To study the effects of Ach, Physostigmine and adrenaline on rabbit intestine

Principle

Rabbit intestine is a smooth muscle which shows regular pendular movement (i.e. continuous contraction and relaxation). Therefore, to study the effect of drugs on intestinal movement rabbit intestine is ideal preparation. Rabbit intestine is supplied by autonomic nervous system. Rabbit intestine contains muscarinic receptors and adrenergic receptors. Muscarinic receptor agonist like Ach produces contraction of rabbit intestine and physostigmine increases the spasm and pendular movements. The muscarinic actions and effects can be blocked by muscarinic blockers.

Adrenaline acts on α and β receptors and exhibit inhibitory effect on pendular movements. The actions of adrenaline are blocked by adrenoceptor blockers viz. α-receptor blockers and β-receptor blockers.

Equipment required

1) Kymograph

2) Student thermostatic organ bath

3) Aeration tube

4) Aerator

5) Aeration tube holder

6) Frontal writing lever

7) Lever holder

8) Screw clip

9) Haemostatic forceps

10) Mariotte bottle

11) Rubber tubes

12) Tuberculine syringe

13) 26 no. needle

14) Scissor

15) Forceps

16) Suturing needle

17) Carbogen gas cylinder with regulator.

Animal Required

Rabbit intestine.

Physiological solution required

Tyrode solution.

Drug solution required

1) Acetylcholine 20µg/ml

2) Physostigmine sulphate 10µg/ml

3) Atropine sulphate 4µg/ml

4) Adrenaline hydrogen tartrate 20µg/ml

5) Propranolol hydrochloride 100µg/ml

Procedure

1) Fast a rabbit for 12 hour period.

2) Set up assembly for rabbit intestine experiment.

3) Balance frontal writing lever using plasticine and apply a tension of 500 mg.

4) Fill the outer jacket of student organ bath with water.

5) Set the thermostat of the student organ bath at 370C and put the switch on.

6) Fill the mariotte bottle with tyrode solution and control the flow of tyrode solution to inner organ bath using haemostatic forceps.

7) When the temperature of the tyrode solution reaches 370C, sacrifice a rabbit by giving a blow on its head and cutting the carotid artery.

8) Open the abdominal region and identify the intestine.

9) Remove the intestine and place it in a petri dish containing tyrode solution at 370C and trim away the mesentry.

10) Cut a piece of intestine (approximately 3-4 cm) and using surgical suturing needle tie a thread at each end, taking care to see that the lumen of the intestine remained open.

11) Tie one end of the thread to the hook of the aeration tube and the other a frontal writing lever.

12) Stabilize the tissue for the 30 minutes period.

13) Aerate the rabbit intestine in the inner organ bath with a mixture of oxygen (95%) and CO2 (5%) (carbogen).

14) Required the normal pendular movement of the rabbit intestine on the drum for 30 seconds. At the end of 30 seconds add 0.1ml of Ach into the inner organ bath and record the spasmogenic effect of the drug for 30 seconds. After recording the response for 30 seconds switch off the kymograph. Immediately open the outlet of the inner organ bath and remove the tyrode solution present in the inner organ bath. Again fill the inner organ bath with fresh tyrode solution and keep it for 60 seconds. Repeat the washing procedure 3-4 times or till the writing point of frontal lever comes to the normal pendular movement base line.

15) Then record the response of 0.1ml physostigmine solution. Give 3-4 washing for recovery.

16) Record the response of 0.1 ml adrenaline solution. Give 3-4 washing for recovery.

17) Add 0.1 ml of atropine sulfate solution into the inner organ bath, allow it to act for 60 seconds then record the response of Ach in the presence of atropine sufate. Give 3-4 washing for recovery.

18) Add 0.1 ml of atropine sulfate solution into the inner organ bath, allow it to act for 60 seconds then record the response of physostigmine in the presence of atropine sufate. Give 3-4 washing for recovery.

19) Add 0.1 ml of propranolol solution into the inner organ bath, allow it to act for 60 seconds then record the response of adrenaline solution in the presence of atropine propranolol. Give 3-4 washing for recovery.

