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KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY,NANDINAGAR CAMPUS, BIDAR-5845401
LABORATORY MANUAL
For:
VETERINARY NEURO PHARMACOLOGY[VPT 321]
DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGYVETERINARY COLLEGE (BIDAR), P.BNO.6; PIN: 585 401
DEPARTMENT OF VETERINARY PHARMACOLOGY AND TOXICOLOGY
1
VETERINARY COLLEGE, BIDAR - 585 401
Certificate
This is to certify that Mr/ Miss…………………………………………………….
bearing I.D.No……………………..,,,studying in IIIrd year B.V.Sc& A.H has
successfully completed the laboratory exercises in Veterinary Neuropharmacology,
VPT-321 (2+1) as per the prescribed norms of Veterinary Council Of India. 2008,
Regulations
Date: Course Teacher
Head of the Department External Examiner
CONTENTS
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Ex.No. Date Title of the Experiment Page No. Remarks
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Ex.No.1 Date: STUDY ON EQUIPMENTS REQUIRED FOR ISLOATED TISSUE EXPERMENTS
Isolated tissue experiments is a traditional experimental design that has been used extensively to investigate the physiology and pharmacology of invitro tissue preparation. This isolated tissue used for experiments have several advantages over intact animals as relatively small amount of the test material is required and several preparation can be tested from a single animal. Intestine (ilium), colon, tracheal rings, arterial rings, cervical strips, vas deferens,diaphragm, oesophagus, smooth and skeletal muscle and rectus abdominus muscle from different laboratory animals are the most common isolated tissues used for the experimental studies.
Organ bath: The tissue bath used to put the animal tissue for studying the drug actions in called student’s organ bath. The organ bath having two units of inner tissue bath is called double unit organ bath. This was first designed by Rudolph Magnus is 1904 and it essentially consists of following parts.
Water bath or outer jacket: It is made up of steel, glass or perspex (transparent thermoplastic resin). It holds water and other parts of organ bath.
Inner organ or tissue bath: It is cylindrical glass with variable capacity. It contains isolated tissue in physiological salt solution.
Heating rod or electrical heater with thermostatic control: It heats the water in outer jacket to a desired temperature
Stirrer: A small electrical stirrer keeps the water in outer jacket circulating at uniform temperature
Glass coil or condenser: A coil made of glass or perspex is connected at one end to the lower end of the organ bath and other to the container having the physiologic salt solution. It usually has double the capacity of inner organ bath to ensure warming of the solution before it enters the organ bath
Oxygen delivery tube: It is made up of a hollow glass tube. It delivers oxygen to the tissue and also serves as tissue holder. Lower end of the tissue is tied with a thread to its S-shaped curved end. The lower end has a small hole through which air or oxygen is supplied to ensure proper aeration of the tissue
Aerator: It is a small electrically operated device, which is connected to the oxygen delivery tube through rubber tubing. It dispenses oxygen ( or carbogen: 5%CO and 95%O) to the tissue in the inner tissue bath. The purpose of aeration is supply of oxygen, maintenance of pH and uniform distribution of drug due to bubbling.
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Writing /Recording levers: They are used to record the contraction or relaxation of the isolated tissue preparation. The recording is done on smoked paper fixed on electrically operated cylinders/recording drums. The writing lever is connected through a thread to upper part of the tissue suspended in the inner tissue bath.
The writing levers are made up of light aluminium, stainless steel, wood (straw) etc. They are usually light and fine but rigid so that while writing they do not bend thereby losing their sensitivity and efficiency.On the basis of location of fulcrum, writing levers are of two types:
i. Class I type: The fulcrum or pivot lies between the writing point and the point of attachment of the tissue.For example, frontal writing lever.
ii. Class II type: In this type, the fulcrum or pivot lies at one end beyond the point of attachment. For example, Starling’s heart lever.
Different types of lever:a) Simple lever (side-way writing): It is the simplest type of lever made up of wood,
stainless steel or aluminium fitted through an axle pivoted between hardened centres mounted on a stout brass frame. A thumbscrew permits the lever to be adjusted for length as required. A celluloid writing tip (stylus) is fitted at one end. The contractions are recorded side way as curved lines.
b) Frontal writing lever: The lever is designed in such a way that the writing point rotates freely about its axle. This helps in reducing the tension between the smoked paper and the recording tip. The contractions are recorded frontally as straight lines.
c) Starling’s heart lever: It consists of a frame carrying a light lever arm with holes and notches supported by a fine adjustable spring attached to an adjustable hook. In this, fulcrum lies at one end beyond the point of attachment. It is used to record the contractions of the heart.
d) Brodie’s universal lever: It is a general utility lever with axis screwed with two nuts between which the interchangeable levers are clamped.
e) 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 levers.
