Int. Med J Vol. 8 No 1 June 2009

Histological Liver Changes in Streptozotocin induced Diabetic Mice

Imad M. Al-Ania; Noman D. Salih Al-Mishadanib; Raad K. Musliha and Salim

R.Hamoodi c

aDept. of Basic Medical Sciences, Kulliyyah of Medicine, International Islamic University Malaysia, Kuantan, Malaysia; bDept. of Biology, College of Science, Almustansiriah Univ. Baghdad Iraq; cDept of pathology, College of Medicine, Baghdad Univ. Baghdad Iraq.

ABSTRACT:

Streptozotocin (STZ) is selectively toxic to cells in the pancreatic islets. It is well

known that STZ causes specific death of B cells and induces diabetes mellitus.

This study has been conducted to detect the histological changes that could be

induced in liver of adult diabetic mice. The experiments were performed using a

single dose of STZ (150mg/Kg. I.P.). Animals were killed two, four and six weeks

after injection. The relative percentage of liver weight and liver histology were

studied. The results showed several morphological and histological alterations in

liver tissues, indicated by increase in the liver percentage of liver weight,

glycogen reduction, associated with lipid deposition, inflammatory cells infiltration

and Kupffer cells hyperplasia. Furthermore, this study illustrated the worsening of

liver histology over a short time in STZ diabetic mice.

INTRODUCTION

Streptozotocin (STZ) is an N-nitros0-n-methylurea derivative of 2-deoxy-d-

glucose, produced by Streptomyces acromogenes, and has been shown in

animal’s model to induce a chronic diabetic state by destruction of B bells in the

pancreatic islet tissue 1. Streptozotocin induced diabetes mellitus in many animal

species has been reported to resemble human hyperglycemic diabetes mellitus

2. It has been reported that STZ selectively damages pancreatic B cells and

produces much less toxic side effect than other chemical diabetogenic agents 3

and induces diabetes similar to poorly treated human diabetes, it develops many

features seen in human patients 4. Diabetes mellitus is a commonly occurring

disease, characterized by elevated plasma glucose concentrations resulting from

insufficient insulin, insulin resistance, or both 5, and it affects carbohydrate, fat

and protein metabolism. Diabetes is a complex and multifarious group of

disorders characterized by hyperglycemia that has reached epidemic proportions

in the present century 6 Diabetes is known to produce substantial changes in

intracellular metabolism in most tissues, including liver 7, 8.

Because of the importance of the liver in carbohydrate metabolism, the present

study is to investigate liver histopathological changes possibly occurring in

diabetic mice at different periods.

MATERIALS & METHODS

The experiments were performed on 200 male Swiss albino mice of the balb/c,

weighing 20-25 grams, in good health housed in plastic cages with free access to

water and food. They were bred in the animal house (Almustansiriah College of

Science), kept at room temperature and fed standard chew of pellets (Al-

Autiafiah Company) and water was provided adlibitum.

The animals were randomly divided into four groups of fifty; the first group was

the control, they were given saline-citrate buffer; the second, third, and fourth

groups were the diabetic groups, after fasting over night, injected with a single

dose of STZ (150mg/Kg, Sigma Chem. Co., USA) dissolved in citrate buffer PH

5.4 In order to prevent hypoglycemia, STZ-treated animals received a solution of

10% glucose instead of normal drinking water over the 24 hours following the

treatment.

The animals were sacrificed at the second, fourth and sixth week. After

sacrificed, the abdominal cavity was opened, the liver was removed to obtain its

weight, and small specimens of the liver were taken for histological examination.

Liver specimens were fixed in Boun’s solution for 12-16 hours. After fixation, they

were washed using many changes of 50%, 70% ethanol to remove the yellow

colour of picric acid, then they dehydrated through graded alcohols and cleared

using two changes of xylene and embedded in paraffin wax. Serial transverse

sections of 4-5 micron thickness were prepared using the microtome. The

sections then floated in a water bath at 50 C, and mounted on slides. The

sections were stained by Harris’s hematoxylin and eosin, others stained by

periodic Acid-Schiff stain (PAS).

RESULTS

A significant increase in the relative percentage of liver weight has been shown in

this study (Fig. 1).

Liver biopsy of the animal of the control group didn’t show any histological

changes during the period of the experiments, the hepatic lobules were seen

normally with polygonal hepatocytes having regular nucleus and cytoplasm (plate

1).

