cell biochemistry and function Cell Biochem Funct (2012) Published online in Wiley Online Library (wileyonlinelibrary.

com) DOI: 10.1002/cbf.2907

Antioxidant and antiapoptotic effects of proanthocyanidin and ginkgo biloba extract against doxorubicin-induced cardiac injury in rats
Noha Ahmed El Boghdady*
Biochemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt

Grape seed proanthocyanidins (GSPE) and ginkgo biloba extract (EGb761) are considered to have protective effects against several diseases. The cardiotoxicity of doxorubicin (DOX) has been reported to be associated with oxidative damage. This study was conducted to evaluate the cardioprotective effects of GSPE and EGb761 against DOX-induced heart injury in rats. DOX was administered as a single i.p. dose (20 mg kg1) to adult male rats. DOX-intoxicated rats were orally administered GSPE (200 mg kg1 day1) or EGb761 (100 mg kg1 day1) for 15 consecutive days, starting 10 days prior DOX injection. DOX-induced cardiotoxicity was evidenced by a signi cant increase in serum aspartate transaminase (AST), creatine phosphokinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), total cholesterol (TC) and triglyceride (TG) activities and levels. Increased oxidative damage was expressed by the depletion of cardiac reduced glutathione (GSH), elevation of cardiac total antioxidant (TAO) level and accumulation of the lipid peroxidation product, malondialdehyde (MDA). Signi cant rises in cardiac tumour necrosis factor-alpha (TNF-a) and caspase-3 levels were noticed in DOX-intoxicated rats. These changes were ameliorated in the GSPE and EGb761-treated groups. Histopathological analysis con rmed the cardioprotective effects of GSPE and EGb761. In conclusion, GSPE and EGb761 mediate their protective effect against DOX-induced cardiac injury through antioxidant, anti-in ammatory and antiapoptotic mechanisms. Copyright 2012 John Wiley & Sons, Ltd. key wordscaspase-3; doxorubicin; ginkgo biloba extract; grape seed proanthocyanidins; heart injury; oxidative stress; tumour necrosis factor-a

INTRODUCTION Doxorubicin (DOX) is a powerful anthracycline antibiotic used to treat a multitude of human neoplasms. However, studies have recognized severe cardiotoxicity after an acute, as well as cumulative, dose of DOX, which compromises the clinical usefulness of the drug.1 The chronic side effects of DOX including the development of cardiomyopathy are irreversible and can lead to a potentially congestive heart failure.2 The cause of DOX cardiotoxicity is multifactorial, but most DOX-induced cardiotoxicity can be attributed to the formation of reactive oxygen species (ROS), which ultimately results in myocyte apoptosis.3 An alternative approach is the use of cardioprotective drugs as adjuvants to DOX therapy. Agents that prevent cardiotoxicity would allow us to exploit the full therapeutic potential of DOX, making a tremendous impact on cancer therapy. Polyphenols, present abundantly in vegetables and fruits, have been recognized as functionally active molecules possessing a novel spectrum of biological, therapeutic and chemopreventive properties.4,5 Proanthocyanidins are the most abundant phenolic compounds in grape seeds and have
*Correspondence to: Noha Ahmed El Boghdady, Biochemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt. E-mail: nohaelbogdady@yahoo.com

been reported to exert antibacterial, antiviral, anticarcinogenic, antimutagenic, anti-in ammatory, antiallergic, and vasodilatory actions.5 Grape seed proanthocyanidins (GSPE) were provided to be highly bioavailable and provide signi cantly greater protection against damage of oxidative stress than vitamins C, E and b-carotene.6 Proanthocyanidins have been reported to inhibit lipid peroxidation, platelet aggregation, capillary permeability and fragility, and also modulate the activity of some enzyme systems including phospholipase A2, cyclooxygenase, and lipoxygenase.5 A large number of reports demonstrated that proanthocyanidins could both enhance the activity of chemotherapeutic agents and diminish their normal tissue toxicity.6,7 Extracts from the leaves of ginkgo biloba have been widely used therapeutically in China and Western countries for years. Standard ginkgo biloba extract (EGb761) contains 24% avone glycosides (kaempferol, quercetin and isorhamnetin) and 6% terpenoid (diterpene lactones, namely, ginkgolides and bilobalide and the bi avones ginkgetin, isoginkgetin, bilobetin).8 The extracts have been found to possess cardioprotective, antiasthmatic, antidiabetic, hepatoprotective and potent CNS activities.911 Furthermore, this extract has been shown to modulate expression of apoptotic related genes, reduce generation of free radicals and increase activity of antioxidant enzymes in various types of animal tissues and cells,12,13 implying
Received 20 June 2012 Revised 22 July 2012 Accepted 10 September 2012