The other types of levers are fixit lever, isometric lever, pendulum auxotonic lever (Patson’s) etc.Sherrington Rotating Drum/Kymograph: The contractions and relaxations are recorded with the help of the writing lever on a smoked paper fixed around an electrically operated recording drum which can be adjusted to any desired speed depending upon the nature of the experiment. For most of the experiments, a speed of one revolution per minute is usually used. This instrument consists of an electrically driven gear box with a vertical spindle carrying a drum. The gear box has clutch and controls which determine the speed of the drum. The clutch must be disengaged before changing the gear, or before moving the drum manually. By convention, the drum rotates in a clockwise direction, so all kymograph records should read from left to right.
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Student’s Physiograph: Student’s physiograph is modern recording instrument that is popularly used in recent times instead of Kymograph. This is electrically operated and consists of writing pens, which record the contractions over moving paper. This instrument thus gives permanent record of the tracings. The adjustments available with the instrument for altering the sensitivity, baseline etc. increase the reliability of the results.
Transducer: The tissue is connected to the transducer on the upper side for proper transmission of signals. The transducer is connected to the physiograph. This helps in the conversion of mechanical signals to electrical signals and makes the pen write on the paper. This is very sensitive equipment and needs very careful handling. While handling care must be taken to ensure that the transducer is not dropped on to floor or ground and not hit against hard surface.
Exercise:
1. Draw neat diagrams of the various appliances used in experimental pharmacology 2. Draw the diagram of isolated tissue organ bath assembly
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Ex.No.2 Date: PHYSIOLOGICAL SALT SOLUTIONS
These are the solutions that immediately bathe the tissues in an isolated organ bath and provide nourishment and ideal conditions for maintenance of the tissue.
These must be carefully prepared with pure materials, e.g., ‘Analar’/ AR grade, and glass-distilled, or deionized water. It is important to select the particular salt solution in which the tissue survives longest. It is also important to aerate the solution with the appropriate gas mixture. For example, on continued aeration by pure oxygen of a solution containing a bicarbonate buffer, it loses carbon dioxide and becomes alkaline.
Physiological salt solutions can be kept for about 24 hours, but they can be good media for the growth of microorganisms and therefore must be chilled. If it is wished to prepare them more than 24 hours in advance of the time when they will be used, they can be stored in the refrigerator for longer periods, provided that the glucose and calcium salts are omitted and added only when the solution is required for use
Composition of some commonly used physiological salt solutions:
Ingredient Function/role Quantities for 10 litres of:Frog-Ringer
Krebs Tyrode Ringer-Locke
De Jalon
NaCl Maintains osmolarity,isotonicity and Contractility of tissue
65 g 69 g 80 g 90 g 90 g
KCl 10 Per cent
Maintains ionic balance
14 ml 35 ml 20 ml 42 ml 42 ml
MgSO4, 7H2O10 per cent
Stabilize the prepration to reduce spontaneous activity
- 29 ml 26 ml - -
NaH2PO4, 2H2O5 per cent
As buffering agent
1.3 ml - 13 ml - -
KH2PO4
10 per centAs buffering agent
- 16 ml - - -
Glucose Provides energy 20 g 20 g 10 g 10 g 5 gNaHCO3 4 g 21 g 10 g 5 g 5 gCaCl2
(Molar)Maintains Contractility of tissue
10.8 ml 25.2 ml 18 ml 10.8 ml
2.7 ml
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Aerating gas Oxygen supply Air O2 + 5% CO2 O2 or air Pure O2 O2+5%CO2
Note: For amphibian tissue use frog –Ringer solution; For mammalian or avian skeletal muscle use Kreb’s solution; For intestine use Tyrode’s solution; For heart muscle use Ringer-Locke solution; For the rat uterus preparation use De Jalon’s solution; Tyrode’s solution is sometimes aerated with O2 + 5% CO2
Exercise:
1. Write a note on different laboratory animals and their physiological parameters 2. Write about the different isolated tissues used for various tissue experiments. 3. Write about the steps/ procedures for preparation of isolated tissues for
experiments
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Ex.No.3 Date:EFFECT OF DRUGS ON GUINEA PIG ILIUM
Introduction:Guinea pig Ileum is a tissue commonly used for carrying out isolated tissue
studies. Acetylcholine, histamine and barium chloride produce contractile effect. These drugs are spasmogenics. atropine, mepyramine and papaverine do not produce any effect of their own but inhibit the responses to acetylcholine, histamine and barium chloride respectively.