Observation and conclusion

Acetylcholine produces contraction in rabbit intestine. Atropine sulfate is a muscarinic receptor blocker; therefore it reduces the spasmogenic effect caused by Ach.

Physostigmine also produces mild spasmogenic effect and increases the peristaltic movement of the intestine. Atropine sulphate blocks the effect of the physostogmine.

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To study the local anaesthetic activity of lignocaine hydrochloride on rabbit cornea (surface anaesthesia method)

Principle

Local anaesthesia is the condition that results when sensory input from a local area to the CNS is blocked. Local anaesthetics are drug which reversibly block conduction of impuses along the nerve axons and excitable membrane.

Equipment required

1) Stopwatch

2) Rabbit cages to keep rabbits in place

3) Dropper

4) Camel hair

Animal required

Albino rabbits.

Albino Rabbit 

Drug solution required

Lignocaine hydrochloride (0.1%, 0.2%, 0.4%, 0.5%, 1% and 2%)

Procedure

1) Select 6 healthy albino rabbits weighing about 1.5 to 2 kg body weight.

2) Trim their eye lashes one day before the experiment.

3) Keep each rabbit separately in a rabbit cage and number the cage.

4) Instill the lowest concentration of the lignocaine hydrochloride solution on the corneal surface of the rabbit, so that the space between eyelids contained a clearly visible film of the solution. Note the time of administration of drug solution.

5) Stimulate the cornea by touching it with a camel hair a minute after instillation.

6) Stimulate the cornea at 60 seconds interval for a period of 30 minutes.

7) Count the number of times animal fails to respond to a corneal stimuli during 30 minutes period.

8) Find out the onset of action, duration of action and degree of anaesthesia.

Observation and conclusion

Local application of lignocaine hydrochloride solution produces loss of sensation at the site of application. The above observation indicates that lignocaine hydrochloride is a surface anesthetic agent.

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To study the effect of mydriatics and miotics on rabbit eye

Principle

Antimuscarinic cause reversible paralysis of constrictor pupillae (circular muscle of iris) and ciliary muscle which leads to mydriasis cycloplegia and increase intraocular pressure. α1 receptor agonists cause contraction of dilator pupllae (radial muscle of iris) and produced mydriasis. α1 receptor agonists do not increase intraocular pressure.

Muscarinic agonists cause contraction of constrictor papillae (the circular muscle of iris) and the ciliary muscle and reduction of intraocular pressure.

Anticholinestrase agents inhibit cholinesterase enzyme and increase the concentration of Ach and thus exhibit muscarinic activity on the eye.

Equipment required

1) Schioetz-Tonometer

2) Pentorch

3) Dropper

Animal required

Rabbits.

Drug solution required

1) Mydriatics (antimuscarinics) Atropine sulphate 1%w/v

2) Mydriatics (α1 receptor agonist) Adrenaline hydrochloride

3) Miotics (Muscarinic receptor agonist) Pilocarpine nitrate 1% w/v

4) Miotic (antichoine esterase agent) Physostigmine sulphate 0.25% w/v

5) Local anaesthetic required for measuring intraocular pressure Lignocaine hydrochloride 4% w/v.

Procedure

1) Select 6 healthy albino rabbits weighing between 1.5-2 kg body weight and do the marking for the identification of each rabbit.

2) Observe the size of the pupil of each rabbit using a petorch.

3) Instill the eye drops of the following drugs as given below:

Name of the drug	Dose	Rabbit no.
Atropine sulphate	2-3 drops	1
Adrenaline hydrochloride	2-3 drops	2
Pilocarpine nitrate	2-3 drops	3
Physostigmine suphate	2-3 drops	4
Normal saline	2-3 drops	5
Lignocaine hydrochloride	2-3 drops	6
After 5 minutes Pilocarpine nitrate	2-3 drops	6

4) Observe the size of the pupil of each rabbit at 30 minutes interval for a period of 2 Hrs (Rabbit No. 1-4)/

5) Adjust the schioetz-tonometer pointer on zero position.

6) Place the tonometer in a vertical position on the centre of the cornea (Rabbit no. 5-6).

