Evaluation of Antioxidant and Neuroprotective Effect of Rhododendron arboreum on Transient Cerebral Ischemia and long term cerebral Hypoperfusion
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Evaluation of Antioxidant and Neuroprotective Effect of Rhododendron arboreum on Transient Cerebral Ischemia and long term cerebral HypoperfusionM. Pharm. Dissertation Protocol Submitted to 
Rajiv Gandhi University of Health Sciences, Karnataka Bangalore – 560 041 By
Under the Guidance of Mr. MANJUNATHA P.M. M. Pharm (Ph.D). Assistant Professor 
2010-11 Department of Pharmacology Acharya & B.M. Reddy College of Pharmacy, Soldevanahalli, Chikkabanavara (Post) Hesaraghatta Main Road, Bangalore – 560 090
Enclosure - I 6. BRIEF RESUME OF INTENDED WORK: 6.1 INTRODUCTION AND NEED OF THE WORK: Stroke is the second leading cause of death throughout the world and considered the most common cause of disability in adults.1 The World Health Organisation (WHO) defines stroke as “rapidly developing signs of focal or global disturbance of cerebral function, lasting longer than 24 h (unless interrupted by death) with no apparent non-vascular cause”. Selective neuronal necrosis (SNN) denotes neuronal death with sparing of glial and vascular elements of the central nervous system. Transient ischemic attacks (TIA) and global ischemia following cardiac arrest are the most common causes of brief cerebral ischemia in humans.2 Transient MCAO model is one of the most widely and successfully used models of ischemic stroke. Under physiological conditions, reactive oxygen species such as superoxide (•O2), hydrogen peroxide (H2O), and hydroxyl radical (•OH), play important roles in signalling and metabolic pathways. Importantly reactive oxygen species levels are checked by endogenous antioxidants which include SOD, glutathione peroxidase, glutathione and catalase. During oxidative stress, pivotal balance of defence mechanism fails and attenuates neuronal death leading to cerebral ischemia. Oxidative stress is now well reported in the pathophysiology of stroke. A plethora of literature indicates that ischemia reperfusion injury causes a significant increase in oxidative stress markers such as; reactive oxygen species, MDA and nitrite concentration. There is also a significant decrease observed in antioxidant enzymes i.e. catalase and SOD activity, in the brain. 3 The role of NO in the progression of ischemic damage has been extensively studied in transient focal cerebral ischemia. The over production of NO after reperfusion may directly influence the activity of enzymes involved, or reacts with superoxide to yield the peroxynitrite anion (ONOO–), which decomposes to highly toxic hydroxyl and nitrogen dioxide radicals. These radicals lead to the loss of protein, nucleic acids and membrane lipids, inhibit the enzymes involved in mitochondrial electron transport, and eventually inhibit the respiration of mitochondria and cause cell deep-injury.4
Present study is focused on the effects of Rhododendron arboreum prevents the oxidative stress during reperfusion injury as well as attenuates the behavioural deficits and histopathological alterations secondary to hypoperfusion.5 The leaves of Rhododendron arboreum were reported to contain Quercetin 3-O- beta -D-glucopyranosyl [1- >6]-O- alpha -L-rhamnopyranoside, pectolinarigenin 7-Orutinoside, 7,2'-dimethoxy-4',5-methylene dioxyflavanone15. Flavonoids, isolated from the leaves of Rhododendron arboreum were found to have potent antioxidant property16, the plant Rhododendron arboreum have been reported for anti-inflammatory. In the absence of reliable neuroprotective drugs in modern medicine, there are numbers of medicinal preparations in the Ayurvedic system of Indian medicine recommended for the treatment of cardiac disorders. Their usage is in vogue since centuries are quite often claimed to offer significant relief. However, no scientific information is available regarding the neuroprotective effect of Rhododendron arboreum. This study addresses the neuroprotective effect of ethanolic extract of Rhododendron arboretum (ERA) in transient partial cerebral ischemia induced BCCA occlusion for 30 minutes followed by reperfusion and on chronic cerebral Hypoperfusion.6 ENCLOSURE – II 6.2 REVIEW OF LITERATURE: Preventive effect of Rhododendron arboreum on cardiac markers, lipid peroxides and antioxidants in normal and isoproterenol-induced myocardial necrosis in rats was reported by Manjunatha PM, Sandip karia, Divakar Goli. 2011.6 Evaluation of Hepatoprotective activity of leaves of Rhododendron arboreum in ccl4 induced hepatotoxicity in rat was reported by T. Praksh, Snehal Dayalal Fadadu, Uday raj Sharma, V. Surendra, Divakar Goli, Perfect Stamina D. Kotresha, 2008.7 Anti- inflammatory activity of flower of Rhododendron arboreum in rat’s hind paw edema induced by various phlogistic agents, reported by shyam SA, Kalpna S, 1988.8 Free radical scavenging activities of Himalyan Rhododendrons was reported by Dhan P, Garima U, Singh BN, Ruchi D, Sandeep K, Singh KK. 2005.9 Flavonidic constituents of Rhododendron arboreum leaves were reported by Kamil M, Shafiullah, Ilyas M. 1995.10 Enclosure – III