The antagonism by atropine and mepyramine to acetylcholine and histamine respectively is specific while that produced by papaverine is nonspecific. It is possible that acetylcholine and histamine act through specific receptors-muscarinic and H2
receptors, respectively. Atropine and mepyramine specifically block these receptors. BaCl2 produces effect by direct action, not involving receptors. Papaverine inhibits this action and hence it is non-specific.
Aim: To study the effect of drugs on guinea pig ileum.
Drugs: Acetylcholine (10mg/ml), histamine (10mg/ml), barium chloride (10mg/ml) mepyramine (1 mg/ml), atropine (1 mg/ml), papaverine (100 mg/ml)
Requirements: Guinea pig, Tyrode’s solution, physiograph with a transducer.
Procedure:1. Set the organ bath assembly.2. Stun the guinea pig that has been fasted overnight by a sharp blow on the head.
Open the abdominal cavity quickly and isolate a piece of ileum and place it in Tyrode’s solution maintained at 37C.
3. Remove the mesentry of the ileum (muscles should be handled with fingers rather than forceps) and wash the interior content with Tyrode’s solution and mount the tissue in an organ bath which is connected to the transducer.
4. Allow the tissue to stabilize for 30 min and take the responses to drugs.5. Add ACh, atropine and ACh in the presence of atropine in the organ bath and
observe the contraction. 6. Add histamine in the presence and absence of mepyramine and observe the
contractile effect of histamine in the presence of mepyramine. 7. Add barium chloride in the presence and absence of papaverine and observe the
contractile effect of barium
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Ex.No.4 Date:DEMONSTRATION OF THE MUSCLE RELAXANT EFFECTS OF DRUGS
AIM Aim: To demonstrate the centrally acting muscle relaxant effect. Apparatus required : Rota rod apparatus, Syringe and needle Drugs / chemicals required : Diazepam Animal required : Mice. Objective: To study the effect of drugs on muscle grip (muscle tone)
PRINCIPLEThe muscle relaxant effect of drugs is studied in animals by using rotating rod and is indicated by loss of muscle grip. The difference in the fall of time from rotating rod is taken as index of muscle relaxation.
PROCEDURE The rota rod apparatus is switched on and the rod is allowed to rotate at a set
speed (20-25 rpm). The time is set to zero. Two mice are taken at a time, weighed, sex noted and are placed on the rotating rod. The time at which these animals are placed on the rod is recorded and the time at which the animal falls down (fall of time) is recorded. The difference in this is considered as the normal reaction time.
Now, each mouse is injected with injection Diazepam at a rate of 2mg/kg i.p and 30 minutes after the injection the animals are placed on the rod once again. The timings are noted down as before and the reaction time is calculated. The fall off time of animals before and after treatment with diazepam is compared.
INFERENCEDiazepam has muscle relaxant effect as indicated by decrease in the fall off time due to loss of muscle grip in treatment animals after its administration
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Ex.No.5 Date:
DEMONSTRATION OF EFFECT OF LOCAL ANESTHETICS
Local Anaesthesia: It is a state in which drugs cause a reversible loss of sensation in a localised area of the body.
The principle of the experiment is to find out how the local anaesthetic effects brings out its effect on administering to a specific area and its responses to a noxious stimuli thereby confirming its effect by temporary loss sensation in that particular area
Types of local anaesthesia
a) Surface anaesthesia: The anaesthesia is induced by application of anaesthetic to the skin or mucous membrane in the form of solution or ointment.
b) Infiltration anaesthesia: The anaesthesia induced by injecting the anaesthetic solution subcutanesously in to the area of operation to block the conduction of nerves supplying that area.
c) Plexus anaesthesia: The anaesthesia induced by deposition of anaesthetics at a convenient point along the course of the desired nerve
Aim: To demonstrate the effect of local anaesthetic in guinea pigs.
Requisites: Animals required: Guinea pig,; Drugs required: Xylocaine 0.5% solution, Instruments required: Dental syringe and needle.
