BIOASSAY OF SKELETAL NEUROMUSCULAR JUNCTION BLOCKER (2) (1).pdf

BIOASSAY OF NEUROMUSCULAR JUNCTION BLOCKER.
D TUBOCURARE BIOASSAY
MUSCLE RELAXANT BIO ASSAY

1. EFFECT OF SKELETAL MUSCLE
RELAXANTS ON FROG RECTUS
MUSCLE
Dr. Chandan Verma
Professor
Department of Pharmacology
SMS Medical College, Jaipur

2. Effects of Skeletal muscle Relaxants
Aim:-
To determine The effects of the skeletal muscle relaxants using the Rectus
Abdominis Muscle of frog.
Background:-
Rectus abdominis muscle is a striated skeletal muscle preparation which is
sensitive to the ACh and curare like substances.
The activity is considered due to presence of nicotinic muscarinic
(NM)-receptor.

3. Identification of frog rectus abdominis muscle
Frog rectus abdominis
muscle is composed of
both Single or
focal-innervated fiber
and Multiple-innervated
fibers, but the
multiple-innervated fibers
dominate and show the
slow contraction.

4. Principle
d- Tubocurarine is skeletal muscle relaxant acting on nicotinic
receptor produces a dose dependent competitive and reversible
antagonism of the acetylcholine.
Thus graded response of d- Tubocurarine in the form of inhibition
of the fixed dose of acetylcholine can be determined.
The response are plotted against log-dose of d- Tubocurarine

5. Materials
Animal/tissue:Frog/rectus abdominis muscle
PSS: Ringer
Lever: Simple writing lever
Magnification :10-15x
Tension: 1-2 gm
Temperature: Room temperature
Air: O2/Carbogen
Drug: d- Tubocurarine

6. Precautions before Experimentation
Frog rectus abdominis muscle is skeletal muscle and requires less
precaution during handling compared to other smooth isolated tissues.
●Clean the organ bath before starting the experiment specially inner
organ bath (chances of presence of previous drug used)
●Balance the simple writing lever tangentially with smoked drum
●Prepare PSS for the experiment with sufficient quantity of chemicals
(1% variability is acceptable)
●Add the calcium chloride at the end of PSS preparation (to avoid any
precipitation: PSS should be clear)
●Try to minimize the handling of tissue (especially at the middle part)
●Maintain the dose cycle properly (tissue sensitivity depend on this
cycle)

7. Method
Step I:Pith the frog and lay it on its back on the frog dissecting
board. Pin the four limbs on the dissecting board
Step II:Remove the abdominal skin and expose the rectus
abdominis muscle (silver grey, distinct from surrounding
tissue, at middle lining).
Step III:Dissect out the muscle on the PSS soaked piece of paper
and spread gently.
Step IV: Cut into two pieces (preserve the longitudinal muscle).

8. Method
Step V: Tie the thread on the one end and tie it to the inner organ
bath and the second end tied to the simple lever in the upright
position under the tension of 1-2 gm.
Step VI: Leave tissue for relaxation for at least 45 min.
Step VII: Meanwhile prepare the standard and test drug serial dilution.
Step VIII:Start the recording of response by using the suitable
experimental design.

9. Procedure
The frog is sacrificed as per CPCSEA recommended guidelines and rectus
abdominis muscle is dissected out and mounted in the organ bath.
The preparation is allowed to stabilize for half an hour during which wash is
given at every 10 minute intervals
To equipment responses to submaximal dose of acetylcholine are recorded. The
drum is moved for 30 seconds and the lowest dose 0.1 ml of the d-
Tubocurarine is added in the bath.
After 2 minute response to the same dose of acetylcholine is taken in the
presence of d- Tubocurarine.
The 5 minutes cycle is followed as usual

10. The responses to acetylcholine are taken after every 5 minutes, till the
recovery to the control height is achieved.
The response to d-tubocurarine in using different doses of standard and of
unknown are recorded in this manner in the case the recovery is not
achieved even after repeated do this then either a large dose of acetylcholine
is given for recovery for the preceding height is taken as control for the next
doses.
The graph is fixed in the colonophony solution the heights of control (H) and
in the presence of antagonist (h) in the presence of d- Tubocurarine are
measured and the percentage of inhibition is calculated as follows:-

%Inhibition = H-h X 100
H

11. Neuromuscular blockade by d-tubocurarine on the acetylcholine-induced muscle twitch response in an
isolated frog rectus abdominis muscle preparation

13. Other methods for the d-TUBOCURARINE
Rabbit head drop method
Principle:
d-TUBOCURARINE hydrochloride is injected into the marginal vein of
rabbits ear till the rabbits neck muscles are relaxed such that the animal
cannot hold his head up.
The total amount of test sample required to produce the end point is
compared with the total amount of standard sample required to produce
similar and point
Selection of rabbit:
Rabbits weighing 2 kg are used. Animal should be free from disease,
obtained from a healthy colony and should be a accustomed with the
experimental procedure.

14. Experimental procedure:
Each rabbit is placed in a holder with his head protruding
outside the head should be freely movable.
Minimum 8 rabbits are used. They are divided into two
groups each containing four 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.

15. Injection should be given at rate of 0.5 ml per minute and should take
about 10 minimum infusion is continued till the rabbit will not be in a
position to hold his head erect or there will be no response by
focusing light on the eyes and the neck gets elongated and toneless
Suitable dose of d-TUBOCURARINE is 0.012% w/v in saline.
During the experiment there is a possibility of respiratory
embarrassment which is treated by injecting neostigmine methyl
sulphate (0.05 mg) and atropine sulphate immediately through the
marginal ear vein.
The rabbit recovers immediately from the effect of curarization.

16. Rabbit head drop method for the bioassay of d-tubocurarine.
(i) : i.v. inj. of d-tubocurarine. (ii) : Head drop after injection.