1.0 TEST SYSTEM DETAILS:
Species : Mus musculus (Mouse)
Strain : BALB/c
Age : 6-8 weeks
Sex : Male
No. of animals : 8 /Group
Total animals : 56 Males
2.0 TEST ARTICLES DETAILS
HF1: Curcuma longa, Acorus calamus, and Herbal Bhasma-based herbs-mineral formulation.
3.0 ALLOCATION OF GROUPS:
|
Group No. |
Group
Description |
Disease
disease-inducing agent administered |
Treatment
administered |
Dose
Volume and Route |
|
G1 |
Normal Control |
PBS administered i.p. on Day 0 and Day 3 |
0.5% MC b.i.d. |
10 ml/kg, p.o. |
|
G2 |
Disease Control |
Cocktail of 5-monoclonal antibodies administered on
Day 0 (1.5 mg/mouse), i.p. + LPS (50 µg/animal) on Day 3 |
0.5% MC, b.i.d. |
|
|
G3 |
Reference Control |
Methotrexate 0.5 mg/kg, p.o., every
alternate day + 0.5 % MC |
||
|
G4 |
Treated with a low dose of HF1 |
HF1: 10-30 mg/kg, p.o.
b.i.d. in 0.5% MC |
||
|
G5 |
Treated with intermediate dose 1 of |
HF1: 30-100 mg/kg, mg/kg, b.i.d. in 0.5% MC |
||
|
G6 |
Treated with intermediate dose 2 of |
HF1: 100-300 mg/kg, b.i.d. in 0.5% MC |
||
|
G7 |
Treated with a low dose of |
HF1: 300-1000 mg/kg, b.i.d. in 0.5% MC |
4.0 METHOD:
• Healthy animals will be selected for the study, randomized based on body weight, and assigned to 7 groups of 8 animals each.
• On day 0, mice will receive a single-dose intraperitoneal (i.p.) injection of a cocktail of 5 monoclonal antibodies to type II collagen (1.5 mg/mouse) followed by i.p. injection of 50 μg of lipopolysaccharide (LPS from Escherichia coli strain 0111:B4) on day 3.
• On day 4, clinical disease activity will be assessed, and based on the score; all the animals will be randomized into different groups. The clinical disease activity (CDA) will be scored on a 4-point scale per paw: 0= normal, 1= erythema and mild swelling confined to the tarsals or ankle joint, 2= erythema and mild swelling extending from the ankle to the tarsals, 3= erythema and moderate swelling extending from the ankle to metatarsal joints, 4= erythema and severe swelling encompass the ankle, foot and digits, or ankyloses of the limb. Thus the maximum score of an animal will be 16.
• 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 the reference drug Methotrexate at the dose of 0.5 mg/kg, every alternate day.
• Animals of group G4-G7 will be treated with HF1, at different dose levels ranging from 10-1000 mg/kg, b.i.d. for 2 weeks
• The animals will continue to receive the treatments for 11 days and CDA scoring will be performed daily.
• Paw thickness will be measured by using a Vernier caliper daily after CDA scoring.
• Animals will be subjected to an assessment of mechanical hyperalgesia by employing the Randall-Sellitto test apparatus on Days 2, 6, 10 and 14
• Further, the animals will be subjected to an assessment of thermal hyperalgesia by utilizing the hot plate test apparatus on Days 3, 7, 11, and 15.
• On Day 16, animals will be subjected to radiological assessment of the knee and ankle joints under isoflurane anesthesia and subsequently 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 to estimate biochemical parameters. Immediately after the animal dies, the right hind limb will be excised and fixed in 10% neutral buffered formalin for histopathological analysis.
2.0 PARAMETERS TO BE EVALUATED:
• Body weight: Twice a week.
• Clinical disease activity (CDA)
• Paw thickness
• Mechanical hyperalgesia
• Thermal hyperalgesia
• Serum cytokines estimation- IL-6, IL-1β, TNF-α
• Radiological assessment of knee and ankle joint
• Histopathology of knee and ankle joint
3.0 REFERENCES:
END OF DOCUMENT
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