), pp. 279-292.

50

8. Smith, H. A., Jones, T. C, and Hunt, R. D., Veterinary


PA, (1974), pp. 21-23.

9. Stowell, R. E., Chang, J. P., and Berembom, M.,Histo-

chemical Studies o£ Necrosis of Mouse Liver Tissue

in Peritoneal Cavity, Lab. Invest. 10, (1961), pp.

111-128.

10. Splitter, G. A., McGavin, M. D.; Sequence and Rate of

Postmortem Autolysis in Guinea Pig Liver; Am. J.

Vet. Res., 12, (1974), pp. 1591-1596.

51

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57

Fig. 1

Fig. 2

Fig. 3. Six hour liver, 4 C. The Kupffer cells have under-gone early pyknosis even at this temperature. H§E stain;x 400.

Fig. 4. 12 hours liver, 4 C. Hepatocyte individualizationis prominent at this magnification. H§E stain; x 100.

59

Fig. 3

Fig. 4

60

Fig. 5. 24 hour liver, 4 C. Nuclear fading is prominentas well as Kupffer cell pyknosis. Note loss of stainingability. H$E stain; x 400.

Fig. 6. 48 hour liver, 4°C. Pyknosis is more distinctand hemolysis more advanced. H§E stain; x 400.

61

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63

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Fig. 8

64

Fig. 9. 3 hours gall bladder, 4 C. Note the incompletedesquamation of the epithelium and very little vacuoliza-tion of the lamina propria. H§E stain; x 400.

Fig. 10. 6 hours gall bladder, 4 C. Note total desquamationof epithelium and vacuolization of lamina propria. H§E stain;x 400.

65

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65a

PAPER III



AUTOLYSIS 24 AND 48 HOURS POSTMORTEM AT -10°C.

66

INTRODUCTION

It is known that postmortem autolysis is slower at

lower than at higher temperatures. It is also known that

tissues are frozen during transportation and storage to

minimize autolytic changes following death. This experi-

ment was designed to study what histologic alterations

occur when porcine liver and gall baldder are kept at

-10°C for 24 hours and 48 hours.


A total of 8 pigs, between 6 to 8 weeks of age and

averaging 17kg, were used. For controls, 2 were electro-

cuted by passing a current of 110 volts for 10 seconds be-

tween 2 electrodes, one placed behind an ear and the other

on the tail. Another set of controls was obtained by ad-

ministering Rompun a intramuscularly and then allowing blood

to flow by cutting the femoral vein while 101 buffered neu-

tral formalin (BNF) was perfused under pressure by connect-

ing a tube containing the solution to the left ventricle.

Perfusion was stopped when clear 101 BNF drained from the

5,6-Dihydro-2- (2 ,6-xylidino) -H-l ,3-thiazine , Haver-LockhartBAYVET Division, Cutter Laboratories, Inc., Shawnee, Kansas66201.

67

femoral vein instead of blood. All four control pigs were

necropsied immediately and liver and gall bladder sections

were taken and fixed in 101 BNF

.

The remaining four test pigs were similarly electro-

cuted and the intact cadavers stored in a freezer maintained

at -10 C. Two pigs each were necropsied at 24 and 48 hours

after freezing, and liver and gall bladder sections taken

and fixed in 101 BNF.

All tissues collected were then dehydrated through

graded ethanols, cleared in xylene, and cut at 6 microns.

Four sections of each liver and gall bladder samples were

cut and stained with hematoxylin and eosin Y (H§E) , Perio-

dic-acid Schiff (PAS), PAS-diatase and Spicer's stains.

Microscopic observations were made under light microscopy.

RESULTS

Gross Findings

Bile imbibition was slight but present, and all four

livers frozen for 24 and 48 hours presented little color

difference compared with those of the controls. The livers

frozen for 48 hours appeared lighter in color than the con-

trols and the 24 hour frozen livers. Shedding of gall

bladder epithelium was observed grossly at 48 hours.

68



The characteristic porcine lobules were retained in

both sets of frozen tissues. Erythrocytes were numerous

4 n the sinusoids and hemolysis was virtually absent even

at 48 hours (Fig. 3). The cord-like arrangement of hepa-

tocytes was retained and individualization was not observed.