7) Read the position of the pointer and calculate the tension in mm Hg (intraocular pressure) by the table.

Observation and conclusion

Atropine sulphate produces dilation of the pupil due to antimuscarinic activity. Adrenaline hydrochloride also produces dilation of the pupil because of α1 receptor stimulation.

Pilocarpine nitrate produces miosis of the pupil and reduces intraocular pressure due to muscarinic receptor activation.

Physostigmine suphate produces effect similar to pilocarpine.

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To study the effects of acetylcholine on rectus abdominis muscle of frog

Principle

Frog rectus abdominis muscle is voluntary muscle. At the neuromuscular junction, a nerve impulse liberates Ach from the nerve ending into the cleft between the muscle and nerve fiber. This Ach causes depolarization of the muscle fiber which in turn sets off a muscle action potential, and contraction of the muscle fiber. The muscle fiber of lower species like frog are multiply innervated and hence nerve stimulation causes persistent depolarization and a prolonged slow contraction of muscle. Local administration of Ach also produces similar effect. Frog rectus abdominis muscle contains nicotinic (N2) receptor. Ach acts as agonist.

Equipment required

1) Student kymograph

2) Student organ bath

3) Aeration tube

4) Aerator

5) Aeration tube holder

6) Frontal writing lever

7) Lever holder

8) Screw clip

9) Haemostatic forceps

10) Mariotte bottle

11) Rubber tubes

12) Tuberculine syringe

13) 26 no. needle

14) Scissor

15) Forceps.

Animal required

Frog.

Physiological solution required

Frog ringer solution.

Drug solution required

Ach 100µg/ml.

Procedure

1) Setup the assembly for the above experiment.

2) Pith the frog by passing a needle through occipito-atlantic junction between the brain and spinal cord. The stretching out of the limbs indicates that the pithing is proper.

3) Place the frog in a tray with the ventral side facing up.

4) Pick up the skin of the abdomen with the help of forceps and make proper incision to expose the abdomen.

5) Cut along the margin of the rectus abdominis muscle and then make a transverse cut through the sternum just above the base.

6) Free the rectus abdominis muscle from the interior abdominal vein.

7) Lift the muscle gently and divide the muscle longitudinally.

8) Tie a long thread on the upper side of the rectos muscle and a short thread on the lower side of the rectus abdominis muscle.

9) Transfer the muscle to a petri-dish containing frog ringer solution.

10) Tie the short thread to the hook of the aeration tube and place the rectus muscle in the inner organ bath containing frog ringer solution.

11) Tie the long thread to the frontal writing lever. The load on the lever should be 1 gm. The magnification should be between 5-7 times.

12) Stabilize the rectus muscle for 55 minutes period.

13) During the stabilization period replace the frog ringer solution in the inner organ bath at an interval of 5 minutes.

14) After stabilizing for a period of 55 minutes, switch on the kymograph and record the normal tracing for a period of 30 seconds. At the ends of 30 seconds period inject 0.1 ml of Ach solution to the inner organ bath and record the tracings for a period of 90 seconds (drug contact time). At the end of 90 seconds switch off the kymograph and give 3-4 washings of rectus muscle with frog ringer solution.

15) Inject 0.1 ml of Ach solution into the inner organ bath once again and record the response for 90 seconds.

16) If two equipotent responses are observed with similar doses of Ach, then record the responses of Ach with higher doses (0.2, 0.4, 0.8 and 1 ml).

17) After fixing the graph measure the height of contraction of the response produced by each dose of Ach and find out the dose which produces maximal response.

18) Plot a graph showing dose of Ach on X-axis and response on Y-axis.

19) Plot another graph showing log dose of Ach on X-axis and response on Y-axis.

20) Plot a third graph showing log molar concentration of Ach on X-axis and percentage on Y-axis

21) From the graph find out the EC50.

22) From the EC50 value finds out the pD2 value.

Observation and conclusions

1) Acetylcholine produces a dose-dependent contraction effect on frog rectus abdominis muscle.

2) A graph plot with a linear contraction scale shows non-sigmoid dose-response curve.

