Juglans regia

JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES

Asadi-Shekaari et al.. Feeding with walnut (Juglans regia) reduces cerebral ischemic damage in rats. Journal of pharmaceutical and biomedical sciences (J Pharm Biomed Sci.) 2013 June; 31(31): 1137-1142. (Article no 11)

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Asadi-Shekaari et al.

Original

article

Feeding with walnut (Juglans regia) reduces cerebral ischemic damage in rats *1Asadi-Shekaari

M, 2Kalantari Pour TP, 3Namazian E, 4Arab Nejad F, 1Zangiabadi N, & 1Afarinesh Khaki MR

1Neuroscience

Research Center, Kerman University of Medical Sciences, Kerman, Iran. and Nursing Faculty, Branch of Kerman, Islamic Azad University, Kerman, Iran. 3Midwifery student, Member of Student Research Committee, Kerman Medical University, Kerman, Iran. 4Medical student, Member of Student Research Committee, Kerman Medical University, Kerman, Iran.

2Midwifery

Abstract: Background & objective for the study: Ischemic stroke is a major cause of death and disability in developed countries. Oxidative stress and inflammation are involved in the pathophysiology of this disease. Walnut kernels (WKs) are a highly nutritious food due to their large amount of phenolic compounds. These compounds have beneficial effects on human health due to their obvious antioxidant and anti-atherogenic properties. The aim of this study was to determine whether or not a WK diet had cerebroprotective effects on the focal ischemic brain. Methods: Male Wistar rats were fed with equal amounts of a WK-enriched diet or a control diet. After 8 weeks of feeding, middle cerebral artery occlusion was induced for 30 min followed by 48 h reperfusion. After behavioural testing, the animals were sacrificed and their brains were removed for histological, ultrastructural, immunohistochemical and biochemical studies. Results: The results showed that the animals which received a WK-enriched diet had a significant reduction in neuronal damage induced by ischemic reperfusion compared to the controls. Conclusion: Our results suggest that pre-treatment with WKs reduces the severity of cerebral ischemic injury.

Key Words: Antioxidant; fatty acids; inflammation; MCAO; Walnut. Introduction:

S

troke or, cerebrovascular disease is one of the leading causes of death and the most common cause of adult disability, especially in the elderly population and in patients undergoing cardiovascular surgery. Neuronal loss due to stroke may result in permanent deficits in visual, sensory, language and motor capabilities. There is no specific treatment for improving functional recovery after stroke except for rehabilitative strategies, which have been found to have limited success [1]. Furthermore, only a few therapeutic options are currently available for the treatment of ischemic stroke, with prophylactic treatment strategies mainly being limited to agents that block platelet aggregation or the coagulation cascade. Although clinical trials have demonstrated the effectiveness of antiplatelet agents in decreasing the incidence of ischemic strokes, these agents, however, do not address the larger issue of reducing cerebral infarct size [2]. Walnut species are the main source of nuts in mild climate zones around the world. In Iran, Juglans regia L. (Juglandaceae) is not just an agricultural product: its fruit, leaves, stems and flowers are all used for different medicinal purposes. Walnut kernels (WK) have

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high concentrations of phenolic compounds, which have positive effects on human health because of their anticonvulsive, neuroprotective, antioxidant and anti-atherogenic effects [3-5]. In addition, walnut kernels are a nutrient-rich food, containing plentiful phospholipids, proteins, unsaturated fatty acids and tocopherols [6]. In addition, WKs are traditionally used to tonify the kidney, relax the bowels and warm the lungs in traditional Chinese medicine [7]. Acute stroke is a progressive neurodegenerative disorder. The generation of free radicals during the reperfusion stage can induce



injury to the brain. It has been shown that pharmacological agents which possess antioxidant and free radical scavenging properties reduce the amount of brain damage during stroke [8]. Several dietary supplements such as carotene, blueberry and date fruit have been reported to have strong antioxidant properties and to reduce the amount of cerebral ischemic damage [9-11]. The aim of this study was to determine whether or not the WK diet has cerebroprotective effects on the focal ischemic brain.