Hepatocyte cytoplasmic basophilia and granularity were per-

sistent even at 48 hours. The cells of the extramedullary

hematopoietic centers were neither altered structurally nor

in their staining ability. Some heaptocyte cell membrane

rupture was observed at both 24 and 48 hours. These obser-

vations were more discernible with the PAS stain and when

predigested with diatase. Occasional freezing artifacts

were present at 48 hours.

Sinuoids

The sinusoids wer-e narrow at both 24 and 48 hours com-

pared with the controls. Erythrocytes were present and

clumped together. The narrowing of the sinusoids were at-

tributed to the swelling of the hepatocytes due to freezing

of intracytoplasmic water which increases in volume when it

forms ice.

Erythrocytes

Erythrocytes were most numerous around the portal areas

There was observable clumping and crenation, as well as

69

anisocytosis (Fig. 3). Hemolysis was not observed through-

out the entire experiment. The acidophilic staining ability

of the erythrocytes was retained for as long as 48 hours.


Cytoplasmic basophilia and granularity was persistent

for 48 hours. Eosinophilic staining was also evident at

both 24 and 48 hours after freezing; it was more intense

at 48 hours than at 24 hours. Clumping of cytoplasmic RNA

as basophilic globules was present but slight. Glycogen

was present in hepatocytes throughout the entire study

though the intensity of staining with PAS decreased with

time postmortem (Fig. 4).

Hepatocyte Nucleus

The nuclei were characteristically large and round.

There was negligible fading in a few hepatocyte nuclei

at 48 hours. Typically only one or two nucleoli were pre-

sent and these were centrally located. Fragmentation and

margination of nuclear chromatin was not observed through-

out the entire study. Pyknosis was also not observed.

Kupffer Cells

The Kupffer cells were swollen, pyknotic and oblong-

shaped both at 24 and 48 hours postmortem. At 48 hours,

many Kupffer cells were almost round. Their nuclei were

round and swollen and the cytoplasm could hardly be de-

lineated.

70

Portal Areas

The endothelial lining of the arteries and veins, and

the bile duct epithelium were preserved as long as 48 hours

The bile duct epithelial cells were swollen, and the endo-

thelial cells of veins were pyknotic and swollen at 48

hours. The walls of the vessels of the portal triad ap-

peared edematous.

Gall Bladder

The epithelium of the gall bladder was swollen and

nuclei were pyknotic at 24 hours after freezing. At 48

hours, the epithelial lining was mostly desquamated and

the wall of the gall bladder edematous. However, the

lamina propria was not vacuolated.

DISCUSSION

Since freezing reduces the rate of autolytic changes,

the observations were less severe than those present at

higher temperatures. Occasional cell membrane rupture oc-

curred in some cells. This may be attributed to freezing

of water content within the cytoplasm. Water, when frozen,

increases in volume and this increase may be responsible

for rupture of the cell membrane.

The Kupffer cells presented more noticeable changes

as they were swollen and pyknotic throughout the entire

test period. The alterations observed might have proceeded

71

before the freezing process involved the visceral organs,

as porcine subcutaneous fat is a good insulator and inter-

feres with heat exchange.

Epithelium of the gall bladder is probably the most

vulnerable structure, as there was separation of the

epithelium from the lamina propria by 24 hours after

freezing. In work by Splitter,1

it was found that the gall

bladder epithelium separated from the lamina propria in

the guinea pig as early as 9 hours at 4 C. This is rapid

in view of the fact that lowering the temperature has a

preservative effect on tissues.

Hepatocyte nuclear changes were minimal. The number of

chromatin remained the same as in the control. Pyknosis

was virtually absent in the hepatocytes.

SUMMARY

Eight pigs were electrocuted to study the sequence of

histologic changes in porcine hepatocyte and gall bladder

at -10°C. For control, two perfused and two non-perfused

samples were utilized. Two pigs were necropsied 24 and 48

hours after freezing and immediately fixed in 10% BNF

.