Copyright 2012 John Wiley & Sons, Ltd.

n. a. e. boghdady that it is a promising cytoprotective agent against a range of exogenous toxic stimuli. Therefore, the present study was undertaken to evaluate the cardioprotective effects of GSPE and EGb761 on cardiac injury induced by DOX in rats via monitoring the changes of different biochemical parameters such as serum aspartate transaminase (AST), lactate dehydrogenase (LDH), creatine phosphokinase isoenzyme (CK-MB), total cholesterol (TC) and triglycerides (TG); endogenous cardiac antioxidants as reduced glutathione (GSH) as well as total antioxidant level (TAO) and lipid peroxides expressed as malondialdehyde (MDA). Moreover, the cardiac tissue damage marker, tumour necrosis factor-a (TNF-a), and the apoptotic marker, caspase-3, were measured. GSPE or EGb761 was administered for the next 5 days. At the end of the experimental period, rats were overnight fasted. Then the animals were sacri ced by decapitation; blood was collected and centrifuged at 800 g at 30  C for 15 min. The separated serum was used to estimate TC, TG, AST, LDH and CK-MB. The hearts were dissected out immediately, washed with ice-cold saline and blotted dry using lter paper. Tissue samples from the heart were taken for histopathology. A 20% of heart homogenate was prepared in ice-cold double distilled water. The obtained heart homogenate was aliquoted and immediately frozen at 80 C for biochemical analysis. Estimation of cardiac function enzymes activities, TG and TC levels Spectrophotometric diagnostic kits were used for the estimation of AST, LDH and CK-MB activities,1618 as well as TG19 and TC20 levels. Serum enzyme activity and lipid pro le were expressed as U l1 and mg dl1 respectively. Estimation of cardiac MDA An aliquot of the homogenate was mixed with equal volume of 2.3% KCl and centrifuged at 600 g for 15 min at 4  C. The supernatant was used for the determination of malondialdehyde (MDA), identi ed as the product of lipid peroxidation that reacts with thiobarbituric acid (TBA) to produce a pink-coloured complex that can be measured spectrophotometrically at 532 nm and 520 nm using tetramethoxypropane as a standard.21 The difference between the two determinations was calculated as TBA value and expressed as nmol mg1 protein. Estimation of cardiac GSH A second aliquot of the homogenate was mixed with equal volume of 7.5% sulfosalicylic acid. The contents were mixed well for complete precipitation of proteins and centrifuged at 600 g at 4  C for 10 min. The protein-free supernatant was used for the estimation of the reduced glutathione (GSH) level based on the reaction of GSH with 5,5-dithiobis-2-nitrobenzoic acid, forming a product that has a maximal absorbance at 412 nm. The results were expressed as mg gm1 tissue.22 Estimation of cardiac TAO Another aliquot of the homogenate was mixed with equal volume of cold phosphate buffer (5 mM potassium phosphate, pH 7.4, containing 0.9% sodium chloride and 0.1% glucose) and subsequently centrifuged at 800 g for 15 min at 4  C. The obtained supernatant was used for the determination of TAO level based on the ability of the antioxidant molecules in the sample to react with a de ned amount of exogenously provided hydrogen peroxide (H2O2) causing its decomposition. The residual H2O2 is determined by an enzymatic reaction that involves the conversion of 3,5-dichloro-2-hydroxybensulphonate to a coloured product measured colourimetrically at 505 nm. TAO level was expressed as mmol mg1 protein.23
Cell Biochem Funct (2012)

