الفهرس 
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Abstract
Methotrexate is a chemotherapeutic drug which is used in the treatment of
many malignant conditions and some other diseases. Its side effects can be
neutralized by antioxidants which reduce reactive oxygen species and prevent
apoptosis. This study was done to evaluate the possible prophylactic and therapeutic
effect of L-carnitine on the submandibular salivary gland of methotrexate treated
rats.
Materials and Methods:
The study was carried out on fifty male Wistar albino rats with an average of
150 -200 grams. Animals were divided randomly into five groups, 10 rats for each
group as following:
▪ group 1 (Negative control): 10 rats received no treatment.
▪ group 2 (MTX treated group):10 rats received a single intraperitoneal
injection of 20 mg/kg MTX (Al-Refai et al., 2014; Fawzy et al., 2020) on day
5.
▪ group 3 (MTX with L-carnitine prophylactic group): 10 rats received:
• L-carnitine as intraperitoneal injection 500 mg / kg per day (days 1:10)
(Şener et al., 2006).
• MTX as single intraperitoneal injection 20 mg /kg on day 5.
• group 4 (MTX with L-carnitine therapeutic group): 10 rats received:
• 20 mg /kg of MTX as single intraperitoneal injection on day 5.
• L-carnitine 500 mg / kg was given as treatment from day 5 till day 10
(Şener et al., 2006; Al-Refai et al., 2014).
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▪ group 5 (L-carnitine treated group): 10 rats received L-carnitine as
intraperitoneal injection 500 mg / kg per day (days 1 :10) (Şener et al., 2006).
At the end of the experiment which lasted for 10 days, all rats were
anesthetized with intraperitoneal dose of ketamine (10%) and xylazine (2%) mixture
and were euthanized by overdose of ether.
After the glands were excised, specimens were fixed in 10% formalin buffered
saline for 48 hours. Then dehydrated, cleared, and infiltrated with molten paraffin
wax, followed by embedding in hard paraffin. Finally, sections of 3-5 microns
obtained and prepared for:
1. Haematoxylin & eosin stain to study the general histological changes.
2. Immunohistochemical localization of:
a) Caspase-3 as a marker of cell apoptosis.
b) BCL-2 as anti-apoptotic marker.
Results:
1. Mortality rate:
The percentage of mortality of both control and treated groups was 0%.
2. Clinical findings:
a)Clinical observation:
The clinical examination revealed that group 2 (MTX treated group)
showed remarkable changes in comparison with negative control group. Hair
loss, diarrhea, abdominal and extremities edema were noticed. Also severe
bleeding, dark red tissue and atrophy in gland was detected during sacrificing.
While the clinical examination of group 3 (MTX with L-carnitine
prophylactic group) and group 4 (MTX with L-carnitine therapeutic group)
showed improvement in clinical signs in comparison with group 2. As these
clinical signs disappeared in group 3 (MTX with L-carnitine prophylactic
Summary
105
group) but in group 4 (MTX with L-carnitine therapeutic group) bleeding
tendency and dark red color still be noticed.
b)Weight assessment:
group 1 (negative control) showed an increase in weight which was near
enough to group 5 (L-carnitine treated group). In group 2 (MTX treated group)
showed significant weight loss in contrary to group 3 (MTX with L- carnitine
prophylactic group) & 4 (MTX with L-carnitine therapeutic group) which
showed an increase in body weight of 15.3% and 11% respectively.
3. Histological results:
Sections of the submandibular salivary glands of the negative control group
revealed normal gland architecture. The gland was encapsulated by fibrous
connective tissue capsule which sent out septa that divided the gland into lobes and
lobules. The L-carnitine treated group showed a picture close to normal control
group with no remarkable difference in collagen content in trabeculae and around
ducts compared to control group.