Procedure:
A guinea pig of either sex can be taken and then shaved on its hind quarters one day prior to the experiment. On the day of experiment the animal is weighed and the sex noted. Then circular markings are made with visible ink on either side of the vertebral column on the shaved area to a diameter of 4cm.
In one of the circles marked the animal is pricked with the tip of a needle or pin six times. The animal will respond to this stimulus either by making a jerky movement or make a scream all the six times. Then 0.1 ml of the 0.5% xylocaine is injected intradermally which will evinced by formation of a swelling. Then remove the needle but do not rub that area after injection. Wait for 10 minutes and then start pricking with tip of the pin six times and observe the response as before and note the response. Like this repeat every 10 minutes till all the effect is lost. When the animal does not respond to any of the pin pricks at one stage is said to be positive for local anesthetic effect. Tabulate the results and give your interpretation. Repeat the experiment in more areas of the same animal and calculate the mean response
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Results :
Dose: Route: Date: Time:
Inference
ID of animals
Sex of the animal
Weight of the animal
Circle Numbers
Total number of pin pricks
Total number of response to pin pricks
Total number of no response to pin pricks
Percentage of response
1 (control)
2
3
4
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Ex.No.6 Date:
DEMONSTRATION OF ANALGESIC EFFECTS OF DRUGS MECHANICAL STIMULUS (USING TAIL CLIP METHOD)
Definition: Analgesics are agents which relieve pain by raising the pain threshold. They may be classified as narcotic and non-narcotic analgesics Eg: Narcotic analgesics- Morphine, Non-narcotic- Aspirin
Pain is induced by a mechanical stimulus method. - A artery clip sleeved with a rubber sleeve is used for application on the base of the tail of a mice to induce pain by mechanical stimulus means - On evincing the pain the animal tries to dislodge the clip which is noted as the reaction timeAim: To demonstrate the analgesic effect of drugs by applying mechanical (physical) stimulusRequisites: Animals: Mice; Drugs required: Injection of morphine sulfate / pentazocine; Instruments : Artery clip with rubber sleevesMechanical Stimulus
Artery clip with rubber sleeves is applied to the base of the tail of a mouse. Due to the pressure of the clip the pain will be induced and the animal will make effort to dislodge the clip. The time taken by the animal to dislodge the clip from the time of application is taken as the end point.
Six mice are used for this study. The animals are weighed and sex is recorded. To these mice the clip is applied to the base of the tail. The time of application of clip is recorded. The time at which the animal tries to dislodge the clip is recorded.
The difference between the two serves as the normal reaction time. To these animals injection morphine / diclofenac / pentazocine is administered at the dose rate of 10mg per kg body weight i.p.
Thirty minutes later the same procedure is repeated. If the animal does not respond and try to dislodge the tail clip even after 30 seconds it is considered positive for analgesic effect.
The timings are recorded and the reaction time is calculated thereafter. If the reaction time is three times the normal reaction time is considered positive
for the analgesic effect and the clip is removed (for example if the normal reaction time is 3 seconds and the reaction time after administration of the drug is 9 seconds it is considered positive
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Results:
Date: Dose: Route: Time: Inference
Animal Number
Sex of the animal
Weight of the animal
Time of application of clip
Time at which animal dislodge the clip
Normal Reaction time
Reaction time
30”
60”
90”
120”
180”
1
2
3
4
5
6
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Ex.No.7 Date:
DEMONSTRATION OF ANALGESIC EFFECTS OF DRUGS DRUGS USING ANALGESIOMETER
Radiant heat is used as a thermal stimulus method. Analgesiometer works based on the principle of application of stimulus which is produced by a nichrome wire.
Analgesiometer The nichrome wire is heated to a temperature of 55 degree celcius which can be
adjusted by milliampere gauge on the instrument which is usually adjusted up to 2.5 mA on the instrument. In order to maintain this temperature the nichrome wire is surrounded by a double jacketed inlet and outlet through free water flow in and out continuously.
The rats are the animal of choice for this experiment. Six rats are weighed and sex of the animal is recorded and markings are also made for easy identification.
The rat is restrained in a rat holder made of stainless steel which is closed at both ends.
At one end it is provided with holes on the lid through with free air flows in for the rat to respire.
