사염화탄소 중독에 의한 백서 간손상에 관한 연구

Abstract

[한글]

Studies on the Carbon Tetrachloride-induced Damages of the Livder in Rats

In Pyo Whang, M.D.

Department of Pathology Graduate School, Yonsei University

(Directors: Prof. Dong Sik Kim and Yoo Bock Lee)

Cirrhosis is relatively common disease in human, and it is known to progress

continuously once developed. The exact cause of the condition has not been

elucidated although many factors have been attributed including nutritional,

infectious and other hepatotoxic agents.

Many methods of induction of hepatic changes similar to human cirrhosis in

animals have been developed. Among which the most common one is carbon

tetrachloride induced hepatic changes in the rat(Davis 1924, Lamson and Wing 1962).

In 1936 Cameron and Karunaratne reported rather comprehensive studies on the

hepatic changes following carbon tetrachloride injections in to the rats. Many

other reports since then have proved that carbon tetrachloride produces various

changes in the liver of rats, from mild fatty degeneration to massive necrosis

leading to the cirrhosis(Wakim and Mann 1942, Ashburn et al. 1947, Glynn and

Himsworth 1944, Ungar 1947). It is also known that the type of changes differ

according to the amount given, route of administration, sex of the animals, and the

duration of administration(Cameron and Karunaratne 1936, Daniel et al. 1952, Reddy

et al. 1962, Reuber et al. 1967).

Recently Leduce and Wilson(1958), Della Porta et al.(1961) and Reuber and

Glover(1967) have reported induction of hepatocellular or cholangiocarcinoma like

lesions in various animals by long term administration of carbon tetrachloride.

However, true neoplastic nature of these lesions is questioned(Stewart and Snell

1957, Sidransky et al. 1963, Reuber and Glover 1967). Noh(1968) also noted marked

biliary proliferation in the liver of rats following carbon tetrachloride

injection.

Clinically, it is well established fat that serum protein alter quantitatively

and qualitatively in cirrhotic patients. However, its mechnism and correlation

between morphologic changes and functional alteration are still being investigated.

The present investigation is aimed to study the process of hepatic damages during

the carbon tetrachloride administration, and the process of healing after the

withdrawal of carbon tetrachloride, namely reversibility of the changes, with

special consideration of biliary proliferation.

Materials and Methods

Male albino rats weighing from 150 to 300 gms were divided into normal control,

carbon tetrachloride injected, and carbon tetrachloride discontinued groups. The

carbon tetrachloride injected and discontinued groups were subdivided into small

and large dosage groups. Carbon tetrachloride was injected subcutaneously once

every 3 days in a dose of 1.0ml. per kg for small and 2.0ml per kg for large dose.

Animals were fed with commercial diet containing more than 15% of protein.

Animals were killed at every 3 day intervals from initial injection until the 10th

injection. Discontinuation group received also 10 injections and then 5 animals

were subsequently killed in every 3 day lasting 30 days after the discontinuation

of carbon tetrachloride injections. All animals were killed by exsanguination from

carotid artery and the blood were saved for serum protein analysis. The liver and

other organs were examined grossly, and sections from the liver, pancreas, and

kidneys were fixed in 10% neutral formalin, which was followed by paraffin

embedding. Sections for microscopic examination were cur in 6u. thickness and all

sections were stained with hematoxylin and eosion. Sections of the liver were

stained also for reticulum, collagen, fat and glycogen. Serum protein on

exsanguinated blood were determined by reflector meter for total protein and

electrophoresis were made with Thomas cabinet model on cellulose polyacetate

strips.

Results and Discussion

Body weight decreased gradually during the administration of carbon

tetrachloride, but increased rather sharply after the discontinuation of carbon

tetrachloride. The first notable gross changes were yellowish dicoloration

associated with friableness of the liver, already at 3 days after the initial

injection. The granular cirrhotic appearance was first noticed at around 15 days

after the initial injection and was most marked at the 6th day after the

discontinuation of carbon tetrachloride, followed by gradual return to normal

smooth appearance at the 30th day after the discontinuation. No notable gross

alteration of the pancreas or kidneys were noted.

the most significant microscopic changes were confined to the liver, which

consisted of necrosis, fatty changes, inflammatory reaction, depletion of glycogen,

karyomegaly, development of hyaline bodies, increase of fibrous connective tissue,

biliary proliferation and regeneration of parenchymal cells.