In the diabetic animals, two weeks after STZ-injection, the liver showed several

alterations including mild degree of fatty changes, cloudy swelling, mild infiltration

of lymphocytes with hemorrhage (plate.2).after weeks, STZ-diabetic animals

showed more progressive changes, sever congestion, necrotic foci, hydropic

changes, aggregation of lymphocytes between the hepatocytes (plate 3).

In the last group, mononuclear inflammatory cells infiltration, sever hydropic

degeneration changes and kupffer cell hyperplasia can be recognized (plate. 4).

Furthermore, alterations in glycogen contents in the hepatocytes were obtained n

specime4ns stained with PAS stain (plates. 5, 6, 7).

DISCUSSION

Attention has long centered on the liver in diabetes mellitus because of the

importance of this organ in carbohydrate metabolism and regulation of blood

sugar. Many studies 9, 10 revealed the occurrence of hepatic changes in some

cases of diabetic patients. Liver biopsy showed an accumulation of fats into the

hepatocytes, lead to a significant increase in liver weight. Liver enlargement has

been indicated in experimental diabetic rats 11 and mice 12.

Diabetes mellitus is one of the most common causes of fatty liver and the

frequent increase in the liver size in patients with diabetes mellitus was

recognized 9, 10. In the present study, there was a significant increase in the

weight of liver tissue in STZ diabetic mice as compared with the control group.

These results are in agreement with those of Cefalu et al 11 and Kume et al 12

and were related to the accumulation of fat into the hepatocytes, fatty changes in

hepatocytes, hydropic changes and cloudy swelling were observed in the present

study. It is well established that if a fatty liver from any cause persists, cirrhosis

will develop 13, 14. In the chronic phase of diabetes, an increase in liver weight or

hepatomegaly might occur due to glycogen deposition or fatty metamorphosis 15.

After four weeks, STZ-diabetic mice showed more progressive changes, there

was severe congestion in the in the portal area with necrotic foci, hydropic

changes and aggregation and infiltration of lymphocytes between hepatocytes,

further more there were hyperplasia of kupffer cells and significant gradual

reduction in glycogen content of the hepatocytes. Hamilton 10 indicated that

diabetes mellitus is one of the most common causes of fatty liver where fats are

accumulated in the hepatocytes; he indicates that fat comprises as much as 40%

of liver weight in patients with diabetes mellitus (It is 5% in normal liver).

Herman et al 16 related the hepatomegaly observed in STZ-induced diabetes in

rats to the hyperplasia in early phase and to the decreased apoptosis in the later

stage. Itoh et al 17 have considered that fatty infiltration of liver as a precursor of

cirrhosis in diabetic patients. Falchuk et al 13 showed hepatic fatty steatosis and

pericentral fibrosis in diabetic patients. They recognized hepatocytes were

markedly swollen and suggested that these abnormalities may represent an

intermediate lesion between fatty steatosis and cirrhosis. Nanji et al 9 mentioned

that the damage was mainly to the plasma membrane of the hepatocytes and

could be attributed to the elevation in aspartate aminotransferases in patients

with fatty infiltrations. The activity of serum alkaline phosphatase, aspartate

aminotransferases, alanine aminotransferases and levels of biluribin and

cholesterol were increased significantly in STZ- diabetic mice 18. Papaccio et al 19

found that STZ interferes with cellular metabolic oxidative mechanisms. Satav

and Katyara 7 studied the effect of STZ-induced diabetes on the oxidative energy

metabolism in rat liver mitochondria and found reduction in respiratory activity.

Lukivskaye et al 20 related the liver pathological changes in alloxan diabetic rats

to the mitochondrial abnormalities. Many authors suggested that liver

mitochondrial dysfunction in diabetes is related to the oxidative stress enhanced

in diabetic animals 21 and patients 22.

The decreased cellularity within the islets of langerhans observed in rats 19 and

rabbits 8 reflect the cytotoxicity of STZ on the B-cells. Whether the present

histopathological finding in the liver of mice indicates that STZ has a direct

cytoxic effect on the liver hepatocytes or it is indirectly through the induced

diabetes mellitus need further investigations.

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Correspondence

Department of BMS,

Faculty of amedicine

International Islamic University Malaysia