At the other end of the lid a slit is provided on the lid for the tail to protrude out. Once everything has been set the base of the tail is placed on the nichrome wire
and the time at which it is placed is noted. The end point of the reaction time is the flick of the tail. Hence, the time at
which the tail is flicked from the nichrome wire is noted. The difference in the timing gives the reaction time.
The animal is then injected with the drug at a dose rate of body weight i.p. After 30 minutes the same procedure is followed and the following timings are
recorded and the reaction time is calculated. An increase of 7 sec in reaction times after administration of drug is indicated for
its analgesic effect. Usually it is not advisable to keep the tail of the rat on the nichrome wire for
more than 15 seconds (cut off time) to avoid injury to tail.
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RESULTS:
Date: Dose: Route: Time:
Animal Number(a)
Sex of the animal(b)
Weight of the animal(c )
Time of application tail on the nichrome wire
(d)
Time at which animal flicks the tail
(e)
Normal Reaction time in seconds
Reaction time in seconds(Difference in time between e and d)
30” 60” 90” 120” 180”
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Ex.No.8 Date:
DEMONSTRATION OF ANALGESIC EFFECT OF DRUGS USING THERMAL STIMULUS (USING MAGNETIC EDDY'S HOT PLATE)
Magnetic Eddy’s Hot plate: - This is a direct thermal stimulus method. - In this method the hot plate is heated upto a temperature of 55 + 5 degree celcius. - The hot plate is covered with a persplex cover with a lid so as to avoid animal jumping from the hot plate once it evinces pain. - Once the instrument is set to the required temperature and switched on.Principle:- The basic principle involved in methods is to elicit reaction of animals to a standardized painful stimuli before and after administration of the test drug. - A significant increase in the reaction time after administration of drug indicates its analgesic effect in animals.
Procedure:
Mice are used as the animal for experiment, weighed and sex noted and then placed on the hot plate.
The end point of the reaction time is either the animal try to jump out from the hot plate or lick the paws.
The time at which the animal is placed on the hot plate is recorded and the time at which the animal tries to lick the paws or try to jump out is noted for the calculation of the reaction time.
The animals are then injected with the analgesic drug and the same procedure is followed and the time is recorded and the reaction time is calculated.
If the reaction time is increased by atleast 7 seconds from the normal reaction time it is considered positive for the analgesic effect
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Results:
Date: Dose: Route: Time:
Animal Number( a)
Sex of the animal(b
Weight of the animal(c )
Time of application introduced into the hot plate
(d)
Time at which animal tries to jump or licks the fore paw
(e)
Normal Reaction time in seconds
Reaction time in seconds(Difference in time between e
and d
30
60
90
120
150
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Ex.No.9 Date:
DEMONSTRATION OF EFFECT OF ANTICONVULSANTS
Anticonvulsants are the drugs used to control the convulsions occurring in epilepsy, tetanus, cerebral hemorrhage, eclampsia and in poisoning with convulsants. The greatest use is in epileptic disorders characterized as petitmal and grandmal and psychomotor seizures.
Epilepsy: According to neurological theory epilepsy is a paraoxysmal, self-limited, cerebral dysrhythmia. It is accompanied by abnormal patterns on the electroencephalograph, and severe seizures may cause loss of consciousness.
Most of the experimental methods for detection anticonvulsant activity involve the artificial induction of convulsions and their inhibition with organic compounds. These tests have another principal use: to detect myorelaxant activity.
CONVULSIONS INDUCED BY CHEMICALS
Pentylenetetrazol:
A drug used as a stimulant of the central nervous system is pentylenetetrazol.
The test substance (anticonvulsant drug) is given in adult rats.
Forty five minutes later an intramuscular injection of pentylenetetrazol is given at a dose of 112 mg/kg.
The convulsion begins with jerks of the head and body, consisting of clonic contractions.
The seizures end fatally or are followed by depression and other seizures before death or recovery finally takes place.
The criterion of anticonvulsant activity is complete protection against convulsions of any kind. Tremors are disregarded. Observations are made for at least 60 minutes after the administration of pentylenetetrazol
Strychnine:
Intraperitoneal injection of 2 mg/kg of strychnine sulfate in mice produces stimulant action and the animal is thrown into convulsion.
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Test substance (anticonvulsant) are introduced at a time interval before the strychnine so that they are at peak activity.
The end-point is the measure of the substance’s ability to prolong life beyond the usual time of death. It would be better to use enough test substance to save the animal’s life and thus demonstrate a more pronounced antagonism to strychnine.