3) A graph plot with a logarithmic concentration scale shows a sigmoid dose-response curve.

4) pD2 value is useful in determination of affinity of a drug for a particular receptor.

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To study the effects of K+, Ca2+, acetylcholine and adrenaline on frog’s heart

Heart is supplied by autonomic nervous system. Adrenaline acts as agonist. It acts on β-receptors and increases heart rate and amplitude. Acetylcholine acts on muscarinic receptors as an agonist and decreases the heart rate and amplitude. Excess concentration of KCl stops the heart beat during diastolic phase. Ca2+ excess concentration stops the heart beat during systolic phase. K+ and Ca2+act on cardiac muscle through non-receptor mechanism of action.

Requirements

Equipment and other items required.

Kymograph, strling heart lever, L-stand T-rod, X-blocks, Syme’s cannula, screw clip, mariotte bottle, rubber tubes, tuberculine syringe, 26 no. needle, surgical instrument box.

Animal required

Frog.

Physiological salt solutions required

Frog ringer solution.

Drug solution required

1. Adrenaline hydrochloride 10µg/ml in distill water.
2. Acetylcholine hydrochloride 10µg/ml in distill water.
3. Potassium chloride 4% in distill water.
4. Calcium chloride 4% in distill water.

Procedure

1. Set up the assembly for above mentioned experiment.
2. Pith the frog by passing the needle through the occipito-atlantic junction between the brain and spinal cord. The stretching out of limbs indicates that the pithing is proper.
3. Place the frog in a tray with ventral side facing up.
4. Make an incision on the skin longitudinally and the expose the rectus abdominal muscle.
5. Make an incision around the rectus muscle without damaging the anterior abdominal vein.
6. Expose the heart after cutting the sternum.
7. Remove the pericardial membrane.
8. Tie one side of the aorta.
9. Put a not around the inferior vena cava then make a small cut for cannulation.
10. After cannulation with Syme’s cannula, cut the other side of aorta and isolate the heart from the body and perfuse with frog ringer solution. Adjust the flow of the frog ringer solution through the horizontal arm of the syme’s cannula.
11. Place a heart-clip on the apex of the heart and connect it to a staling lever.
12. Record the normal heart beat on the paper of the drum.
13. Inject 0.05-0.1ml of adrenaline solution into Syme’s cannula. Immediately switch on the kymograph and record the effect of adrenaline for 2 minutes period. After 2 minutes switch off the kymograph till the heat beat and amplitude comes to the normal. Observe the onset and duration of action of adrenaline.
14. Inject 0.05-0.1ml of acetylcholine solution into Syme’s cannula. Immediately switch on the kymograph and record the effect of acetylcholine for 2 minutes period. After 2 minutes switch off the kymograph till the heat beat and amplitude comes to the normal. Observe the onset and duration of action of acetylcholine.
15. Inject the 0.1 ml KCl solution into Syme’s cannula. Immediately switch on the kymograph and record the effect of KCl for 2 minutes period. After 2 minutes switch off the kymograph till the heat beat and amplitude comes to the normal. Observe the onset and duration of action of KCl.
16. Inject the 0.1 to 0.4 ml CaCl2 solution into Syme’s cannula. Immediately switch on the kymograph and record the effect of CaCl2 for 3 minutes period. After 3 minutes switch off the kymograph till the heat beat and amplitude comes to the normal. Observe the onset and duration of action of CaCl2.

Procedure

Observation and conclusion

1. Adrenaline  increases  the  heart  rate  and  amplitude.  Heart  contains  β-receptors.
2. Adrenaline stimulates β-receptors and increases the heart rate and amplitude. Based upon this experimental observation, adrenaline 1 in 10,000 solution is recommended in a dose of 10 ml by i.v. infusion for cardiac arrest. Drugs which block β-receptors (propranolol, atenolol etc) are clinically used in the hypertension and tachycardia.
3. Acetylcholine decreases the heart rate and amplitude. This effect is similar to the effect produced by vagus nerve stimulation. This effect is mediated through muscarinic receptors. Hence, muscarinic blockers (atropine, belladonna extract) are used to reduce vagal tone and muscarinic actions.
4. Potassium excess concentration decreases the heart rate and amplitude.
5. Calcium concentration in excess stop the heart beat during systolic phase, an effect similar to digoxin poisoning. Therefore, EDTA (ethylene diamine tetra-aceticacid) is used in digitalis poisoning. Calcium channel blockers like verapamil, diltiazem, amlodipine etc. are used as antihypertensive agents.

