Experiments on isolated skeletal muscles mukunda

Dr. Mukunda Bharat Bargade,
JR1, Dept. of Pharmacology,
IGGMC, Nagpur.
9 April 2015 1Dr.M. B. Bargade

 Introduction
 Purpose of experiments in pharmacology
 Bioassay
 Experiments on isolated skeletal muscle
preparations
 Conclusion

In pharmacology, assay on animals and animal
tissue preparations are needed because
 1. Number of drugs obtained from plant and
animal sources are not in chemically pure form.
 2. Screening of new drugs
 3. To handle minute quantity of the drugs
eg. Estimation of drug metabolite

 1. Qualitative experiments to analyze activity
 2. Quantitative experiments to estimate amount
of active drug in plant or animal extract
 3. Assay of agonist
 4. Comparison of activity of different agonist
 5. Assay of antagonist

•Based on physical
characteristicsPhysical
•Estimation of concentration
of active principleChemical
•Using various biological
methodsBiological

 Definition- Bioassay is the estimation of the
potency of an active principle in the given
quantity of the given preparation using
biological method.
 Specific bioassays-
A. Bioassay on whole animal
B. Bioassay on isolated tissue
C. Bioassay on cells
D. Bioassay on microorganism9 April 2015 6Dr.M. B. Bargade

A. The phrenic nerve-diaphragm preparation of rat
B. The rectus abdominis muscle of frog
C. The dorsal muscle of leech
D. The chick biventer cervicis preparation

 Commonly used- Ratus norvegicus
 Preferred – wistar rat & sprague dawley rat
 First described by Bulbring
 Study of drug effects on twiches of striated
muscle

• Sacrifice the rat by stunning on head
• Mount on dissecting board
• Open the thorax and expose the thoracic cavity
• Cut the ribs from base of sternum, half thorax is cut & removed
• Identify the Phrenic nerve, running from diaphragm to thymus
• Dissect out phrenic nerve, at diaphragm & base of thymus

• Transfer the preparation to a dish containing Krebs’ solution
• Tie thread- on apex of the piece of diaphragm & with another
color to the end of nerve
• Transfer the preparation to the organ bath of 30-40 mm dia.,
vol- 40 ml. at 37* celcius
• Contractions recorded with a light spring-loaded lever with a
sideways-writing point
• Nerve stimulated at a rate of 12 shocks/min. by rectangular-
wave pulse of 0.5 msec.

 Tubocurarine-
 Estimation of the concentration of unknown
solution
 Standard solution- 2*10-4 M
 Doses of standard & unknown which produce
roughly 30% & 50% inhibition of contraction
 Perform four-point assay of Latin square
design

 Solution required –
 Krebs’ solution- minimum 5 lit.
 Tubocurarine chloride- standard 2*10-4 M
unknown- stronger than 5*10-5 M
Stock- 10-3 M
 Time required-
 For full 16 dose assay- 4 hours or more
 Cycle-
0 min- Start kymoghraph
1 min.- Add drug
6 min.- Stop kymoghraph & wash preparation
8 min.- wash preparation
10 min.- start kymoghraph9 April 2015 14Dr.M. B. Bargade

 Effect of following drugs on preparations-
1] Ach 10-2 M
2]Decamethonium
6*10-3 M or
suxamethonium 2*10-
3 M
3] Hexamethonium
2*10-2 M
4] Tubocurarine 2*10-4 M or
Gallamine triethiodide 5*10-3
M
5] Tubocurarine or Gallamine
(as in 4) f/b 3 min after without
wash, 0.2 ml Physostigmine 10-4
M or neostigmine 10-4 M

6] Decamethonium 6*10-3
M or suxamethonium
2*10-3 M f/b 3 min after
without wash, 0.2 ml
Physostigmine 10-4 M or
neostigmine 10-4 M
7] 0.2 ml Physostigmine
10-4 M or neostigmine 10-4
M f/b 1min after without
wash O.2 ml Ach 10-2 M
8] 0.2 ml Lignocaine 3*10-2
M