The main objective of the present study is to determine the,
7 . materials and methods: Plant materials Leaves of the plant R. arboreum will be collected (in the month of June) from the surrounding fields of Meghalaya. Authentification and herbarium already done. Plant extract Freshly collected R. arboreum L. Whole plant will be dried under shade and the dried material was milled to obtain a coarse powder. The ethanolic extract of powder will be prepared by the process of continuous extraction (Soxhletion), extract will be stored in desiccators for further study. Acute toxicity studies Earlier acute oral toxicity studies were carried out for ethanolic extract of R. arboreum using acute toxic class method, according to OECD guidelines No. 423. Based on toxicity data two dose of 150 and 300 mg kg-1 will be selected (OECD, 1996)6. Drugs: Ethanolic extract of Rhododendron arboreum will be suspended in distilled water using Tween 80 (1% v/v). Two doses of ERA (150 and 300 mg kg-1) will be chosen for oral administration route based on our earlier studies6. Chemicals: 1,1,3,3-tetraethoxyproprane, NADH, nitroblue tetrazolium (NBT), phenazine methosulphate will be procured from Sigma- Aldrich chemicals Ltd, St. Louis, USA. 5, 5’-Dithiobis (2-nitrobenzoic acid) (DTNB), reduced glutathione were obtained from Himedia Laboratories, Mumbai. All the other chemicals to be procured from Merck laboratories, nice chemicals, Loba chemie, Sd. fine chemicals will be of analytical grade. Animals: 80 healthy Wistar albino Rats (200-250g) of either sex will be procured from registered suppliers. The animals will be housed in standard environmental condition & provided with food & water ad libitum. All experimental animals will be conducted in accordance with the guidelines of CPCSEA Goverment of India. ENCLOSURE IV 7.1 SOURCE OF DATA: Data will be obtained from experiments which involves
Enclosure – V 7.2 METHOD OF COLLECTION OF DATA 7.2.1 Experimental protocolAnimals will be divided into four groups of 10 rats each. To obtain maximum data from small number of animals, each group will be subjected to Locomotor activity, Beam Walking test and Hanging wire test before focal cerebral ischemia and after 24 h reperfusion; finally subjected to examine cerebral infarct volume, biochemical parameters and histopathological studies. The first group will sham operated [rats were subjected to surgical procedure, but did not suffer middle cerebral artery occlusion (MCAO), except for exposure of right internal carotid artery (ICA) and right external carotid artery (ECA)]. The second group served as control group (vehicle treated) i.e., rats were orally administered vehicle [simple syrup I.P. + Tween 80 (1%, v/v), 10 ml/kg] for 7 days (pre-treatment) before subjecting to 30 min MCAO followed by reperfusion for 24 h. Third and fourth group of rats, orally received 150 mg/kg and 300 mg/kg, dose of ERA respectively. Vehicle or drugs were fed once daily for 7 consecutive days prior to the experiment. 7.2.1 A. Induction of transient cerebral ischemia: Transient cerebral ischemia was produced by following the method of iwasaki et al. (1989). Rats will be anesthetized by giving Thiopentone sodium (40 mg/kg) i.p. surgical technique and placed in dorsal recumbency. A longitudinal incision of 1.5cm in length was made in the midline of the ventral cervical skin. The right common carotid artery, ICA and ECA were exposed and carefully isolated. A nylon monofilament (40mm in length and 0.24mm in diameter), its tip rounded by flame-heating, was inserted from the lumen of the ECA to that of the right ICA to occlude the origin of the right middle cerebral artery (MCA). The right MCA was occluded for 30 min, and there after brain was allowed to be reperfused with blood by withdrawing the of the nylon thread. 24 h after reperfusion, rats were decapitated. Temperature was maintained at 37±0.5 ◦C throughout the surgical operation.11 7.2.1 B. Induction of chronic hypoperfusion: For chronic hypoperfusion experiments, animals will be divided into four groups (ten animals each). The first group served as sham-operated control. In the second group, ERA (300 mg/kg/day p.o. once in a day) for entire experimental period was administered in sham-operated animals (ERA per se). Animals in the third group were subjected to permanent BCCA occlusion for 15 days and received vehicle only (hypoperfusion group). In the fourth group, ERA (300 mg/kg orally) was administered 60 min before permanent BCCA occlusion. ERA (300 mg/kg/day p.o. once a day) was then continued up to the 15th postsurgical day. On day 15 (60 min after last dose of ERA), all animals were subjected to behavioural assessment to Locomotor activity, Beam Walking test and Hanging wire test. Then, under overdose of Thiopentone sodium, animals were sacrificed by decapitation and brain samples were collected for histopathological analysis, biochemical estimation and infarct size determination.12