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To Study the conventional equipments used in experimental pharmacology

Organ Bath 

Organ bath is an assembly for recording either contraction or relaxation of isolated tissues. The design of organ bath varies from laboratory to laboratory. It may be a single unit as designed by Rudolph Magnus or double unit as designed by Sir Gaddum. 

Organ Bath

The various parts of organ bath are as follow – 

      A rectangular outer bath made up of Perspex or Glass. A thermostate and heating element are also fixed with the outer bath. An electrically operated stirrer is also fixed in the outer organ bath of the newly designed single unit organ bath.

      An inner organ bath made of glass with varying capacity (10, 50, 100 ml) for suspension of tissue in physiological salt solution. The inner organ bath has inlet and outlet for the physiological solution. The inlet side tube is connected to a glass coil. The outer end of the coil is connected to the mariotte bottle through polythene tube.

      A small electrical stirrer which keeps the water in the bath circulating for uniform heating. 

      Two small perpendicular rods attached both at left side and right side of the outer organ bath are used for fixing the frontal writing lever and aeration tube respectively.

      The hook of the aeration tube also act as a tissue holder. 

Mariotte Bottle

Mariotte bottle is used to keep the physiological solution used in the experiment.

Mariotte bottle

Levers

The levers mostly used in isolated tissue work are made up of light aluminum or stainless steel. Types of levers used in the experimental pharmacology –

Lever

Simple lever

It is pivoted between hardened centers mounted on a stout brass frame the screw located on the axel midway between the centers permits the lever to be adjusted for length as required. A light celluloid point is fitted at one end.

Frontal writing lever 

The lever is made up of an aluminum trip fixed to a wheel which rotated freely about its axel. A long frontal writing point made of glass capillary is attached to the end of strip by means of a hinge. By this lever the actual shortening of the muscle is magnified in linear proportion on the drum. 

Sterlings heart lever

It consists of a frame carrying a light lever arm with holes and notches supported by a fine adjustable nickel-silver spring attached to an adjustable hook.

Universal lever or Brodies lever

In this lever the axis is screwed with two nuts between which interchangeable levers are clamped.

Gimbal Lever

In this type the pressure of the stylus is effected by the force of gravity, thus giving careful adjustments necessary in case of ordinary levels. 

Sherington’s recording drum

It is constructed with kymograph drum which is detachable and the recording device body with shaft on which cylinder i.e. kymograph drum rotates.

The recording device shows gear arrangements with fast, slow, neutral gear and its catch.

Kymograph is an electrical device consists of gear box with provision for carrying drum. The speed of the drum can be controlled by clutch and gear.

Sheringtons-recording-drum

Smoking

The cylinder is passed over a rod which is fixed in a smoking rack, below it is placed a special fine shaped smoke flame. The drum is rolled uniformly at minimum speed of one revolution per second. This ensures a uniform deposit of soot on the glazed surface. Smoking is continued till an even brown or black surface is produced. 

Sooty flame

A sooty flame can be prepared by burning a wick in kerosene oil lamp with deficient air.  A mixer of benzene and kerosene is a ratio 1:9 also given a satisfactory sooty flame. 

Cutting

After the record is taken the paper is cut at proper place to get a continuous tracing. No record should be taken on the joint of paper.

Fixing or varnishing

This is done to make the record permanent. It is done by dipping the paper in solution of resin in methylated spirit.

150 gram of resin dissolved in 2 litres of spirit.

OR

Varnish diluted 10 times with alcohol can be used as fixing solution.

OR

A saturated solution of colophony in ethylalcohol or methylalcohol can be used as a fixing solution.

Result – we study the all parts of organ bath assembly. 

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