 Convinient to arrange the experiments in pairs-
 One preparation for Decamethonium,
tubocurarine chloride, Physostigmine
 Other for Suxamethonium, Gallamine
triethiodide, Neostigmine

 Solutions required-
 Krebs’ solution- 1 lit. for each drug
 Other drugs as mentioned in expt.
 Time required- in pair- 2 hr, without pair- 3 hr
 Time cycle-
1 min.- Add drug
10 min.- start kymoghraph

 Usually made from Rana temporaria, Rana
pipiens, Rana esculenta
 Any frog about 20 gm
 Robust & easily set up
 Originally described by Burn

•Stunning of frog
•Double pithing of frog
•Pin four limbs on dissecting board, ventral part facing above
•Remove abdominal skin & expose the rectus abdominis muscle
•Take out muscle on dish with ringer lactate, spread gently

• Cut into two longitudinaly & transfer to Frog ringer solution.
• Thread is attached at each end of preparation
• Preparation is mounted in organ-bath of capacity 5-10 ml at
room temperature
• Contractions are recorded with a simple sideways-writing
lever
• Contractions are obtained by administrating drugs to the
organ bath

 6 min cycle to test responses to ACh
 Log dose response curve
 Dose for 30% of maximum response
 Doubling the conc. will give about 50-60 %
response
 Test doses for unknown solution
 Select 2 suitable conc. & perfom four-point
assay with Latin square method

 Frog-Ringer – 1 lit. for two preparations
 ACh- Stock – 5*10-3 M ( 1mg/mL)
Standard- 5*10-5 M (10ug/mL )
Unknown- not less than 2*10-5 M (1ug/mL)
 Time required-
 Four point assay (16 Dose)- 2 hr
 Three-point assay- 90 min.
 Time cycle- 0 min.- raise 1gm wt & start kymoghraph
2 min.- Add ACh
3.5 min.- Stop kymoghraph, lower wt, wash
6 min.- Raise wt & start kymoghraph

 Response to ACh 2*10-6 M obtained
 Conc. which gives 50% of maximum response
selected
 Serum is added to ACh solution & immediately
test done- Gives same response
 Serum is added to ACh solution & incubated
for 5 min- test gives much smaller/ no
response
 Physostigmine added to serum f/b ACh-
incubated for 5 min.- test gives bigger response

 Frog-Ringer – 1 lit. for two preparations
 ACh– 5*10-5 M (10ug/mL )
 Physostigmine- 3*10-4 M (100ug/mL)
 Serum- 1mL or 2 fold diluted serum
 Time required-
 1hr

 Choline esters-
 Relationship between chemical structure &
biological activity
 Comparison is made in presence of
Physostigmine (10ug/mL)
 Comparison results obtained are compared
with those of comparison of same esters on
guinea-pig ileum

 Onium salts-
 Comparison with ACh requires 1 hr
 30 min to show antagonistic activity of
tetraethylammonium & n-
pentyltriethylammonium
 Results compared with those obtained on isolated
guinea-pig ileum

Choline ester Conc. Likely to be
effective (M)
Stock solution
(M)
Acetylcholine 2*10-6 5*10-5
Carbachol 8*10-6 2*10-4
Propionylcholine 8*10-7 2*10-5
Butyrylcholine 2*10-6 5*10-5
Suxamethonium 2*10-6 5*10-5
Methacholine 4*10-4 10-2

Onium salt Conc. Likely to be
effective (M)
Stock solution
(M)
Tetramethylammonium 2*10-5 5*10-4
n-Butyltrimethylammonium 10-5 2*10-4
n-Pentyltrimethylammonium 2*10-5 5*10-4
n-hexyltrimethylammonium 4*10-5 10-3
Decamethonium 8*10-6 2*10-4

 Agonist conc. giving 30% & 60% of maximum
responses are obtained
 Ringer then replaced by Frog-Ringer solution
with tubocurarine chloride 10-6 M
 Agonist now gives much smaller response

