الفهرس 
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Abstract
Cyclophosphamide is a cytotoxic prodrug that requires hepatic bioactivation to the biologically active species, phosphoramide mustard
and acrolein. It has been used in the treatment of Hodgkin’s disease,
lymphomas, leukemias, Wegener’s granulomatosis, severe rheumatoid
arthritis, and lupus erythematosus. It is also used in combination with
other drugs to treat breast cancer, leukemia, and ovarian cancer. The drug
also has immunosuppressant action when it has used in smaller doses.
Although CP plays an important role as a chemotherapeutic agent, many
adverse effects have been recorded on different body organs including
testes, kidney, and liver.
Ginkgo biloba (Ginkgoaceae) is one of the oldest living trees with a
long history of use in traditional Chinese medicine. The extract of a dried
leaf is popular for their uses as a diet supplement and herbal medicine in
various countries including USA, Europe, Japan, as well as South Korea.
Ginkgo biloba leaves extract is known for its specific actions on
improvement of blood flow, protective actions against damage by free
radicals, and anti-inflammatory effects. It has been used in the treatment of
cerebrovascular and peripheral vascular disorders, asthma, bronchitis,
heart disease, eye disease, short term memory loss, brain trauma and
depression.
The current study evaluated the protective potential of EGb on CP
induced histological, immunohistochemical, histochemical and
biochemical alterations in liver and testes of mice.Animal groups:
Ninety six adult male mice were used in the current study. They
were divided into 4 groups, each consist of 24 mice.
· First group:
They served as a control (non-treated) group.
· Second group:
Animals in this group were orally given EGb at a dose level of 100
mg/kg body weight/day, for 3 or 4 weeks.
· Third group:
Animals in this group were orally given CP at a dose level of 6.5
mg/kg body weight/day, for 3 or 4 weeks.
· Fourth group:
In this group, animals were orally given CP (6.5 mg/kg body
weight), then after 2 hours they were orally given EGb (100 mg/kg
body weight) daily for 3 or 4 weeks.
Twelve animals were dissected from each group after 3 weeks of the
treatment and the rest of animals were dissected after 4 weeks of the
experiment. Animals were dissected using excessive ether anesthesia
technique.
Slices of liver and testicular tissue from each group were
immediately removed after sacrificing, fixed in alcoholic Bouin’s fluid (for
histological study, general carbohydrates and total protein demonstration),
or in 10% formalin (for immunohistochemical demonstration of Bax and
Bcl-2). Paraffin sections (5μm thickness) were cut, stained with Ehrlich’s
hematoxylin and counter stained with eosin for histopathological
examination. For immunohistochemistry, antibodies directed against Bax
and Bcl-2 were used. For histochemical purposes, carbohydrates were
Summary
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demonstrated by periodic acid Schiff’s (PAS) technique, and total protein
visualized by mercury bromophenol blue method.
For biochemical analysis, liver transferases, ALP activities, LH and
testosterone levels were determined in sera of all groups. On the other
hand, activity of SOD and CAT, and MDA level were detected in liver and
testicular tissue homogenates.
The results can be summarized as follows:
I-Liver:
There was no histological change detected in livers of animals
treated with EGb (100 mg/ kg/ day) either for 3 or 4 weeks.
Animals treated with CP for 3 weeks showed many pathological
changes in liver including multiple scattered focal areas of inflammation,
degenerated hepatocytes, and enlarged nuclei. More pronounced
pathological alterations were observed in liver sections obtained from
animals treated with CP for 4 weeks. In these specimens, the majority of
hepatocytes showed fatty degeneration and enlarged nuclei. Different
nuclear abnormalities including nuclear inclusions, irregularity in shape,
and chromatin condensation were also seen. The portal venous channels
were congested and dilated, while the hepatocytes that surround the portal
areas showed foamy degeneration.
When animals were treated with both CP and EGb, gradual
improvement was detected in liver of these animals. This improvement
was more pronounced after 4 weeks. Most of hepatocytes were normal and
the nuclei were normal in size and in chromatin distributed. The nuclear
cytoplasmic ratio of the hepatocytes was significantly decreased as
compared to CP treated group.