MATERIALS AND METHODS Chemicals and kits DOX (AdriblastinaW) was obtained from Pharmacia (Milan, Italy). GSPE, commercially known as NoxylifeW, was obtained from Pharco Pharmaceuticals Company (Alexandria, Egypt). GSPE is a standardized waterethanol extract from grape seeds. The extract was supplied in the form of standardized 95% oligomeric proanthocyanidins. EGb761, commercially known as TanakanW, was obtained from Amriya for Pharmaceutical Industries (Alexandria, Egypt). The biochemical kit for estimation of serum AST was supplied by Quimica Clinica Aplicada (Spain). Serum TC, TG, LDH and CK-MB kits were purchased from Stanbio (Texas). Cardiac TAO level was measured by using a commercial kit supplied by Bio-diagnostic (Egypt). Cardiac TNF-a ELISA kit and caspase-3 colourimetric assay kit were purchased from Quantikine, R&D Systems (Minneapolis, MN). All other chemicals used in the experiment were of the nest analytical grade and obtained from Sigma-Aldrich Co. (USA). Animals and experimental design Thirty-two adult male Wistar albino rats weighing 200250 g were used in this study. They were obtained from the animal house of the Faculty of Medicine, Cairo University, Egypt. They were kept under controlled conditions, fed standard chow diet, and provided with free access to food and water. The study was approved by the Ethical Committee for Animal Experimentation of the Faculty of Pharmacy, Cairo University. After 1 week of acclimatization, animals were randomly divided into four groups of eight rats each. Group I (NC): Normal control untreated rats received orally an equivalent volume of normal saline based on body weight. Group II (DOX group): Rats were injected with a single dose of DOX dissolved in normal saline (20 mg kg1 i.p.). This dose is well documented to induce cardiotoxicity in rats.14 Group III (DOX + GSPE): Rats were administered GSPE (200 mg kg1 day1), orally7 dissolved in normal saline for 10 days. Group IV (DOX + EGb761): Rats were administered EGb761 (100 mg kg1 day1), orally15 for 10 days. Following a single dose of DOX on the 10th day of pre-treatment, either
Copyright 2012 John Wiley & Sons, Ltd.

modulation of doxorubicin cardiotoxicity Estimation of cardiac TNF-a and caspase-3 levels A portion of the heart was weighed (~50 mg) and homogenized in 0.8 ml lysis buffer, pH 7.4. The lysate was centrifuged at 10 000 g for 15 min at 4  C, and the supernatant was taken for estimation of the TNF-a and caspase-3 levels. TNF-a was measured by solid phase sandwich ELISA using two kinds of high speci c antibodies. Tetramethyl benzidine was used as chromogen. The strength of colour measured at 450 nm was proportional to the quantities of rat TNF-a, which was expressed as pg mg1 protein.24 The caspase-3 colourimetric assay was based on the spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the labelled substrate DEVD-pNA (acetyl-Asp-Glu-Val-Asp p-nitroanilide). The pNA can be quanti ed using a spectrophotometer at 405 nm.25 The caspase level was expressed as optical density.
Table 1.

Estimation of protein in cardiac homogenate The protein content of the different fractions, resulting from ultracentrifugation of heart homogenate was determined by the method of Lowry et al.26 using bovine serum albumin as the standard. Heart histological examination Heart specimens were xed in 10% formaldehyde and subsequently embedded in paraf n and sliced into slices of 4-mm thickness followed by staining with hematoxylin and eosin (H&E) and examined under light microscope.27 Statistical analysis Statistical analysis was performed by GraphPad InStat software. Means and standard error of means were calculated, and statistical signi cance was tested by one-way ANOVA.

Effect of GSPE and EGb761 on serum AST, LDH, CK-MB, TC and TG in DOX-induced cardiac injury in rats NC DOX 8.5 0.42 ** 472 42.7 *** 101 8 *** 153 10.9 *** 124 10.8 *** DOX + GSPE 6.75 0.25 ## 265 19.99 ### 46 4.2 ### 64 4.4 ### 48 3.9 ### DOX + EGb761 6.88 0.35 ## 243 23.09 ### 46 3.8 ### 69 7.4 ### 53 4.3 ###

AST (U l1) LDH (U l1) CK-MB (U l1) TC (mg dl1) TG (mg dl1)

6.5 0.27 247 17.5 49 4.5 56 6.7 54 5.4

Data represent the means SEM (n = 8). **P 0.01, as compared with the normal control group. ## P 0.01, as compared with DOX-intoxicated rats. ***P 0.001, as compared with the normal control group. ### P 0.001, as compared with DOX-intoxicated rats.