The necrosis was centrilobular and characteristic findings in larger dose group

while the animals is smaller dose group showed only centriobular fatty changes. The

glycogen depletion was pararelled with fatty changes or necrosis at centrilobular

area, and when the fatty changes became diffuse the glycogen also disappeared

completely form the hepatic lobule. Either necrosis or fatty changes led into

collapse of hepatic lobules and followed by increase of fibrous tissue, first by

condensation or actural increase of reticulum and then by increase of collagen

fibers.

Markedly enlarged and hyperchromatic nuclei were frequently observed during and

after the cessation of carbon tetrachloride injections. These changes were

considered to be an expression of regeneration. Intracytoplasmic hyaline bodies

were numerous after the discontinuation of carbon tetrachloride injection, and it

was thought to be a process of intracellular sequestration of partially damaged

hepatic cells during regenerative phase.

The biliary proliferation started much later than necrosis or fatty changes. At

first it was an aggregation rather than actual proliferation in the area of

collapse of lobules after the necrosis or fatty changes. The surrounding reticulum

framework at this stage was loose while it became very dense at later stage. The

most marked biliary proliferation was noted in large dose group at the 12th day

after discontinuation producing a picture of adenoma like lesion. It is thought

that these biliary proliferations are mainly result of aggregation following the

collpase of hepatic lobules and partly actual proliferation. No encapsulation or

evidence of malignant transformation is noted.

After the discontinuation of carbon tetrachloride, hepatic parenchymal cells

regenerated to restore hepatic lobules which almost completed at the 30 days after

the discontinuation. As the regeneration of hepatic cells progress fibrous septal

tissue became compressed and finally disappeared indicating reversibility of

fibrosis in carbon tetrachloride induced cirrhosis of rats. Proliferated biliary

ductules also reduced to almost normal numbers.

The most significant changes of serum protein was continuous decrease of serum

albumin during the carbon tetrachloride administration associated with marked

increase of gamma globulin fraction, while they returned gradually to normal after

the cessation of carbon tetrachloride injection. These changes correlated closely

with morphologic alterations, namely decrease of albumin concommitant with

hepatocellular damage and increase of gamma globulin with mesenchymal

proliferation. In summary; large dose of carbon tetrachloride produced massive

necrosis and postnercrotic type of cirrhosis while small dose induced fatty changes

and Laennec type of cirrhosis. Biliary proliferation was partly apparent

aggregation and partly true hyperplasia. All of these changes were reversible after

the cessation of carbon tetrachloride injection.

[영문]

Cirrhosis is relatively common disease in human, and it is known to progress continuously once developed. The exact cause of the condition has not been elucidated although many factors have been attributed including nutritional, infectious and other hepatotoxic agents.

Many methods of induction of hepatic changes similar to human cirrhosis in animals have been developed. Among which the most common one is carbon tetrachloride induced hepatic changes in the rat(Davis 1924, Lamson and Wing 1962).

In 1936 Cameron and Karunaratne reported rather comprehensive studies on the hepatic changes following carbon tetrachloride injections in to the rats. Many other reports since then have proved that carbon tetrachloride produces various changes in the liver of rats, from mild fatty degeneration to massive necrosis

leading to the cirrhosis(Wakim and Mann 1942, Ashburn et al. 1947, Glynn and Himsworth 1944, Ungar 1947). It is also known that the type of changes differ according to the amount given, route of administration, sex of the animals, and the duration of administration(Cameron and Karunaratne 1936, Daniel et al. 1952, Reddy et al. 1962, Reuber et al. 1967).

Recently Leduce and Wilson(1958), Della Porta et al.(1961) and Reuber and Glover(1967) have reported induction of hepatocellular or cholangiocarcinoma like lesions in various animals by long term administration of carbon tetrachloride.

However, true neoplastic nature of these lesions is questioned(Stewart and Snell 1957, Sidransky et al. 1963, Reuber and Glover 1967). Noh(1968) also noted marked biliary proliferation in the liver of rats following carbon tetrachloride

injection.

Clinically, it is well established fat that serum protein alter quantitatively and qualitatively in cirrhotic patients. However, its mechnism and correlation between morphologic changes and functional alteration are still being investigated.

The present investigation is aimed to study the process of hepatic damages during the carbon tetrachloride administration, and the process of healing after the withdrawal of carbon tetrachloride, namely reversibility of the changes, with

special consideration of biliary proliferation.