The locomotor activity will be record by using actophotometer (inco pvt.ltd. ambala, India) before locomotor task; animals were placed individually in the activity meter for 2 min for habituation. Thereafter, locomotor activity was recorded using actophotometer for a period of 5 min (kulkarni 1999).13
Beam walking test will be used to evaluate gross vestibule motor function. The apparatus consisted of a rod 120 cm in length and with a diameter of 2.3 cm. A wooden box (20 cm X 20 cm X 10 cm) was set at one end of the rod as a nest for motivating the animal to cross the beam. The apparatus will be suspended 50 cm above a cushion, which protected the animals against fall injury. Rats were trained twice daily for 2 days before BCCA occlusion and assessed for motor coordination after 24 h of reperfusion. The time taken to traverse the beam was recorded. The cut-off time was taken as 120 s (Song et al. 2006; Yan et al. 2007).14
This Hanging wire test will be used to measure forelimb grip strength of the rats. In this test, animals were suspended by the forelimbs on a wire (45 cm long and 0.3 cm diameter) stretched between two posts 40 cm above a foam pillow. The time(s) until the animal fell will be recorded. The cut-off time will be taken as 90 s (Hunter et al. 2000).15
1. Measurement of Total Protein: The protein content of the brain homogenate was determined by Lowry’s method (Lowry et al. 1951) using bovine serum albumin as standard the level of protein will be expressed as mg/protein/g of tissue.16 2. Measurement of Lipid Peroxidation: The extent of lipid peroxidation was measured by estimating the amount of malanodialdehyde (MDA) formed, as described by Akhtar et al (2008). Briefly, to 0.1 ml homogenate, 1 ml of 10% (w/v) trichloroacetic acid (TCA) and 1 ml of 0.67% (w/v) thiobarbituric acid were added and placed in a boiling water bath for 30 min. Then the mixture was placed in crushed ice for 10 min followed by centrifugation at 40009g for 10 min. The absorbance of the clear pink-colored supernatant was measured at 532 nm and the results to be expressed as n.mol MDA/mg protein.17 3. Measurement of Total Thiols: The total thiol content was determined by the method described by Sedlak and Lindsay (1968), with slight modifications. To a mixture of 0.2 ml of homogenate and0.36 ml of buffer, 0.04 ml of 10 mM DTNB and 1.5 ml of methanol were added and mixed well. The mixture was centrifuged at 15009g for 5 min at -40c. Then the intensity of the yellow color developed was measured at 412 nm and the results to be expressed as n.mol/mg protein.18 4. Measurement of Glutathione: Glutathione-S-transferase (GST) activity was measured as described by Habig et al. (1974). Briefly, 0.85 ml of Phosphate buffer (pH 7.4), 0.05 ml homogenate and0.05 ml of 10 mM GSH were added, to which 0.05 ml of 1 mM CDNB was added to initiate the reaction. The rate of formation of GSH-CDNB complex was monitored for5 min at 340 nm and the results will be expressed as n.mol of CDNB conjugate formed/min/mg protein.19 5. Measurement of Brain Infarct Area: For the measurement of infarct area, two animals from each group were decapitated under deep anaesthesia and the brains were removed and sliced coronally into 2-mm-thick sections, incubated in phosphate-buffered saline (pH 7.4) containing 2% of 2,3,5-triphenyltetrazolium chloride (TTC) for 30 min at 370C and fixed in 10% neutral-buffered formalin overnight20 expressed as a percentage of the total measured brain area.
A section of the brain was fixed with 10% formalin, embedded in paraffin wax and cut into sections of 5-lmthickness. The sections will be stained with hematoxylin and eosin dye for histopathological observations.21
The data obtained from the above study will be subjected to statistical analysis using analysis of variance (ANOVA) followed by Dunnets test. Total duration for the completion of whole project will be 9 months.
ENCLOSURE – VI 7.3 Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly. The above study requires investigation on 80 Wistar albino rats of either sex for neuroprotective activity of Rhododendron arboreum on cerebral ischemia. 7.4 Has ethical clearance been obtained from your institution in case of 7.3? Applied ENCLOSURE – VII LIST OF REFERENCES:
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