(C)

Figure 1. Effect of GSPE and EGb761 on cardiac (A) MDA, (B) GSH and (C) TAO levels of normal control, DOX-intoxicated, DOX + GSPE and DOX + EGb761 rats. Data represent the means SEM (n = 8). *P 0.05, **P 0.01, and ***P 0.001, as compared with normal control group. #P 0.05, ##P 0.01, ###P < 0.001, as compared with DOX-intoxicated rats Copyright 2012 John Wiley & Sons, Ltd. Cell Biochem Funct (2012)

n. a. e. boghdady The strength of association between pairs of variables was assessed by Pearson correlation coef cient. The level of signi cance was set at P 0.05. RESULTS Biochemical results Testing of the cardiac function and the lipid pro le following a single injection of DOX (20 mg kg1 i.p.) in normal rats revealed signi cant elevations in serum AST, LDH, CK-MB, TC and TG, which were restored by either GSPE or EGb761 treatment (Table 1). A considerable elevation of lipid peroxides (MDA) and TAO levels in cardiac tissue was demonstrated following DOX injection. The elevated MDA was ameliorated, but the elevated TAO was apparently reduced following treatment with either GSPE or EGb761 (Figure 1). Further evidence for DOX-induced toxicity in the cardiac tissue was the profound reduction in the GSH level. GSPE and EGb761 have succeeded in elevating the reduced cardiac GSH level (Figure 1). Both TNF-a and caspase-3 levels showed about four- and 1.5-fold elevation, respectively, following DOX injection. These rises were signi cantly ameliorated by GSPE and EGb761 (Figure 2). Histopathological results As shown in Figure 3, the heart of normal rats revealed no histopathological alteration. Normal histological structure of the myocardium was recorded (A). By contrast, heart sections from rats receiving DOX showed hyalinization of the myocardium in a focal manner (B and C), in association with in ammatory cells in ltration in pericardium with oedema in both pericardium and in between the myocardium (D). Concurrent administration of GSPE (E) and EGb761 (F) improved the alterations in heart morphology as evidenced by decreased and even abolished myocardial hyalinization in GSPE and EGb761, respectively. However, congestion in myocardial blood vessels and mild vacuolization in the myocardial cells were still detected in GSPE and EGb761, respectively (Table 2). DISCUSSION Effective anticancer therapy with DOX and other quinine anthracyclines is severely limited by acute and chronic side effects such as myelosuppression and cardiotoxicity.28 The present study was planned to investigate the cardioprotective effects of GSPE and EGb761 against DOX-induced cardiomyopathy and apoptotic changes in rats. Interestingly, the cytotoxic activity of DOX is partly related to its quinine structure. DOX is converted into its semiquinone form in the cardiac myocyte by myocardial cytochrome P450 and avin monoxygenases. The semiquinone form is a toxic, short-lived metabolite and interacts with molecular oxygen and initiates a cascade of reaction, producing ROS.29 Another reported mechanism of DOX-induced oxidative stress is the formation of an anthracycline-iron (Fe2+) free radical complex.30 The latter reacts with hydrogen peroxide to produce hydroxyl (OH) radical. ROS react with lipids, protein and other cellular constituents to cause damage to mitochondria and cell membranes of the heart muscle cells. The DOX-induced cardiotoxicity, which was observed 5 days after treatment, was documented by the signi cant increments in the activities of serum AST, LDH, and CKMB, which may attributed to the myocardial membrane damage produced by DOX and the leakage of these enzymes from cardiac cytosol into blood. The current results were consistent with previous studies.14,31 These changes were corrected to normalcy upon oral administration of either GSPE or EGb761 to DOX-intoxicated rats. The DOX-induced hyperlipidemia seen in this study has also been reported by others.32,33 Hyperlipidemia was indicated by approximately twofold and threefold increases in serum TG and TC, respectively, in the DOX group. DOX is reported to cause the development of chronic glomerulonephritis, leading to progressive glomerulosclerosis associated with the nephrotic syndrome.33 Typically, nephrotic syndrome is characterized by the presence of persistent proteinuria, hypoalbuminemia, hyperlipidemia, and lipiduria.32,33 Thus, it is likely that the hyperlipidemia observed due to DOX treatment is basically the result of the DOX-induced nephrotic syndrome.33,34 It is suggested
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###