Materials and Methods

Male albino rats weighing from 150 to 300 gms were divided into normal control, carbon tetrachloride injected, and carbon tetrachloride discontinued groups. The carbon tetrachloride injected and discontinued groups were subdivided into small

and large dosage groups. Carbon tetrachloride was injected subcutaneously once every 3 days in a dose of 1.0ml. per kg for small and 2.0ml per kg for large dose.

Animals were fed with commercial diet containing more than 15% of protein. Animals were killed at every 3 day intervals from initial injection until the 10th injection. Discontinuation group received also 10 injections and then 5 animals were subsequently killed in every 3 day lasting 30 days after the discontinuation

of carbon tetrachloride injections. All animals were killed by exsanguination from carotid artery and the blood were saved for serum protein analysis. The liver and other organs were examined grossly, and sections from the liver, pancreas, and kidneys were fixed in 10% neutral formalin, which was followed by paraffin

embedding. Sections for microscopic examination were cur in 6u. thickness and all sections were stained with hematoxylin and eosion. Sections of the liver were stained also for reticulum, collagen, fat and glycogen. Serum protein on exsanguinated blood were determined by reflector meter for total protein and

electrophoresis were made with Thomas cabinet model on cellulose polyacetate strips.

Results and Discussion

Body weight decreased gradually during the administration of carbon tetrachloride, but increased rather sharply after the discontinuation of carbon tetrachloride. The first notable gross changes were yellowish dicoloration associated with friableness of the liver, already at 3 days after the initial injection. The granular cirrhotic appearance was first noticed at around 15 days

after the initial injection and was most marked at the 6th day after the discontinuation of carbon tetrachloride, followed by gradual return to normal smooth appearance at the 30th day after the discontinuation. No notable gross alteration of the pancreas or kidneys were noted.

the most significant microscopic changes were confined to the liver, which consisted of necrosis, fatty changes, inflammatory reaction, depletion of glycogen, karyomegaly, development of hyaline bodies, increase of fibrous connective tissue,

biliary proliferation and regeneration of parenchymal cells.

The necrosis was centrilobular and characteristic findings in larger dose group while the animals is smaller dose group showed only centriobular fatty changes. The glycogen depletion was pararelled with fatty changes or necrosis at centrilobular

area, and when the fatty changes became diffuse the glycogen also disappeared completely form the hepatic lobule. Either necrosis or fatty changes led into collapse of hepatic lobules and followed by increase of fibrous tissue, first by condensation or actural increase of reticulum and then by increase of collagen

fibers.

Markedly enlarged and hyperchromatic nuclei were frequently observed during and after the cessation of carbon tetrachloride injections. These changes were considered to be an expression of regeneration. Intracytoplasmic hyaline bodies were numerous after the discontinuation of carbon tetrachloride injection, and it

was thought to be a process of intracellular sequestration of partially damaged hepatic cells during regenerative phase.

The biliary proliferation started much later than necrosis or fatty changes. At first it was an aggregation rather than actual proliferation in the area of collapse of lobules after the necrosis or fatty changes. The surrounding reticulum framework at this stage was loose while it became very dense at later stage. The most marked biliary proliferation was noted in large dose group at the 12th day after discontinuation producing a picture of adenoma like lesion. It is thought that these biliary proliferations are mainly result of aggregation following the

collpase of hepatic lobules and partly actual proliferation. No encapsulation or evidence of malignant transformation is noted.

After the discontinuation of carbon tetrachloride, hepatic parenchymal cells regenerated to restore hepatic lobules which almost completed at the 30 days after the discontinuation. As the regeneration of hepatic cells progress fibrous septal tissue became compressed and finally disappeared indicating reversibility of fibrosis in carbon tetrachloride induced cirrhosis of rats. Proliferated biliary ductules also reduced to almost normal numbers.

The most significant changes of serum protein was continuous decrease of serum albumin during the carbon tetrachloride administration associated with marked increase of gamma globulin fraction, while they returned gradually to normal after

the cessation of carbon tetrachloride injection. These changes correlated closely with morphologic alterations, namely decrease of albumin concommitant with hepatocellular damage and increase of gamma globulin with mesenchymal proliferation. In summary; large dose of carbon tetrachloride produced massive necrosis and postnercrotic type of cirrhosis while small dose induced fatty changes and Laennec type of cirrhosis. Biliary proliferation was partly apparent aggregation and partly true hyperplasia. All of these changes were reversible after the cessation of carbon tetrachloride injection.