Ameliorative Effect of Dark Chocolate on Brain and Heart of Chronic Restraint Stressed Male Albino Rats

Research Article

Austin J Anat. 2018; 5(1): 1076.

Ameliorative Effect of Dark Chocolate on Brain and Heart of Chronic Restraint Stressed Male Albino Rats

Salama RM and El-Sherif NM*

Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Egypt

*Corresponding author: El-Sherif NM, Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Egypt

Received: December 11, 2017; Accepted: January 04, 2018; Published: January 11, 2018


Objective: The aim was to investigate for the first time, the ameliorative effect of Dark Chocolate (DC) against oxidative stress and apoptosis in heart and brain frontal cortex of rats subjected to Chronic Restraint Stress (CRS).

Methods: 24 male albino rats were divided into four experimental groups of six rats each: Group I (Control rats), Group II (Control rats treated with DC) Group III (stressed rats) exposed to CRS by placing the rats in restrainer (20 cm × 7 cm plastic tubes) for 2 h/day for 21 days and Group IV (Treated rats).

After 21 days, the rats were sacrificed; blood sample was taken by cardiac puncture for biochemical analysis of Lipid Peroxidase (LPO), Superoxide Dismutase (SOD) & Catalase (CAT). Heart & brain tissues were removed carefully for histological (H&E staining) and immune histochemical analysis of oxidative stress and inflammatory marker (inducible Nitric Oxide Synthase (iNOS) and apoptotic marker (Caspase 3).

Results: Administration of dark chocolate by stressed rats improves their antioxidant status by reducing the indices of lipid peroxidation (LPO, SOD & CAT). Also DC attenuates the restraint stress-induced histopathological changes in brain and heart tissues and resulted in a significant decrease in the mean area % of iNOS and Caspase 3 immunoreactivity.

Conclusion: The results suggest that dark chocolate had antioxidant and antiapoptotic effects, able to block the stress-induced oxidative stress and apoptosis in the heart and brain tissues of rats. These results may be attributed to anti-oxidant flavonoids contained in dark chocolate.

Keywords: Dark chocolate; Chronic restraint stress; iNOS & caspase 3


Stress has become an increasingly popular and widely applied term in our everyday languages. People have witnessed the physical damage of stress that can work on the body [1]. Stress disturbs the normal physiological equilibrium which induces emotional and physical strain which has been considered the basic factor in the changes in the different organ systems, including the cardiovascular system [2], gastro-intestinal system [3] and Central Nervous System (CNS) [4,5].

Chronic Restraint Stress (CRS) model is an easy, suitable and most commonly employed in laboratory animals, from the various stress models available for investigation of oxidative stress– related tissue changes, as it effectively mimics chronic exposure to psychological stress in humans that results in increased oxidative stress and resultant tissue damage [6].

It has been suggested that CRS results in the creation of Reactive Oxygen Species (ROS) e.g hydrogen peroxide (H2O2), hydroxyl radical and superoxide anion radical that cause lipid peroxidation, especially in membrane, playing an important role in tissue injury [7] and induce apoptosis [8].

Dark Chocolate (DC) with its high cocoa content, contains a large percentage of antioxidant molecules, mainly flavonoids, that has several beneficial health effects on the brain by inducing widespread stimulation of brain perfusion, provoking angiogenesis, neurogenesis and changes in neuron morphology, mainly in regions involved in learning and memory. Also chocolate induces positive effects on mood and is often consumed under emotional stress [9]. Consumption of dark chocolate are thought to possess cardioprotective properties and lowers cardiovascular mortality due to the contained high levels of polyphenolic flavonoids that exhibit anti-oxidant health benefits preventing cardiovascular disease [10,11]. Also Sentürk and Günay, [12] reported that coronary artery disease, hypertension, heart failure, hyperlipidemia, inflammation and oxidative stress were the favorable effects of flavonoid-rich dark chocolate.

The present study aims to review the effects DC on oxidative stress and apoptosis induced in heart and brain tissues of rats subjected to CRS.

Materials and Methods

Drugs & chemicals

Dark chocolate were purchased from local market, Cairo, Egypt.

All markers were purchased from local distributer (Sigma chemical) Cairo, Egypt.

Induction of chronic restraint stress

Restraint Stress was induced in rats by putting them in 20 cm × 7 cm plastic tubes for 2 h/day for 21 days [13]. There are several 3 mm holes at the both ends of the tubes which allow sufficient air for breathing and the rats were unable to move.

Experimental design

Twenty four adult male albino rats weighing 180 to 200 g were used in this study. All rats were maintained under constant room temperature and standard conditions, with ad libitum access to water and commercial food and were placed in individual plastic cages with soft bedding.

Animals were randomly divided into four experimental groups of six rats each:

Group I: (Control rats): Rats were not subjected to CRS.

Group II: (Control rats treated with DC): DC was administered at a dose of 500 mg/Kg body weight per day, as a solution in water using an intragastric tube, for 21 days to rats which were not subjected to CRS.

Group III: (Stressed rats): Rats were exposed to CRS for 2 h/day for 21 days.

Group IV: (Treated rats): Rats were exposed to CRS for 2 h/day & post-orally treated with DC at a dose of 500 mg/Kg body weight per day, as a solution in water using an intragastric tube, for 21 days.

Following each stress session, rats were returned to home cages and were able to access food and water freely for the reminder of the day.