Figure 2. Effect of GSPE and EGb761 on cardiac (A) TNF-a and (B) caspase-3-activity of normal control, DOX-intoxicated, DOX + GSPE and DOX + EGb761 rats. Data represent the means SEM (n = 8). ***P 0.001, as compared with the normal control group. ##P 0.01, ###P 0.001, as compared with DOX-intoxicated rats Copyright 2012 John Wiley & Sons, Ltd.

modulation of doxorubicin cardiotoxicity

(A) (B) (C)

(D)

(E)

(F)

Figure 3. Photomicrographs of heart sections from (A) normal control rats (H&E, 80): normal histological structure of the myocardium (my). (B, C and D) DOX-treated rats (H&E, 80, 160, 160 respectively): hyalinization of the myocardium in focal manner (h), in ammatory cells in ltration in pericardium (m) with oedema in both pericardium (o) and in between the myocardium (o). (E) DOX + GSPE (H&E, 160): congestion of myocardial blood vessels (v) with focal hyalinization in the myocardium in few manner (h). (F) DOX + EGb761 (H&E, 160): mild vacuolization in some myocardial cells (!)

Table 2. The severity of the reaction in heart according to the histopathological alterations in different groups Gp 1 Control Myocardial hyalinization In ammatory cells in ltration in pericardium Oedema in myocardium Oedema in pericardium Congestion in blood vessels Myocardial vacuolization Gp 2 DOX +++ ++ +++ +++ Gp 3 DOX + GSPE + + Gp 4 DOX + EGb761 +

++++, very severe; +++, severe; ++, moderate; +, mild; , nil.

that hyperlipidemia is deleterious for heart function and appears to contribute to the DOX-induced heart failure. By treating rats with GSPE or EGb761, TG and TC levels were decreased to a level near that of normal control group. Similar studies have found that EGb761 lowers circulating free cholesterol in ageing rats35 and inhibits the elevation of serum TG in high cholesterol diettreated rats.36 The hypolipidemic action of EGb761 seems to be principally derived from the avonoids. Epidemiological data showed that total intake of avonoids, or a single quercetin component, was inversely correlated with the plasma TC and lowdensity lipoprotein cholesterol (LDL-C).37 Moreover, as a major avonoid component of EGb761, quercetin has been identi ed as a potent hypolipidemic in experimental
Copyright 2012 John Wiley & Sons, Ltd.