Histological examination of the submandibular salivary glands of MTX
treated group revealed marked degeneration and disorganization in the parenchymal
elements including the acini and ducts in comparison to the controls with increase
of connective tissue septa spaces between the lobes. The serous acini showed a lot
of cytoplasmic vacuolization and pyknotic nuclei. The granular convoluted tubule
and striated ducts also showed loss of configuration. The excretory ducts show loss
of the pseudo stratification of their lining and most of ducts showed dilatation in
their lumens with secretion stagnation. Also dilatation of congested blood vessels,
apoptotic bodies and cellular infiltration were detected.
While sections of group 3 (MTX with L-carnitine prophylactic group) &
group 4 (MTX with L-carnitine therapeutic group) showed improved and partial
improvement in comparison with group 2 (MTX treated group) respectively.
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106
group 3 showed no widening of connective tissue septa spaces between the
lobes. Acinar cells and granular convoluted tubules (GCTs) showed almost normal
texture with no or little cytoplasmic vacuolization. The striated ducts regained their
basal striations. The excretory ducts showed pseudo stratification of their lining with
no stagnations in lumens. Normal blood vessels were detected.
While group 4 (MTX with L-carnitine therapeutic group) showed an increase
in fibrous CT content surrounding ducts and interstitial connective tissue. Few
Acinar cells and ducts of submandibular glands of the therapeutic group rats showed
almost normal architecture with little cytoplasmic vacuolization. Shrunken acini and
ducts still be noticed with minimal stagnation and cytoplasmic vacuolization. Some
of striated ducts regained their basal striations but the granular convoluted tubules
still sub normal. Some excretory ducts showed pseudo stratification of their lining
with no stagnations in lumens. Dilatation of blood vessels were still detected with
RBCs engorgement.
4. Immuonohistochemical results:
a. Caspase-3
The expression of apoptotic marker caspase-3 showed significantly increase
in group 2 (MTX treated group) which showed strong cytoplasmic reaction of acini
and ducts in comparison to group 1 (negative control group) which showed negative
to mild cytoplasmic reaction of caspase-3.
Submandibular salivary glands of L-carnitine treated group showed a slight
decrease in caspase-3 expression in comparison to negative control group which
showed negative cytoplasmic reaction of caspase-3.
Significant decrease in caspase-3 expression of group 3 (MTX with Lcarnitine prophylactic group) and group 4 (MTX with L-carnitine therapeutic group)
in comparison with group 2 (MTX treated group) as MTX with L-carnitine
Summary
107
prophylactic group showed showing negative cytoplasmic reaction of acini and
moderate cytoplasmic reaction of ducts of caspase-3. Methotrexate with L-carnitine
therapeutic group showed slight increase in caspase-3 expression compared to MTX
with L-carnitine prophylactic group which showed negative cytoplasmic reaction of
acini and moderate to strong cytoplasmic reaction of caspase-3.
b) BCL-2:
The expression of anti-apoptotic marker BCL-2 showed significantly decrease
in group 2 (MTX treated group) which showed negative cytoplasmic staining
reaction in comparison to group1 (negative control group) which showed mild
cytoplasmic staining reaction of acini and strong cytoplasmic staining reaction of
ducts.
Submandibular salivary glands of L-carnitine treated group showed a slight
increase in BCL-2 expression in comparison to negative control group which
showed mild cytoplasmic staining reaction of acini and myoepithelial cells and
strong cytoplasmic staining reaction of ducts of BCL-2.
Significant increase in BCL-2 expression of group 3 (MTX with L-carnitine
prophylactic group) and group 4 (MTX with L-carnitine therapeutic group) in
comparison with group 2 (MTX treated group) as MTX with L-carnitine
prophylactic group showed strong cytoplasmic staining reaction of acini and ducts
of BCL-2. Methotrexate with L-carnitine therapeutic group showed slight decrease
in BCL-2 expression compared to MTX with L-carnitine prophylactic group which
showed negative cytoplasmic staining reaction of acini and strong cytoplasmic
staining reaction of ducts