Concerning the immunohistochemical findings, there was no
difference in Bax staining ability of liver sections between EGb treated
group and control group where cytoplasm of most of the hepatocytes was
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135
negatively stained for Bax expression. In animals treated with CP, multiple
scattered Bax positively stained hepatocytes were detected. When mice
treated with both CP and EGb, liver sections revealed marked reduction in
the number of Bax-positive stained hepatocytes as compared to CP treated
group. On the other hand, when liver sections were stained for Bcl-2
expression EGb treated mice revealed multiple scattered positively stained
hepatocytes all over the section and there was no statistical difference in
the mean of Bcl-2 positive cells of this group and the control group. In CP
treated group, marked disappearance of Bcl-2 positively stained
hepatocytes was detected while, the combined treatment with CP and EGb
succeeded to retain normal Bcl-2 expression in hepatocytes.
Histochemical observations revealed that liver sections of EGb
treated animals showed normal carbohydrate and protein contents. After
CP treatment, marked reduction in both carbohydrate and protein content
in liver sections was observed. Combined treatment of animals with both
CP and EGb showed gradual restoration of carbohydrate and protein
content and obvious improvement was detected after 4 weeks of treatment.
II- Testis:
Histological observations of testes of animals treated with EGb
revealed the same picture as in the control group. Examination of testicular
sections of mice treated with CP for 3 weeks showed marked pathological
alterations including irregularity in the basement membrane of many
seminiferous tubules, less compact and loosely arranged spermatogenic
cells separated from each other with vacuoles, hyaline bodies, cytoplasmic
vacuolization of spermatogonia, and degeneration in Leydig cells.
Significant reduction in the height of spermatogenic layer was recorded
while no statistical difference in diameter of seminiferous tubules was
detected between CP treated group and control group. All these changes
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increased with time and they were more obvious in testes of animals
treated with CP for 4 weeks. Animals treated with EGb in combination
with CP for 3 weeks revealed moderate pathological alterations as
compared to CP treated group at the same duration as regard to epithelial
height, compact of spermatogenic cells, Leydig cells, vacuolization, and
venous congestion. When animals coadministered with CP and EGb for a
longer period (4 weeks), they exhibited advanced degree of improvement
in their testicular tissues.
Concerning sperm count and abnormalities, CP treatment either for
3 or 4 weeks induced marked reduction in sperm count and elevated the
mean of sperm head and tail abnormalities. Treating animals with both CP
and EGb significantly elevated sperm count and reduced sperm
abnormalities over that in CP treated group.
Immunohistochemical observations revealed no statistical difference
in the mean of Bax-positive Leydig cells and Bcl-2 positively stained
Leydig cells of the control group and EGb treated group. Significant
increase in the number of Bax-positive Leydig cells per intertubular area
was detected after CP administration compared to the control group.
Coadministration of CP and EGb succeeded to decrease Bax expression in
Leydig cells as compared to CP treated group. On the other side, testicular
sections of animals treated with CP showed that most Leydig cells were
nearly negative for Bcl-2 immunostaining. Examined testicular sections of
mice treated with both CP and EGb showed upregulation in the expression
of Bcl-2 in Leydig cells as compared to CP treated group.
Histochemically, no visible changes were seen in carbohydrate and
protein content in all the testicular tissue of EGb treated animals when
compared to the control group. Slight increase in carbohydrate content was
detected in testicular tissue after CP administration which was gradually
reduced in the combined treated group with CP and EGb. Animals treated
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with CP showed gradual reduction in protein content in the cytoplasm and
nuclei of the majority of spermatogenic cells as well as in the intertubular
connective tissue. This protein reduction was more obvious after 4 weeks
of treatment. When animals treated with CP and EGb for 3 weeks, the
examined testicular sections showed partial restoration of the protein
content compared to CP treated group while after 4 weeks of the same
treatment, the testicular tissue reflected marked restoration of the protein
content.
Biochemical analysis:
Cyclophosphamide treated animals either for 3 or 4 weeks showed
significant increase in serum ALT, AST, and ALP activities when
compared to control group at the same durations. In addition, CP
administration for 3 or 4 weeks showed significant reduction in serum
level of LH and testosterone in a time dependent manner when compared
to control group at the same duration. On the other hand, animals treated
with CP and EGb managed to elevate serum ALT, AST, and ALP
activities as well as LH and testosterone levels as compared to CP treated
mice.
Concerning the antioxidant potential of EGb, combined treatment
with CP and EGb succeeded to elevate the antioxidant enzymes (CAT and
SOD) and reduce MDA level in liver and testicular tissue homogenate
compared to CP treated animals.
Finally, EGb has a protective effect on the histological,
immunohistochemical, histochemical, and biochemical changes induced
by CP in the liver and testicular tissue of albino mice.