The cytoplasmic basophilia and granularlity was per-

sistent for 48 hours. Clumping and crenation of erythro-

cytes was marked and hemolysis absent. Hepatocyte nuclear

fragmentation and margination was not observed and pyknosis

72

was not prominent. The Kupffer cells were swollen, pyknotic

and oblong. Bile duct epithelial cells were swollen, while

the endothelial cells of the hepatic vein were swollen and

their nuclei pyknotic.

The gall bladder epithelium presented swollen, pyknotic

nuclei at 24 hours and desquamation of the epithelial cells

was observed at 48 hours. Concentration of erythrocytes

was highest around the portal triad. The typical porcine

hepatic lobulation was preserved. Fibrous connective tissue

is most resistant to autolysis. This may be attributed to

the fact that they contain few lysosomes and a corresponding

low level of hydrolytic enzymes.

73

REFERENCES

1. Splitter, G., Sequential Changes in the Guinea Pig Liver

and Gall Bladder Undergoing Postmortem Decomposition:

M.S. Thesis, KSU, 1970.

73a

GENERAL DISCUSSION

74

The most consistent observations made in this series

of experiments include loss of cytoplasmic granularity and

basophilia, sloughing and separation of gall bladder epithe-

lium from the lamina propria, fragmentation of nuclear

chromatin, nuclear fading and hepatocyte individualization.

Cytoplasmic granularity and basophilia are lost early

following death. In these experiments, it was observed

that loss of cytoplasmic basophilia was noted after 3 hours

postmortem at 26°C; 6 hours postmortem at 4 C, and 24

hours after freezing at -10°C (Table I). These findings .

were similar to those of Splitter and McGavin, who first

noted loss of cytoplasmic basophilia centrilobularly in the

guinea pig liver at 0.75 hours, and total loss of baso-

philia at 3 hours when the guinea pigs were kept at 20 C.

Sloughing and separation of gall bladder epithelium also

occurred early. It can be concluded that the gall bladder

is useful for histopathologic study after 3 hours postmortem

only if frozen, and even then, it is useful only if studies

are made before 24 hours after freezing.

Nuclear changes were fairly consistent. Fragmentation

and margination of nuclear chromatin were observed only at

26 C. These changes were not observed at 4 C and -10 C,

indicating that they were temperature dependent. Typical

nuclear changes were fading or lysis and to a lesser extent

pyknosis. The Kupffer cells in contrast to hepatocytes had

pyknosis and swelling, and fading was only secondary, even

under freezing conditions.

75

Hemolysis, which was observed at 26 C before 12 hours

was replaced at 4°C and -10 C by anisocytosis and crenation.

These findings were similar to the findings of Willot ejt al .

who noticed distortion and anisocytosis in guinea pig liver

erythrocytes after 4 hours postmortem. Although glycogen

was observed in all samples throughout the entire experiment,

midzonal loss of glycogen was most prominent at 26 C, while

at lower temperatures, glycogen loss had no particular pat-

tern. This is in agreement with the findings of Morrione

2and Mamelock, who found no relationship of glycogen dis-

appearance to hepatic lobule topography. At 4 C , however,

in this study more lobules had centrolobular loss of gly-

cogen than either midzonal or periportal.

Fibrous connective tissue is the most resistant to

autolysis, as there was persistence of interlobular con-

nective tissue throughout the entire experiment. This

reflects the fact that they have few lysosomes and a corres-

1 3ponding low level of hydrolytic enzymes. '

76

REFERENCES

Anderson, W. A. D. , and Kissane, J. M., Pathology;

7th Ed., C. V. Mosby, St. Louis, MO (1977), p. 94.

Morrione, T. G. and Mamelok, H. L., Observations on

the Persistence of Hepatic Glycogen After Death.

,

Am. J. Path., 28, (1952): pp. 497-502.

Smith, H. A., Jones, T. C. and Hunt, R. D., Veterinary

Pathology, 4th Ed., Lea § Febiger, Philadelphia, PA,

1972.

Splitter, G. , and McGavin, M. D., Sequence and Rate of

Postmortem Autolysis in Guinea Pig Liver; Am. J.