Material and method: Animals Healthy male Wistar rats were used in this study, which was performed in accordance with Neuroscience Research Centre guidelines for the care and use of laboratory animals (EC/KNRC/88/16). All efforts were taken to reduce discomfort and pain to the animals. We chose a simple random sampling that all samples at a range 220-290 g weight have an equal chance of being selected from the entire population. Experimental design The animals were individually caged and divided into two groups according to the diet: (A) control diet (n=12) and (B) WK-enriched diet (6% of food intake for 2 months) (n=10). A subgroup of controls was selected as the sham group (n=4). Middle cerebral artery occlusion (MCAO) Right middle cerebral artery was occluded using the intraluminal filament model and the method described by Longa et al. (1989)[12]. In brief, the animals were anaesthetized with chloral hydrate (400 mg/kg) and paced in supine position. A midline ventral incision was made and the right common carotid artery (CCA) was exposed and carefully separated from the vagus nerve. All branches of the external carotid artery (ECA) and extra cranial of the internal carotid artery (ICA) were blocked. Then, a 4-0 nylon suture was inserted into the ICA and advanced intracranially to block the blood flow into the MCA. After 30 min of ischemia, the suture was withdrawn to restore the blood flow (reperfusion). The rectal temperature was maintained at 37 ± 0.5 °C using a thermistor coupled to a heated blanket during surgery. The animals were returned to their cages once they had recovered from the anaesthesia. After full recovery a neurological study was carried out to ensure that MCAO had occurred, and the animals with no clinical signs were excluded from the experiment.

Sample preparation method Light microscopy The animals were perfused intracardially with 10% formalin in phosphate-buffered saline (pH 7.4, M 0.1%) whilst under deep anaesthesia. The brains were removed and immersed in the same fixative overnight at 4 °C. Following this, the brains were processed for light microscopy studies according to routine procedures. Coronal sections 1.6-2.8 mm posterior to the bregma were cut at a thickness of 8 µm using a rotary microtome. Neuronal injury in the

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cortex was assessed by staining sections with haematoxylin and eosin, using the method described by Sakurai-Yamashita et al. (2006)[13]. Electron microscopy The rats were sacrificed by transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer solution (pH 7.4). The brains were removed and immersed in 4% buffered glutaraldehyde overnight. A small piece of the cortical area was cut and fixed in the same fixative for an additional 24 h. The specimens were post-fixed in 1% (w/v) OsO4/1% (w/v) of phosphate buffer. After dehydration in ethanol, the slices were embedded in Epon 812 resin. A section (400 nm) was stained with toluidine blue in order to locate the areas of interest. Afterwards 70-80 nm sections were cut and stained with lead citrate and uranyl acetate stain. The sections were examined with a Philips (EM 300, Eindhoven, Netherlands) transmission electron microscope. Rotarod activity The rats were assessed in terms of balance and grip strength using the rotarod. Each animal was given a training session before the start of therapy in order to acclimatize them to the rotarod apparatus (Technical and Scientific Equipment, GmbH, Germany). The animals were placed on the rotating rod with a diameter of 7 cm (speed 20 rpm). Three trials were performed for each animal with 10 min intervals and a cut-off time of 300 s throughout the trial. The results were recorded as average time to fall [14]. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling histochemistry




The terminal deoxynucleotidyl transferase (TdT)-mediated in situ dUTP nick end-labelling (TUNEL) assay was used on the brain sections with the cell death detection kit POD (Roche; Indianapolis, IN). A dark brown colour indicates the development of DNA breaks after incubation with DAB (3-3΄diamonobenzidine tetrachloride) and hydrogen peroxide. This method was used to verify cell death in the cortical area[15].

Statistics The statistical analysis was performed using SPSS software version 13.5. All values are expressed as the mean ± SEM. Differences in the parameters measured between the different groups were analysed by one-way ANOVA followed by a post-hoc Tukey test. A P-value of P