 Again repeated with tubocurarine 10-5 M and
10-4 M
 Dose-ratios of agonist for corresponding conc.
of antagonist calculated
 Graph of dose-ratio minus one versus conc. of
antagonist is ploted

 Solution require-
 Frog- ringer- 1 lit. for each preparation
 ACh- 5*10-3 M, 5*10-4 M, 5*10-5 M
 Tubocurarine chloride- 10-3 M, 10-4 M, 10-5 M
diluted 1 in 10 in frog-ringer
 Time required-
 About 2 hr

 Responses are obtained with ACh,
Suxamethonium, Decamethonium
 Conc. of all 3 giving fairly-equal size response
are calculated
 Effect of Tubocurarine chloride or Gallamine
triethiodide evaluated
 Rough value of dose-ratio and affinity constant
of the antagonist can be estimated

 Solution required-
 Frog-ringer- 1lit. For 2 preparations
 ACh- 5*10-5 M
 Suxamethonium- 5*10-5 M
 Decamethonium- 2*10-4 M
 Tubocurarine chloride- 10-4 M
 Gallamine triethiodide- 10-3 M
 Time required-
 To test one agonist-antagonist takes about 1 hr9 April 2015 40Dr.M. B. Bargade

 Conc. of Suxamethonium which gives between
30% & 60% of maximum response
 Serum is added to Suxamethonium solution &
immediately test for response- Gives same
response
 Serum to Suxamethonium solution incubated
at 37* for 10 min. & 30 min.- much smaller/ no
response
 Physostigmine /Neostigmine added to serum
f/b Suxamethonium- incubate 30 min.- test
gives bigger response

 Frog-ringer – 1 lit. for 2 preparations
 Suxamethonium- 10-4 M
 Physostigmine- 3*10-4 M
 Neostigmine- 3*10-4 M
 Serum/plasma- 8 ml for each preparation
 Time required-
 2 hr

 Leeches – Hirudo medicinalis
 Contains slow fibres
 When treated with Physostigmine, most
sensitive tissue to ACh
 Can estimate less than 50 picomoles of ACh
 Active & plentiful cholinesterases present

• Leech is pined on two end over cork board
• Cut- along 2 pale lateral line from mouth to tail
• Remove internal organs
• Dorsal muscle pinned out & divided longitudinally
• Thread is attached on both ends

•Tissue is mounted in organ bath of 5-10 ml capacity at room
temp.
•Frog-ringer or Locke’s solution used
•Contraction are recorded with Simple sideways-writing lever
•Drugs are tested by administrating into organ bath

 Dose-response curve with ACh using Frog-
ringer
 Physostigmine 25 mL 3*10-4 M in 250 mL of
Frog-ringer
 Frog-ringer containing Physostigmine is added
to organ bah & incubate for 20 min.

 Dose-response curve with ACh obtained again
 Degree of potentiation for Physostigmine
calculated from comparison of above 2 dose-
response curves
 Unknown solution of ACh in frog-ringer
containing Physiostigmine is tested
 A simple bracketing assay

 Solution required-
 Frog-ringer or Locke’s solution- 5 lit. for 2
preparations
 ACh- 10-4 M, 10-6 M & unknow ( stronger than
3*10-7 M)
 Physostigmine- 3*10-4 M
 Time required-
 About 2 hr
 Time cycle- 0 min.- start kymograph
2 min.- add ACh
3.5 min.- stop kymograph & wash
10 min.- wash
15 min.- start kymoghraph9 April 2015 50Dr.M. B. Bargade

 About 3 weeks old chick is used
 Contains both slow & twitch fibres
 Twitch responses are simillar to obtained with
rat-diaphragm
 Contracture of slow fibres simillar to response
of frog-rectus

•Chick is euthanized with Phenobarbitone or Chloroform
•Clear the feathers
•Midline incision on back from skull to base of neck
•Biventer cervicis muscle is identified on either side of midline of neck
just below the surface
•Thread is attached to upper end & muscle is cut from bottom, and
then from other end

• Transfer the preparation to a dish containing Krebs’ solution
• Lower end tied with thread & upper end attached to fix pin at
the bottom of electrode holder
• Transfer the preparation to the organ bath of 30-40 mm dia.,
vol- 40 ml. at 37* celcius
• Contractions recorded with a light sprung lever
• Nerve stimulated at a rate of 12 shocks/min. by rectangular-
wave pulse of 0.5 msec.