Vet. Res. 35, 12 (1974), pp. 1591-1596.

Willot, M. , Debarge , A., and Muller, P.; Aspects histo-

logiques: I'Autolyse du foie de cobaye. Acta Med

Leg et Soc (Liege), 20, (1967): pp. 21-64.

77

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Fig. 2. 24 hour liver, -10 C. The cells of the extra-medullary hematopoietic center are still more hyperchromaticthan the hepatocytes and Kupffer cells. Note also the redblood cells in the sinusoids. H§E stain; x 400.

80

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81

Fig. 3. 24 hours liver, -10°C. Erythrocytes are crenatedin sinusoids and portal vein. H§E stain; x 40.

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82

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82a

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IN PORCINE LIVER AND GALL BLADDER

UNDERGOING POSTMORTEM AUTOLYSIS

BY

MICHAEL ADEKUNLE SALAKOD.V.M., Ahmadu Bello University, Zaria, Nigeria, 1975

AN ABSTRACT OF A THESIS

submitted in partial fulfillment of the

requirements for the degree

MASTER OF SCIENCE

Department of Pathology

KANSAS STATE UNIVERSITY

Manhattan, Kansas

1978

The aim of this study was to determine the sequence

and rate of histologic changes resulting from postmortem

autolysis in the porcine liver and gall bladder, as well

as determining the effect of temperature on these changes.

Thirty-two pigs between 7 and 8 weeks of age with an

average weight of 17kg were used in the study. Thirty

were electrocuted with 110 volts for 15 seconds and 2 were

immediately necropsied and their liver and gall bladder

fixed in 101 buffered neurtal formalin (BNF) as controls.

Another set of control was obtained by perfusing 2 pigs

with 10% BNF after administering rumpum intramuscularly

and their liver and gall bladder were removed and fixed in

101 BNF. Twelve were maintained at 26°C and 12 at 4 C.

From these two test groups, 2 pigs were necropsied at 3, 6,

12, 24, 48 and 96 hours postmortem (PM) , respectively. The

remaining 4 pigs were frozen at -10°C and 2 were necropsied

at 24 and 48 hours PM, respectively. Sections cut from the

livers and gall bladder were stained with hematoxylin and

eosin.

The earliest changes were observed 3 hours PM at 26 C

and included sloughing of gall bladder epithelium, and loss

of hepatocyte basophilia and granularity. Fragmentation and

margination of nuclear chromatin were prominent at 12 hours.

Fading was the most typical nuclear change. Glycogen loss

was rapid but presisted as long as 96 hours. Lobular pattern

was retained.

Abstract 2

At 4 C, the changes were less rapid. Fragmentation

of nuclear chromatin was not observed and hepatocyte baso-

philia and granularity was significantly delayed till 6 hours

PM. Sloughing of gall bladder epithelium was observed at

6 hours. Hepatocyte individualization was marked at 12

hours. It was noted that changes at 4 C occurred half as

rapidly as they did at 26 C. Glycogen loss was noted to be

less rapid and to follow no particular pattern. Hemolysis

was less severe than at 26 C and clumping, crenation and

anisocytosis were observed.

At -10 C, hemolysis was absent. Clumping of erytho-

cytes and anisocytosis were prominent. There was pyknosis

of Kupffer cells and rupture of some cell membrane walls

due to freezing. The gall bladder epithelium was preserved

at 24 hours but had sloughed at 48 hours after freezing.

Fragmentation and pyknosis of hepatocytes were not observed.

The most rapid alterations observed in this study were

loss of cytoplasmic basophilia and sloughing of gall bladder

epithelium. However, following death, the Kupffer cells

lost their elongated, slender form and became swollen and

pyknotic at the temperatures employed. Bearing these al-

terations in mind, good histologic studies of the liver can

be made within 24 hours postmortem at 4°C and up to 48 hours

at -10°C.

Documents

Sequence of histologic alterations in porcine liver and ... · process,suchasproteolysis,intheaffectedcells. Berembom etal. ' stated that autolysis and necrosisresult from isolating