Bioassay of D-Tubocurarine
1. Rabbit Head-drop Method :
Principle: d-Tubocararine hydrochloride is injected into the marginal vein of a rabbit’s ear till the rabbit’s neck muscles are relaxed such that the animal cannot hold its head up. The total amount of test sample required to produce the endpoint is compared with the total amount of the standard sample required to produce similar endpoint.
Selection of Rabbits: Rabbits weighing 2 kg. are used. Animals should be free from disease, obtained from a healthy colony and should be accustomed with the experimental procedure.
Experimental Procedure: Each rabbit is placed in a holder with its head protruding outside. The head should be freely movable. Minimum 8 rabbits are used. They are divided into two groups each containing 4 rabbits. First group will receive standard sample and the second group will receive the sample under test. d-Tubocurarine solution is injected at a constant speed by infusion apparatus through the marginal vein.
Injection should be given at a rate of 0.4 ml/min and should take about 10 min. Infusion is continued till the rabbit will not be in a position to hold its head erect or there will be no response by focussing light on the eyes and the neck gets elongated and toneless.
Suitable dose of d-tubocurarine is 0.012% w/v in saline. Rabbits recover immediately from the effect of curarization. During the expt. there is a possibility or respiratory embarrassment which is treated by injecting neostigmine methyl sulphate (0.05 mg.) and atropine sulphate immediately through the marginal ear vein.
Cross-over test is carried out to minimise biological error due to animal variation. Those rabbits which received the standard sample on the first day will be given test sample on the second day of expt. and vice versa. Mean dose which produces head drop of the test sample is compared with the mean dose of standard preparation.
2. Frog’s Rectus Abdominis muscle Preparation:
A frog is pithed and laid on its back on a cork covered board to which it is pinned. The skin covering the abdomen is cut away and the rectus abdominis muscle of one side is dissected from the pelvic girdle to its insertion in the cartilage of the pectoral girdle. The muscle is then pinned to the cork by four pins to keep its normal length while a thread is sewn through each end. It is then mounted in the organ bath containing frog’s Ringer solution which contains : NaCl, 6.5gm.; KCl, 0.29 gm.; CaCl2, 0.24 gm.; NaHCO3, 0.4 gm.; glucose, 1.5 gm. and distilled water 2000 ml. Oxygenation is carried out to keep the tissue alive. The muscle is stabilized for 30-45 min. in order to get critical quantitative response. The responses are recorded using isotonic frontal writing lever with 1 G. tension.
Two similar contractions with the same concentration of acetylcholine are obtained. Three doses of the standard sample and one intermediate dose of the test sample are selected and the reduction in height of contraction induced by acetylcholine is noted down.
Acetylcholine contraction is recorded on slow moving drum for 90 sec. d-Tubocurarine is allowed to act for 30 sec. The percentage reduction at each dose levels is calculated and log dose response curve of the standard drug is plotted. A linear response will be obtained. The potency of test sample is calculated from the standard curve.
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