 Effect of following drugs on preparations-
1] Ach 10-2 M
2]Decamethonium
10-4 M or
suxamethonium 10-4
M
3] Hexamethonium
10-2 M
4] Tubocurarine 2*10-3 M or
Gallamine triethiodide 10-3
M
5] Tubocurarine or Gallamine
(as in 4) f/b 3 min after without
wash, 0.2 ml Physostigmine 10-4
M or neostigmine 10-4 M

6] Decamethonium 10-4 M
or suxamethonium 10-4 M,
f/b 3 min after without
wash, 0.2 ml
Physostigmine 10-4 M or
neostigmine 10-4 M
7] 0.2 ml Physostigmine
10-4 M or neostigmine 10-4
M f/b 1 min after without
wash O.2 ml Ach 10-2 M
8] 0.2 ml Lignocaine 3*10-2
M

 Convinient to arrange the experiments in pairs-
 One preparation for Decamethonium,
tubocurarine chloride, Physostigmine
 Other for Suxamethonium, Gallamine
triethiodide, Neostigmine

 Krebs’ solution- 1 lit. for each drug
 Other drugs as mentioned in expt.
 Time required- in pair- 2 hr, without pair- 3 hr
 Time cycle-
1 min.- Add drug
10 min.- start kymoghraph

 “In the era of sophisticated physical and
chemical assay techniques, isolated
preparations of skeletal muscles are still the
important available methods to assay various
agents acting at neuromuscular junctions.”

 Dept. of pharmacology University of Edinburgh.
Pharmacological expt. on isolated preparations:
skeletal muscle preparations. 2nd ed. Churchill
Livingstone, Edinburgh, Great Britain. 1976. pp.
30-57.
 Ghosh MN. Fundamentals of experimental
pharmacology: study on isolated muscle
preparations. 3rd ed. Bose printing house, Kolkata,
India. 2005. pp. 106-9.
 Medhi B, Prakash A. practical manual of
experimental & clinical pharmacology:commonly
used expt. Animals, Identification & collection of
tissue/muscle. 1st ed. Jaypee brothers medical
publishers (p) ltd. 2010. pp. 6-15, 140-9.

 Satoskar RS, Rege NN, Bhandarkar SD.
Pharmacology and pharmacotherapeutics: drug
invention; new drug development; and drug assay.
23th ed. Popular prakashan pvt. Ltd., Mumbai.
2013. pp. 77-80.
 Vogel HG. Drug discovery & evaluation:
Pharmacological assay; Effect on peripheral nerve
function. 3rd ed. Springer, Berlin, Germany. 2008.
pp.973-81.
 Sheth UK, Dadkar NK, Kamat UG. Selected topics
in experimental pharmacology; Autonomic
pharmacology. 1st ed. The Kothari book depot,
Mumbai, India. 1972. pp. 40-77.

 Sharma HL, Sharma KK. Principles of
Pharmacology: Bioassay of drugs. 2nd ed. Paras
publications, Hyderabad. 2011. pp. 920-6.
 Rang HP, Dale MM, Ritter JM, Flower RJ,
Henderson G. Rang and Dale’s Pharmacology:
Method & measurment in pharmacology. 7th ed.
Elsevier Churchill Livingstone, Spain. 2012. pp.89-
91.
 Satoskar RS, Rege NN, Bhandarkar SD.
Pharmacology and pharmacotherapeutics: drug
invention; new drug development; and drug assay.
23th ed. Popular prakashan pvt. Ltd., Mumbai.
2013. pp. 77-80.

Experiments on isolated skeletal muscles mukunda

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