studies.38,39 Quercetin promotes an increase in faecal sterols, which in turn leads to a decreased absorption of dietary cholesterol, as well as lower plasma and hepatic cholesterol by inactivating b-hydroxy-methyl-glutaryl CoA (HMG CoA) reductase, a rate-limiting enzyme in cholesterol biosynthesis.39 On the other side, GSPE was found to inhibit the elevation of serum TC and TG in high cholesterol diet treated rats.40 Previous research showed that the micellar solubility of cholesterol in vitro is reduced signi cantly by proanthocyanidins, suggesting that proanthocyanidins inhibit the absorption of cholesterol.41 Oxidative stress injury was monitored by MDA, TAO and GSH levels. In the present study, DOX caused a considerable increase in myocardial levels of MDA and TAO capacity along with a marked depression of GSH. These results were in agreement with several studies.14,42 A close association of GSH depletion and lipid peroxides formation has been reported previously.43 The signi cant increase in cardiac lipid peroxides of DOX-treated rats explains the observed leakage of cellular AST, LDH and CK-MB to circulation due to cardiac injury. On the other hand, the superoxide anions generated by the semiquinone structure of DOX, stimulated the activity of antioxidant enzymes, as evidenced here by the obvious stimulation of TAO in cardiac tissue, leading to activation of glutathione peroxidase (GSHPx) in response to the accumulated peroxides, which can subsequently lead to the formation of hydroxyl radicals in the presence of metal ion catalysts.44 The latter can react with polyunsaturated phospholipids that ultimately lead to the formation of a great variety of
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n. a. e. boghdady diffusible aldehydes represented by MDA.45 The stimulation of GSHPx and the overproduction of free radicals can be detoxi ed by the endogenous antioxidants, causing their cellular stores to be depleted.46 This is in agreement with our results as revealed by GSH depression. The oral administration of GSPE and EGb761 prior to the DOX injection signi cantly reduced the extent of lipid peroxidation and TAO and increased GSH levels, suggesting that these agents scavenged free radicals and decreased cellular injury. These outcomes were consistent with histopathologic scores. Because GSPE and EGb761 are avonoids, it is known that avonoids have a potential function in the transfer of electrons between their isoalloxazine group and cell reactants in oxidationreduction reactions to render them less reactive.4,47 They can also chelate metals like iron involved in free radical formation47,48 and inhibit many enzymes, including cyclooxygenase, lipoxygenase,49 and phospholipase A2.4,50 In addition, the molecular structure of avonoids allows them to accumulate at lipid interfaces50 and/or to penetrate membranes,48 so there are decreased lipid peroxidation and subsequent protection of cells. Moreover, in vitro investigation of antioxidant potential of GSPE shows that it is a strong antioxidant, up to ve times more effective than vitamin C or E.51 In order to evaluate the cardiac in ammation and apoptosis, TNF-a and caspase-3 were measured. DOX, in the present study, caused marked increase in myocardial TNF-a and caspase-3 levels, which were consistent with previous reports.52,53 It is possible that increased oxidative stress following DOX administration caused expression of TNF-a. Oxidative stress is known to activate P38-mitogen activated protein kinase and nuclear factor kappa B and thus plays a role in the sequence of signalling events involved in myocardial TNF-a production.54,55 On the other hand, increased oxidative stress caused disturbances in mitochondrial membrane permeability, causing leakage of free radicals and cytochrome c from the mitochondria to the cytosol. Once cytochrome c is released into the cytosol, it binds to another protein, Apaf-1, and promotes activation of the caspase cascade, leading to cell death.56 From the results of the present study, it was seen that alteration in lipid pro le, increased free-radical generation and lipid peroxidation caused damage to the myocardium. These changes affect the cellular membrane, thereby releasing mitochondrial cytochrome c into the cytosol and activating caspase-3, which may further lead to apoptosis. GSPE as well as EGb761 treatment signi cantly reduced the elevated levels of myocardial TNF-a and caspase-3 observed in the DOX group an effect that might be attributed to reduction in oxidative stress. Histopathological effects of DOX show severe hyalinization of the myocardium, associated with moderate focal in ammatory cells in ltration in the pericardium, as well as severe oedema in both pericardium and myocardium. Thus, histopathological analysis has con rmed that administration of a single dose of DOX (20 mg kg1 i.p.) produced signi cant cardiomyopathy in rats. This is in line with previous literature.57,58 Evidence suggests that EGb761
Copyright 2012 John Wiley & Sons, Ltd.

(100 mg kg1 day1) pre-treatment signi cantly protects the myocardium from DOX-induced toxicity while GSPE (200 mg kg1 day1) partially protects the myocardium, as evidenced by fewer hyalinized myocardium as compared to the DOX group. There are some limitations in the present study. First, GSPE and EGb761 are speci c and complex products prepared from grape seed and ginkgo leaves respectively. Grape seeds extract contained other avonoids as gallic acid, catechin, epicatechin, gallocatechin, and epigallocatechin besides the proanthocyanidins. EGb761 contained 24% ginkgo- avonole glycosides and 6% terpenoids. Which component produces the protective effect or does more work in protecting against DOX-induced cardiac injury in the present study remains to be elucidated. Second, data relating to arterial blood pressure and heart rates were not recorded. Finally, more prospective studies are needed to evaluate the ef cacy and optimum dosage of GSPE and EGb761 to be used as a protective agent against the side effects of chemotherapeutics. In conclusion, the present study indicates that GSPE and EGb761 have a protective effect on cardiac injury induced by DOX, which may be related to antioxidative, anti-in ammatory and antiapoptotic properties. They appear to be an effective protective agent against cardiomyopathy, although their mechanisms remain to be elucidated.

CONFLICT OF INTEREST The author has declared that there is no con ict of interest.

ACKNOWLEDGEMENT The author is thankful to Dr. Adel Bakeer, Histology Department, Faculty of Veterinary Medicine, Cairo University, for performing the histopathological examination.

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