PROTOCOL
evaluation of TEST FORMULATIONS to PARKINSON’S DISEASE USING ROTENONE INDUCED
ANIMAL MODEL
1.0 TEST SYSTEM DETAILS:
Species : Rattus Norvegicus (Rats)
Strain : Albino Wistar
Age : 6-8 weeks
Body Wight : 200-250 g
Sex : Male or Female
No. of animals : 7 /Group
Total animals : 35+ 7 extra = 42
2.0 ALLOCATION OF GROUPS:
|
Groups |
Treatment |
Dose; ROA |
Number
of Animals |
|
G1 |
Normal
Control |
Normal
saline or 0.25% Na-CMC |
7 |
|
G2 |
Negative
Control (Rotenone) |
with vehicle DMSO + Miglyol
812N 1ml/kg
i.p. |
7 |
|
G3 |
Reference
Drug- (L-Dopa) |
10 mpk;
p.o. |
7 |
|
G4 |
Test Formulation-1 |
A1
mpk; p.o. |
7 |
|
G5 |
Test
Formulation-2 |
A1
mpk; p.o. |
7 |
7 extra animals will be taken extra in
each study for the randomization purpose
3.0 METHOD:
· Healthy
animals will be selected, randomized based on body weight and divided into 5 different
groups consisting of 7 animals each.
ROTENONE INDUCED
PARKINSONISM DISEASE (PD)
·
The Rotenone solution
thus prepared will be administered at 1ml/kg body weight/day intraperitoneally to
all the groups till 9th day, except for the normal control group
which receives only the vehicle (DMSO+Miglyol 812N).
·
The respective treatment
groups will receive their treatment from the day of starting of rotenone
injection.
·
During daily handling, animals
will be observed for the emergence of PD phenotype.The animals will be assessed
for their motor and behavioral scores on day 6, day 9 and day 11.
·
The trial and standard
drug treatment will be continued till the animals develop debilitating phenotype
or till the 11th day.
·
Animals developing
debilitating phenotype – limiting their mobility, feeding or grooming; will be
sacrificed and brain samples will be collected from them for further analysis.
4.0 END POINT PARAMETER(S):
· Motor
& Behavioral Assessment
a)
Open field test
b) Pole
test
c)
Rearing behavior
d) Postural
instability test
e)
Rotarod test
f)
Tail suspension test
· Time taken for the
development of debilitating PD phenotype
· Oxidative stress
assessment
· a)
Measuring the extent of lipid peroxidation (LPO) in brain homogenates
· b)
Measurement of reactive oxygen species (ROS) generation in the brain regions
· Estimation of striatal
Dopamine levels
· Brain histopathological
examination.
5.0 REFERENCE(S):
1. Correa M, Wisniecki A, Betz A, Dobson DR, O’Neill
MF, O’Neill MJ, et al. The adenosine A2A
antagonist KF17837 reverses the locomotor suppression and tremulous jaw
movements induced by haloperidol in rats: Possible relevance to
parkinsonism. Behav Brain Res. 2004;148:47–54.
2. Conceição IM, Frussa-Filho R. Effects of a single administration of buspirone on catalepsy, yawning
and stereotypy in rats. Braz J Med Biol Res. 1993;26:71–4.
3. Fernagut PO, Chesselet MF. Alpha-synuclein and transgenic mouse models. Neurobiol Dis.
2004;17: 123–30.
4. Fleming SM, Delville Y, Schallert T. An intermittent, controlled-rate, slow
progressive degeneration model of Parkinson's disease: antiparkinson effects of
Sinemet and protective effects of methylphenidate. Behav Brain Res.
2005;156:201–13.
5.
Fleming SM, Zhu
C, Fernagut PO, Mehta A, DiCarlo CD, Seaman RL, Chesselet MF. Behavioral and immunohistochemical effects
of chronic intravenous and subcutaneous infusions of varying doses of rotenone.
Exp Neurol. 2004; 187:418–29.
6. Woodlee MT, Kane JR, Chang J, Cormack LK, Schallert T. Enhanced
function in the good forelimb of hemi-parkinson rats: compensatory adaptation
for contralateral postural instability? Exp Neurol. 2008;211:511–7.
7. Fleming SM, Mulligan CK, Richter F, Mortazavi F, Lemesre V, et al.
(2011) A pilot trial of the microtubule-interacting peptide (NAP) in mice
overexpressing alpha-synuclein shows improvement in motor function and
reduction of alpha-synuclein inclusions. Mol Cell Neurosci 46: 597–606.
8. Duan W, Mattson MP (1999) Dietary restriction and 2-deoxyglucose
administration improve behavioral outcome and reduce degeneration of
dopaminergic neurons in models of Parkinson's disease. J Neurosci Res 57:
195–206.
9. Cryan JF, O'Leary OF, Jin SH, Friedland JC
et al (2004) Norepinephrine-deficient mice lack responses to
antidepressant drugs, including selective serotonin reuptake
inhibitors. Proc Natl Acad Sci U S A 101:8186–8191.
10. Ohkawa H, Ohnishi N, Yagi K. Assay
for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal
Biochem 1979;95:351-58.
11. Driver AS, Kodavanti PS, Mundy WR. Age related changes in reactive oxygen species production in rat brain
homogenates. Neurotoxicol Teratol. 2000;22:175-81.
12. Shinomol GK, Muralidhara. Differential induction of oxidative
impairments in brain regions of male mice following subchronic consumption of khesari dhal (Lathyrus sativus) and detoxified khesari dhal. Neurotoxicol 2007;28:798-806.
13. Dalpiaz A, Filosa K, de Capraris P, Conte G, Bortolotti F, Biondi C,
Scatturin A, Prasad PD, Pavan B. Molecular mechanism involved in the transport
of a prodrug dopamine glycosyl conjugate. Intl J Pharmaceutics 2007;336:133-9.
14. Na Zhang, Deqiang Dou, Xiaoku Ran and Tingguo Kang. Neuroprotective
effect of arctigenin against neuroinflammation and oxidative stress induced by
rotenone. RSC Adv., 2018, 8, 2280
END OF DOCUMENT
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