Strain : Balb/c or Swiss Albino
Age: 4-5 weeks
Sex: Male
No. of animals: 10 /Group
Total animals: 70
2.0 TEST ARTICLES DETAILS
RS1: HB-1
3.0 ALLOCATION OF GROUPS:
|
Group No. |
Group
Description |
Sleep
disrupting agent administered |
A sleep-inducing agent administered |
Treatment
administered |
Dose
Volume and Route |
|
G1 |
Normal Control |
Normal Saline administered by intraperitoneal route
(i.p.), |
Pentobabitone-45 mg/kg, i.p., single dose |
0.5% MC, p.o., b.i.d. |
10 ml/kg, p.o. |
|
G2 |
Disease Control |
Caffeine-50 mg/kg, i.p.,
single dose |
Pentobabitone-45 mg/kg, i.p., or thiopentone-50
mg/kg, i.p., single dose |
0.5% MC, p.o., b.i.d. |
|
|
G3 |
Reference Control |
Alprazolam 0.3 mg/kg, once only |
|||
|
G4 |
Treated with a low dose of HB1 |
HB1: 10-30 mg/kg, b.i.d. in 0.5% MC |
|||
|
G5 |
Treated with intermediate dose 1 of HB1 |
HB2: 30-100 mg/kg, mg/kg, b.i.d. in 0.5% MC |
|||
|
G6 |
Treated with intermediate dose 2 of HB1 |
HB3: 100-300 mg/kg, b.i.d. in 0.5% MC |
|||
|
G7 |
Treated with high dose of HB1 |
HB4: 300-1000 mg/kg, b.i.d. in 0.5% MC |
Abbreviations: MC-Methyl Cellulose, p.o.-per os. q.d.: quaque die; bid: bis in die, i.p.-Intraperitoneal
4.0 METHOD:
· Healthy animals will be selected for the study, randomized based on body weight, and assigned to 7 groups of 10 animals each.
· Animals of Group G1 will be designated as normal-control and administered 0.5% MC, p.o., b.i.d.
· Disease control animals (assigned to group G2) will receive 0.5% MC, p.o., b.i.d.
· Animals of group G3 will be treated with reference drug alprazolam at the dose of 0.3 mg/kg, p.o.
· Animals of group G4-G7 will be treated with HB1 at different dose levels ranging from 10-1000 mg/kg, b.i.d.
· Compound administration will be initiated 3 days before the sleep test.
· On day 3 the animals will be kept fasting for 24 hours
· On day 4, half an hour after administration of test articles, caffeine will be injected into the animals at the dose of 50 mg/kg, through the intraperitoneal route
· Subsequently, after 30 minutes of caffeine administration mice will either receive pentobarbitone at the dose of 45 mg/kg injected intraperitoneally or thiopentone administered intraperitoneally at the dose of 50 mg/kg administered intraperitoneally.
· Sleep latency (time taken for the loss of righting reflex) and sleep duration will be recorded for all the groups.
· Any animal that does not sleep within 15 minutes post barbiturate injection will be excluded from the study.
· After completion of the sleep test, animals will be sacrificed under an overdose of thiopentone anesthesia. Immediately after the animal dies, the brain will be excised, weighed, and stored at -80°C for molecular analysis.
5.0 PARAMETERS TO BE EVALUATED:
· Body weight
· Brain weight
· Evaluation of sleep latency and sleeping time
· Gene expression analysis by real-time PCR: GABAA, GABAB, and 5HT1A in brain tissue.
6.0 REFERENCE(S):
1. List of All SOPs and Documents for the Microbiology Laboratory
2. List of All SOPs and Documents for In-vitro Laboratory
3. List of All SOPs and Documents for the In-vivo Laboratory.
4. List of All SOPs and Documents for Clinical Research
5. List of Instruments and Equipment Used In Laboratory Animal Facility
6. Animal Facility Design - Small Laboratory Animals (Rat, Mice, Rabbit, Guinea Pig)
7. List of Chemicals Needed for In-vivo Laboratory
8. List of Physiological Data for Small Laboratory Animals
9. Leading Lab-Diet Suppliers in India and Beyond
10. List of All SOPs and Documents for Animal House Facility
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