A STUDY ON NEUROPROTECTIVE EFFECT OF SHARK
SQUALENE IN BILATERAL COMMON CAROTID ARTERY
OCCLUSION INDUCED GLOBAL CEREBRAL ISCHEMIA IN MICE
M. Pharm Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore– 560041
By
Mr. RAHUL ANIL GAJARE B.Pharm
Under the Guidance of
Dr.A.R. KULKARNI M.Pharm, Ph D.
Department of Pharmacology
S.E.T’s COLLEGE OF PHARMACY
S. R. Nagar, Dharwad–580002
2010-2011
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA
ANNEXURE –II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERATION
1.
NAME OF THE CANDIDATE AND
RAHUL ANIL GAJARE
ADDRESS
DEPT. OF PHARMACOLOGY,
SET’s COLLEGE OF PHARMACY,
S.R.NAGAR,
DHARWAD-580002.
2.
NAME OF THE INSTITUTION
SET’s COLLEGE OF PHARMACY,
S. R. NAGAR,
DHARWAD-580002.
3.
COURSE OF STUDY AND SUBJECT
MASTER OF PHARMACY IN
PHARMACOLOGY
4.
DATE OF ADMISSION TO THE
JUNE-2010
COURSE
5.
TITLE OF THE TOPIC
“ A STUDY ON NEUROPROTECTIVE EFFECT OF SHARK SQUALENE
IN BILATERAL COMMON CAROTID ARTERY OCCLUSION
INDUCED GLOBAL CEREBRAL ISCHEMIA IN MICE”
1
6.0 BRIEF RESUME OF THE INTENDED WORK :
6.1 Need for the Study :
The brain is particularly vulnerable to oxidative stress because of its high metabolic
rate and low antioxidant defenses.1 Neuroprotection within the nervous system protects neurons
from apoptosis or degeneration, for example following a brain injury or as a result of chronic
neurodegenerative diseases. Currently, there is a broad interest in how apoptosis and
neuroprotection affect the brain during development and after the onset of CNS diseases
(stroke, schizophrenia, Parkinson's disease).
Neurodegeneration is the term for the progressive loss of structure or function of
neurons, including death of neurons. Many neurodegenerative diseases including Parkinson’s,
Alzheimer’s and Huntington’s occur as a result of neurodegenerative processes.2 Cerebral
ischemia is a syndrome characterized by rapid onset of neurological injury due to interruption
of blood flow to the brain and it leads to various pathophysiological modalities such as reactive
oxygen species (ROS), calcium overload, mitochondrial damage, neuronal cell death and also
associated with oxidative stress and DNA fragmentation.3-5 The most common form of cell
death in neurodegeneration is through the intrinsic mitochondrial apoptotic pathway. This
pathway controls the activation of caspase-9 by regulating the release of cytochrome c from the
mitochondrial intermembrane space.6 Over production of ROS is a central feature of all
neurodegenerative disorders. Reperfusion after cerebral ischemia further adds to the
complications of stoke by releasing various mediators such as proinflammatory cytokines and
free radical generation. This increases the oxidative stress to the brain and ultimately leading to
neuronal cell death.7
Shark squalene combats cancer at the earliest stages, squalene’s preventive and
therapeutic possibilities are extremely promising. Squalene’s powerful antioxidant and
cytoprotective effects are very significant. A research team from Toronto’s Hospital for Sick
Children demonstrated that squalene has selective cytoprotection in in vitro and in vivo
models.8 To best of our knowledge there is no scientific data regarding the neuroprotective
activity of Shark squalene. Hence we undertaken this project to evaluate and investigate the
neuroprotective effect of shark squalene bilateral common carotid artery occlusion induced
global cerebral ischemia in mice.
