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

                                                                  STUDY PROTOCOL

EVALUATION OF THE 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/Female

No. of animals        : 10 /Group

Total animals         : 60

 

1.0   ALLOCATION OF GROUPS:

Group No.	Group Description	A 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	Normal saline,	0.5% MC, p.o.,	5 ml/kg, p.o.
G3	Reference Control	Normal saline,	Piracetam-400 mg/kg, q.d.in 0.5% MC	5 ml/kg, p.o.
G4	Treated with a low dose of TF		TF-X1 mg/kg,  in 0.5% MC	5 ml/kg, p.o.
G5	Treated with an intermediate dose of TF		TF-X2 mg/kg, in 0.5% MC	5 ml/kg, p.o. 5 ml/kg, p.o.
G6	Treated with a high dose of TF		TF-X3 mg/kg, in 0.5% MC	5 ml/kg, p.o.

Abbreviations: MC-Methyl Cellulose, p.o.-per os. q.d.: quaque die; X1, X2, X3, and X4 are defined as the incremental doses of the Test formulations. 

1.0  METHOD:

· Healthy animals will be selected, randomized based on body weight, and allocated into 6, different groups consisting of 10an animals each.

· Animals will be adapted and trained for neurobehavioral tests like an 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 with 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 be 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 the reference drug Piracetam at the dose of 400 mg/kg, p.o.,     q.d.

· Animals of groups G4-G6 will be treated with TF at different dose levels.

· 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, a Novel object recognition test will be conducted on days 44,47, 50, 53, 56, and 59. Additionally, the 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 an 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, 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, and biochemical and molecular parameters.

2.0 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.

· Endpoint parameters:

Ø HPLC in the hippocampus for estimation of acetylcholine.

Ø Oxidative stress parameters: Superoxide dismutase, oxidized and reduced

glutathione, malondialdehyde, 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. appendiculate 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.

 

 

 END OF DOCUMENT

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5. List of Pharmacopeial Tests Quality Control Tests or Batch Testing Parameters (In-vivo)

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