EVALUATION OF EFFICACY OF TEST FORMULATIONS IN MOUSE MODEL OF
MPTP AND PROBENECID-INDUCED PARKINSON’S DISEASE
1.0
TEST SYSTEM DETAILS:
Species :
Mus musculus (Mice)
Age :
10 weeks
Sex : Male/Female
No. of animals :
10/Group
Total animals :
70
2.0 ALLOCATION OF GROUPS:
|
Group No. |
Group
Description |
Disease
Induction agent administered |
Treatment
administered |
Dose
Volume and Route |
|
G1 |
Normal Control |
5% sodium bicarbonate solution, ten injections
distributed over 40 days by
intraperitoneal (i.p.) routes and normal saline, ten injections
distributed over 40 days by subcutaneous (s.c.) route |
0.5% MC, p.o., q.d. |
5 ml/kg, p.o. |
|
G2 |
Disease Control |
Probenecid-250 mg/kg, i.p., ten injections
distributed over 40 days and MPTP-25 mg/kg, s.c., ten injections distributed
over 40 days |
0.5% MC, p.o. |
|
|
G3 |
Reference Control |
Selegiline-3 mg/kg,
q.d.in 0.5% MC |
||
|
G4 |
Treated with low dose of Test Formulation |
Test Formulation -X1 mg/kg, q.d. in 0.5% MC |
||
|
G5 |
Treated with intermediate dose of Test Formulation |
Test Formulation -X2 mg/kg, q.d. in 0.5% MC |
||
|
G6 |
Treated with high dose of Test Formulation |
Test Formulation -X4 mg/kg, q.d. in 0.5% MC |
Abbreviations: MC-Methyl Cellulose, p.o.-per os. q.d.:
quaque die; X1, X2, X3, X4 are defined as the incremental doses of the Test
formulations. The dose range will be from 10 mg/kg to 1000 mg/kg, q.d.
3.0
METHOD:
· Healthy animals will be selected, randomized based on body
weight and allocated into 6, different groups consisting of 10 animals each.
·
Animals will be adapted and trained for neurobehavioral tests
like narrow beam walk test, pole test and open field test, after the completion
of acclimatization. The training session for each test will be conducted on
three different days and the animals will be trained for each test at least
three times.
·
Prior to initiation of disease induction, the neurobehavioral
assessments will be performed (Day 0).
· Normal Control group (G1) will be injected 5% sodium
bicarbonate solution by intraperitoneal route and thirty minutes later, normal
saline will be administered by subcutaneous route. Animals will receive a total
of 10 injections distributed over a period of 31.5 days (Interval between two
consecutive injections will be of 3.5 days).
·
Animals allocated to G2-G6 will be administered probenecid (250
mg/kg) dissolved in 5% sodium bicarbonate and thirty minutes later, MPTP (25
mg/kg) will be administered by subcutaneous route. Animals will receive a total
of 10 injections distributed over a period of 40 days (Interval between two
consecutive injections will be of 4 days).
·
Animals allocated to Group G1 will serve as Normal-control and administered 0.5% MC, p.o., q.d.
·
Disease control (G2) animals will receive 0.5% MC, p.o., q.d.
·
Animals of group G3 will be
treated with reference drug Selegiline at the dose of 3 mg/kg, p.o., q.d.
·
Animals of group G4-G6 will be treated with Test Formulation
at different dose levels ranging from 10-1000 mg/kg, q.d.
·
Compound
administration will be initiated 5 days prior to and continued till the end of
the experiment.
· 2 days after the last
injections, neurobehavioral test will be performed. Subsequently, they will be sacrificed
under overdose of thiopentone anaesthesia. After suitable anaesthesia but
before the animal dies, blood will be collected from the retro-orbital plexus
for the estimation of biochemical parameters. Immediately after the animal
dies, the brains of the mice will be dissected out, substantia nigra and corpus
striatum will be separated, weighed and sectioned into two halves each. One
half of both the tissues will be transferred to containers filled with 10%
neutral buffered formalin, for the ensuing histopathological analysis,
respectively. The remaining half will be snap frozen in liquid nitrogen and
immediately stored at -80°C for the subsequent analysis of neurotransmitters, biochemical and
molecular parameters.
4.0
PARAMETERS TO BE EVALUATED:
·
Clinical observation
·
Body weight: Twice a week.
·
Neurobehavioral parameters: Narrow beam, open field and pole
test.
·
End point parameters:
Ø HPLC in brain striatum for
estimation of Dopamine, DOPAC and HVA.
Ø Immunohistochemistry
analysis in brain:
v
SN: TH positive dopamine neuronal cells
v
Striatum: TH positive dopamine neuronal terminals,
α-synuclein positive cells, GFAP positive cells, Iba-1 positive cells.
Ø
Quantitative real time reverse transcriptase polymerase chain
reaction (qRTPCR) in brain SN: Gene expression analysis of ND-1, TNF-α, IL-1β,
Bax, Bcl-2, Nrf-2, HO-1 and NQO-1.
5.0
REFERENCE(S):
1)
Xuan He, Shuangshuang Yang, Rui
Zhang, Lina Hou, Jianrong Xu, Yaer Hu, Rang Xu, Hao Wang and Yongfang Zhang.
Smilagenin Protects Dopaminergic Neurons in Chronic MPTP/Probenecid-Lesioned
Parkinson’s Disease Models. Front Cell Neurosci. 2019 Feb 5;13:18. doi:
10.3389/fncel.2019.00018.
2) Govindasamy Pushpavathi Selvakumar, Udaiyappan Janakiraman, Musthafa Mohamed Essa, Arokiasamy Justin Thenmozhi, Thamilarasan Manivasagam. Escin attenuates behavioral impairments, oxidative stress and inflammation in a chronic MPTP/probenecid mouse model of Parkinson's disease. Brain Res. 2014 Oct 17;1585:23-36. doi: 10.1016/j.brainres.2014.03.010.
3) Nataraj J, Manivasagam T, Justin Thenmozhi A, Essa MM. Neurotrophic Effect of Asiatic acid, a Triterpene of Centella asiatica Against Chronic 1-Methyl 4-Phenyl 1, 2, 3, 6-Tetrahydropyridine Hydrochloride/Probenecid Mouse Model of Parkinson's disease: The Role of MAPK, PI3K-Akt-GSK3β and mTOR Signalling Pathways. Neurochem Res. 2017 May;42(5):1354-1365. doi: 10.1007/s11064-017-2183-2.
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