2
6.2 Review of Literature :
Review of literature revealed that the shark liver oil is a rich source of alkoxyglycerols,
chemicals that have healing properties. It is commonly known as shark squalene. Squalene is an
isoprenoid hydrocarbon with six nonconjugated double bonds, or isoprene units. It has an
effective and stable antioxidant configuration.9
It is promoted as dietary supplement used to boost the immune system,10 fights off
infections and heal wounds and to treat cancer. It also possesses cell protecting ability.11
Compare to other fish oils, Shark liver oils contains high amount of alkoxyglycerols and
squalaminne which promotes healing properties. Because of their immune-boosting effects they
are also claimed to help against colds, flu, chronic infections, asthma, psoriasis, arthritis and
AIDS. Most of the scientific studies with shark liver oil have focused on its possible benefits
against cancer and infections.12
Shark squalene has been used for over 40 years as both a therapeutic and preventive
agent. Some of the other fish oils are found to produce significant cytoprotective properties.11
Shark liver oil decreases inflammation in our bodies. Shark liver oil is an excellent in skincare
products. It has a component called squalene that is particularly effective for skin repair. It adds
moisture to your hands if used in a hand ointment. It is high in antioxidants and it attacks free
radicals that are attempting to start the formation of skin cancer.8
6.3 Objective of study :
The present study was designed to investigate the neuroprotective effect of shark
squalene, precursor of vitamin D on acute phase changes in mice model of cerebral ischemia
induced by Bilateral Common Carotid Artery Occlusion (BACCAO) :  Brain protein assay will be undertaken in three groups.
 Estimation of Glutathione peroxidase will be carried by Beutler et al method.
 Lipid Peroxidation Assay will be carried by method of Ohkawa et al.
 Microscopic Evaluation (Histopathology) of brain.
3
7.0
MATERIALS AND METHODS :
7.1 Source of Data :
The source of data are

Laboratory experiments on animals

Literature survey

Books and Journals

Internet
7.2 Method of collection of Data :
Animals :
Swiss albino mice of either sex weighing 20-30g will be used. The animals will be
maintained under controlled conditions of temperature (23 ± 2C) and 12-h light-dark cycles.
The animals are randomized into experimental and control groups and housed separately in
sanitized polypropylene cages containing sterile paddy husk as bedding. They will have free
access to standard pellets as basal diet and water ad libitum.
Methods :
INDUCTION OF CEREBRAL ISCHEMIA BY BILATERAL COMMON CAROTID
ARTERY OCCLUSION (BACCAO)13: Mice will be anaesthetized with chloral hydrate (400mg/kg, ip). A midline incision will
be made in the region between neck and sternum and trachea is exposed. Both the right and left
common carotid arteries will be located lateral to sternocleidomastoid, freed from surrounding
tissues and vagus nerve separated. Cerebral ischemia will be induced by clamping both the
arteries with the help of aneurysm clips.14 After 10 min of cerebral ischemia, the clips will be
removed from the arteries to allow the reflow of the blood through carotid arteries. The incision
is sutured back in layers with surgical suture.15 While performing the surgical procedure, the
body temperature is maintained at 370C by heated IR lamp. All surgical instruments used in
procedure will be sterilized prior to use.
4
Animals are divided into 3 groups: Sham group
 Surgery group
 Shark squalene group
7.3
DOES
THE
INTERVENTIONS
STUDY
TO
BE
REQUIRE
CONDUCTED
ANY
ON
INVESTIGATION
PATIENTS
OR
OR
OTHER
HUMANS/ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY.
Yes. The investigation involves swiss albino mice as an experimental animal.
7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION
IN CASE OF 7.3?
The copy of the ethical clearance certificate obtained from Institutional Animal Ethical
Committee (IAEC) is attached.
5
8.0
REFERENCES :
1. Reynolds A, Laurie C, Mosley RL, Gendelman HE. Oxidative stress and the
pathogenesis of neurodegenerative disorders. Int Rev Neurobiol 2007;82:297-9.
2.
Standaert DG, Young AB. Treatment of central nervous system degenerative disorders.
In: Brunton LL, Lazo JS, Parker KL, editors. Goodman and Gilman’s The
Pharmacological Basis of Therapeutics. 11th ed. New York (NY): McGraw Hill; 2006.
p. 527-9.
