EFFICACY OF TEST FORMULATIONS IN MOUSE MODEL OF D-GALACTOSE INDUCED ACCELERATED SENESCENCE-ASSOCIATED LEARNING AND MEMORY IMPAIRMENT

                                                                     STDUY PROTOCOL

 EFFICACY OF TEST FORMULATIONS IN MOUSE MODEL OF D-GALACTOSE INDUCED ACCELERATED SENESCENCE-ASSOCIATED LEARNING AND MEMORY IMPAIRMENT

TEST SYSTEM DETAILS:

Species                   :Mus musculus (Mice)

Age                        :10 weeks

Sex                         : Male

No. of animals        :10/Group

Total animals          : 60 

1.0   ALLOCATION OF GROUPS:

Group No.	Group Description	Disease Induction agent administered	Treatment administered	Dose Volume and Route
G1	Normal Control	Normal saline, administered by subcutaneous route × 42 days	0.5% MC, p.o.	5 ml/kg, p.o.
G2	Disease Control	D-galactose (400 mg/kg), administered by subcutaneous route × 42 days	0.5% MC, p.o.
G3	Reference Control		Piracetam-400 mg/kg, q.d.in 0.5% MC
G4	Treated with low dose of Test Compound		X1 mg/kg, in 0.5% MC
G5	Treated with intermediate dose of Test Compound		X2 mg/kg, in 0.5% MC
G6	Treated with high dose of Test Compound		X3 mg/kg, in 0.5% MC

Abbreviations: MC-Methyl Cellulose, p.o.-per os. q.d.: quaque die; bid: bis in 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.

· 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 elevated plus maze, Morris water maze and novel object recognition test, after the completion of acclimatization. The training session for each test will be conducted on nine different days and the animals will be trained for each test at least three times.

· Normal control group (G1) will be injected normal saline by subcutaneous route for 42 days.

· Animals allocated to G2-G6 will be administered D-galactose dissolved in normal saline for 42 days.

· Animals allocated to Group G1 will serve as Normal-control and administered 0.5% MC, p.o.

· Disease control G2 animals will receive 0.5% MC, p.o.

· Animals of group G3 will be treated with reference drug Piracetam at the dose of 400 mg/kg, p.o., q.d.

· Animals of group G4-G6 will be treated with Test Compound at different dose levels ranging from 10-1000 mg/kg

· Compound administration will be concurrent with the disease induction agent.

· One day after the last injection, neurobehavioral tests will be performed according to the below mentioned schedule: Elevated plus maze test will be performed on days 43, 46, 49, 52, 55 and 58. Further, Novel object recognition test will be conducted on days 44,47, 50, 53, 56 and 59. 

Additionally, Morris Water Maze test will be performed on days 45, 48, 53, 56, 59, 60 and 61. On day 63 they will be sacrificed under overdose of thiopentone anesthesia. 

After suitable anesthesia 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, cerebral cortex and hippocampus 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.

3.0  PARAMETERS TO BE EVALUATED:

·     Clinical observation

·     Body weight: Once a week.

·     Neurobehavioral parameters: Elevated plus maze test, Morris water maze test and novel object recognition test.

·     End point parameters:

Ø  HPLC in hippocampus for estimation of acetylcholine.

Ø  Oxidative stress parameters: Superoxide dismutase, oxidized and reduced 

     glutathione, malondialdehye and catalase.

Ø   Acetylcholinesterase activity in the hippocampus.

Ø  Histopathology of Cortex & Hippocampus and determination of neuronal density

REFERENCE(S):

1. Lieh-Ching Hsu,1 Yu-Jen Ko,1 Hao-Yuan Cheng,2 Ching-Wen Chang,1 Yu-Chin Lin,3 Ying-Hui Cheng,1 Ming-Tsuen Hsieh,1 and Wen Huang Peng. Antidepressant-Like Activity of the Ethanolic Extract from Uncaria lanosa Wallich var. appendiculata Ridsd in the Forced Swimming Test and in the Tail Suspension Test in Mice. Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 497302, 12 pages doi:10.1155/2012/497302

2. Dinesh Dhingra and Varun Kumar. Memory-Enhancing Activity of Palmatine in Mice Using Elevated Plus Maze and Morris Water Maze. Hindawi Publishing Corporation Advances in Pharmacological Sciences Volume 2012, Article ID 357368, 7 pages doi:10.1155/2012/357368

3. Stone WS, Croul CE, Gold PE. Attenuation of scopolamine-induced amnesia in mice. Psychopharmacology (Berl). 1988;96(3):417-20.

4. Kelley Bromley-Brits, Yu Deng, and Weihong Song. Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice J Vis Exp. 2011; (53): 2920.

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