After the experimental period, blood sample was taken from rats by cardiac puncture for biochemical analysis of Lipid Peroxidase (LPO), Superoxide Dismutase (SOD) & Catalase (CAT). Then all rats were anesthetized and sacrificed by cervical decapitation. Ventricular wall of the heart and frontal cortex of the brain were removed carefully for Histopathological (H& E staining) and immunohistochemical analysis of inducible Nitric Oxide Synthase (iNOS) and caspase 3.

Biochemical analysis

Serum was obtained from cardiac blood samples by centrifugation. The concentration of Lipid Peroxidase (LPO), Superoxide Dismutase (SOD) & Catalase (CAT) in blood are used as an indicator of lipid peroxidation (a biomarker of protective oxidative injury).

Histopathological examination

The heart and brain were removed and fixed in 10% formalin solution and processed routinely by embedding in paraffin and then the heart and frontal brain cortex were sectioned into 5 μm sections and stained with hematoxylin & eosin stain for L/M study.


Analysis of inducible Nitric Oxide Synthase (iNOS): Kits used: Ready to use target retrieval solution (S1700, Dakocytomation), primary antibody rabbit polyclonal IgG specific for iNOS enzyme in mouse and rat (M-19/Sc 650, Santa Cruz Biotechnology) with no cross reactivity for nNOS or eNOS enzymes and ready to use antibody diluent with background reducing components (S3022, Shireen A. Mazroa et al. 237 Dakocytomation). Universal detection kits (K 0673, Dakocytomation) based on a modified avidin-biotin (LAB) technique in which a biotinylated secondary antibody forms a complex with peroxidase conjugated streptavidin molecule 29. Steps of iNOS immune-staining: Sections of heart and brain tissues were dewaxed in xylol for 20 minutes (two changes) and hydrated in descending grades of alcohol down to distilled water. They were immersed into preheated target retrieval solution to 95- 99°C (without boiling) in water bath for 40 minutes, removed from the bath and allowed to cool for 20 minutes at room temperature. Sections were rinsed 3 times with Phosphate Buffered Saline (PBS). Excess liquid was tapped off the slides. Enough hydrogen peroxide was applied to cover the specimen for 5 minutes, then slides were rinsed gently with PBS and excess liquid was tapped off. Enough amount of primary antibody (dilution 1: 100) was applied on specimens and was incubated for two hours in humidity chamber at room temperature. Slides were rinsed in PBS. Biotinylated link was applied on specimens for 10 minutes and sections were rinsed in PBS. Streptavidin HRP reagent was applied on specimens for 10 minutes and sections were then rinsed in PBS. Freshly prepared DAB substrate chromogen solution (1 drop of DAB chromogen /1 mL of substrate buffer) was removed from 2-8°C storage and applied on specimens for 10 minutes. Slides were rinsed gently in distilled water, immersed in haematoxylin for 1/2 minute and were rinsed in tab water until blue. Slides were dehydrated in ascending grades of alcohol, cleared in xylol, mounted by Canada balsam and covered with a cover slip. Negative control slides were prepared by the same steps except they were incubated with the antibody diluent instead of primary antibody. Positive reaction appeared brown in color [14,15].

Analysis of caspase 3

Immunohistochemical detection of caspase-3 was performed using primary rabbit anti-rat caspase-3 antibody. The avidin–biotin complex technique was used. Serial frozen heart and brain sections (5 μ\m) were allowed to equilibrate to room temperature and exposed to acetone for 10 min before starting the streptavidin–biotin-staining technique (Vectastain Universal Quick Kit; Vector Laboratories). Sections then were preblocked with 10% horse serum/PBS + 0.2% Tween 20 for 20 min at room temperature followed by elimination of excess serum from the session and incubation with specific antibody and isotype-matched control antibody at dilutions (1:500 dilution) [16]. After washing, bound antibodies were detected by a universal biotinylated antibody prediluted in TBS at room temperature for 20 min followed by incubation for 10 min with a peroxidaseconjugated streptavidin. ABC (3-amino-9-ethylcarbazole) was used as colorimetric substrate. Sections were rinsed, counterstained, and mounted in aqueous mounting medium. Analysis of tissue sections was performed by light microscopy.

Positive reaction for caspase 3 was visualized as brown coloration of the cytoplasm of the cardiac and neural cells. Negative controls were done using the same steps except that phosphate buffered saline was applied instead of the primary antibodies [17].

Statistical analysis

Statistical analyses were carried out with the Statistical Package for the Social Sciences for Windows 11.5 (SPSS Inc., Chicago, IL, USA). All values were given as mean ± standard deviation. Differences between groups were statistically analyzed by Kruskal–Wallis test (ANOVA) and chi-square test. Statistical significance was accepted asP < 0.05.


Biochemical results

Dark chocolate administration in (Group IV) ameliorated the changes in antioxidant and oxidative stress indices in stressed rats (Group III). The heart and brain tissue of stressed rats (Group III) had significant greater levels of enzymatic activities of the antioxidants SOD, CAT and LPO (as an index of lipid peroxidation) compared to the control rats (Groups I & II). In parallel, dark chocolate caused a significant reduction in the SOD and CAT, as well as the levels of LPO as compared to stressed rats (Tables 1 and 2). These results demonstrate that dark chocolate antioxidant capability is able to block the stress-induced oxidative stress in the heart and brain tissues of rats.