3. Baker K, Marcus CB, Huffman K. Synthetic combined super oxide dismutase/ catalase
mimetics are protective as a delayed treatment in a rat stroke model: A key role for
reactive oxygen species in ischemic brain injury. J Pharmacol. Exp Ther 1998;284:2157.
4. Easton JD, Hansen SL, Martin JB. Cerebrovascular diseases. In: Fauci AS, Braunwald
E, Isselbaker KJ, Wison JD, Martin JB, Kasper DL, et al, editors. Principles of internal
medicine. 14th ed. New York(NY): McGraw Hill; 1998. p. 2325-8.
5. Kaundal RK, Iyer S, Kumar A, Sharma SS. Protective effects of pioglitazone against
global cerebral ischemic-reperfusion injury in gerbils. J Pharmacol Sci 2009
Mar;109(3):361-7.
6. Rang HP, Dale MM, Ritter JM, Moore PK, Pharmacology. 6th ed. New Delhi:Elsevier
Publication;2007. p. 77-9.
7. Osvaldo Camilo MD, Larry B, Goldstein MD. Seizures and epilepsy after ischemic
stroke. Stroke 2004;35:1769-1.
8. Preez HD. Squalene antioxidant of the future. Natural Medicine 2008;(33):109-10.
9. Lewkowicz N, Lewkowicz P, Kurnatowska A, Tchórzewski H. Biological action and
clinical application of shark liver oil. Pol Merkur Lekarski 2006 May;20(119):598-601.
10. Reddy LH, Couvreura P. Squalene: A natural triterpene for use in disease management
and therapy. Adv Drug Deliv Rev 2009 Dec 17;61(15):1412-26.
6
11. Hajimoradi M, Hassan ZM , Pourfathollah AA, Daneshmandi S, Pakravan N. The effect
of shark liver oil on the tumor infiltrating lymphocytes and cytokine pattern in mice. J
Ethnopharmacol 2009 Dec 10;126(3):565-70.
12. Smith TJ. Squalene: potential chemopreventive agent. Expert Opin Investig Drugs 2000
Aug;9(8):1841-8.
13. Medhi B, Aggarwal R, Chakrabarti A. Neuroprotective effect of pioglitazone on acute
phase changes induced by partial global cerebral ischemia in mice. Indian J Exp Biol
Aug 2010;48:793-9.
14. Richard JT. Animal models of focal and global cerebral ischemia. Institute Lab Animal
Res 2003;44(2):85-6.
15. Homi HM, Mixco JM. Severe hypertension is not essential for isoflurane
neuroprotection against forebrain ischemia in mice. Anaesthesiology 2003;99:85-7.
7
9
SIGNATURE OF THE
CANDIDATE
10
REMARK OF THE GUIDE:
The above information and literature has been extensively investigated, verified and was
found to be correct. The present study will be carried out under my supervision and
guidance.
11
11.1 NAME AND DESIGNATION
OF GUIDE
Dr. A R. KULKARNI. M.Pharm., Ph.D.
PROFESSOR,
DEPT.OF PHARMACOLOGY,
S E T’s COLLEGE OF PHARMACY,
S. R. NAGAR, DHARWAD-580002
11.2 SIGNATURE
11.3 HEAD OF THE
Dr. A. R. KULKARNI. M.Pharm., Ph.D.
DEPARTMENT
PROFESSOR,
DEPT.OF PHARMACOLOGY,
S E T’s COLLEGE OF PHARMACY,
S. R. NAGAR, DHARWAD-580002.
11.4 SIGNATURE
12
12.1 REMARKS OF THE
The above mentioned information is correct and
PRINCIPAL
I recommend the same for approval.
Dr. V H. KULKARNI. M.Pharm., Ph.D.
PROFESSOR AND PRINCIPAL,
S E T’s COLLEGE OF PHARMACY,
S. R. NAGAR, DHARWAD-580002.
12.2 SIGNATURE
8