Calculate the percentage of protection given by the test substanace ( anticonvulsant)
Results: Date: Route:
S.No Sex of the animal
Weight of the animal
Dose of the test substance
Time of administration of test substance
Status of animal survival / death
% of protection
1
2
3
4
5
6
% of protection = - (Experimental/Control) X 100
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CONVULSION INDUCED BY ELECTROSHOCK
The animals of choice for this experiment are adult male rats or mice.
At some interval before electroshock a test substance (anticonvulsant) dissolved in a suitable vehicle is given by the subcutaneous or the oral route or intraperitoneal route.
The maximal electroshock test is a measure of the ability of an anticonvulsant drug to abolish the tonic, extensor component of the hind limb in the maximal seizure pattern induced by 50 mA of current (mice) or by 150 mA (rats) delivered for 0.2 second.
Substances are compared in groups of 5 to 6 animals.
Phenobarbital (10 to 30 mg/kg) may be employed to demonstrate anticonvulsant activity.
Results:
Date: Route:
S.No Sex of the animal
Weight of the animal
Dose of the test substance
Time of administration of test substance
Maximal electroshock administered in mA
Status of animal survival / death
% of protection
1
2
3
4
5
Inference
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Ex.No.10 Date:
DEMONSTRATION OF CENTRAL NERVOUS SYSTEM DEPRESSION USING TRANQUILIZERS OR ATARATIC DRUGS
Principle:
The substances depressing the CNS have a variety of manifestations. Since the mildest of these is a tranquillization, the tests for CNS depressants and for ataractic agents overlap. Even those ataractic agents that cause no clear loss of alertness potentiate soporific activity of CNS depressants such as barbiturates.
The following tests are among the most commonly used: the righting reflex, motor activity.
RIGHTING REFLEX
Groups of 10 female mice are injected subcutaneously with a test substance.
After 15,30,60,120,180,240 and 300 minutes each animal is placed gently on its back and an undulated surface and kept up to 30 seconds.
If the animal remains on the back for 30 seconds, loss of righting reflex is said to occur.
MOTOR ACTIVITY
A photocell (Actophotometer) counter is used .Mouse is housed in individual cages for 1 or 2 hours prior to intraperitoneal
dosing. Fifteen minutes, later the mice are placed in groups in counting boxes. A group consists of 5 mice which receive equal doses. Interruption of a light beam passing through the box activates a photocell relay
and a summation counter Counting is continued for 90 minutes. The count for a group is recorded as a percent of the value for a control group Mice are given CNS stimulants / depressaants like picrotoxin at a dose rate of 10
mg/kg and Phenobarbital at a dose rate of 40 mg/kg. In case of mouse given stimulant the count will be more and with the
depressants it will be less than the normal animal
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ROTAROD TEST
Mice are placed on the horizontal rotating wooden rod / iron ros with serrations having a diameter 30 mm and rotating at the rate of 5 rotations per minute.
Circular sections divide the linear space of the rod into 10 lengths, so that 10 mice are tested together.
The control mouse is able to remain on the rod for 3 or more minutes in two successive trials are selected and placed in groups of five.
Each group is injected with a drug and placed on the rod at intervals of 30, 60, 90,120 and 150 minutes after dosage.
If the animals fails more than once to remain on the rod for 3 minutes, the test is considered positive, which is motor incordination is present.
TRACTION TEST
Mice are suspended by means of their forepaws to a metallic wire, stretched horizontally.
The normal mouse effects a better posture in less than 5 seconds. The number of animals that are incapable of touching the wire with at least one of the hind paws.
Thus the animals being inhibited from touching the wire with a hind paw is within 5 seconds after the placement on the wire is taken as positive and 30 minutes after the injection of a drug.
23
Ex.No.11 Date:
DEMONSTRATION OF EFFECT OF INHALANT ANESTHETICS
Inhalant anaesthetics induce general anesthesia by CNS depression. Their potency and efficacy are compared by noting various parameters,onset and duration of general anaesthesia.
Animal : Rat
Apparatus required: Glass jar/glass funnel, cotton
Drug: Anaeshtetic ether
Procedure:
The rats are weighed, numbered and the sex of the animal noted. Respiratory rate and heart rate are recorded.
A small piece of cotton is soaked in anaesthetic ether and put inside the jar/funnel. After ensuring that the jar is filled with anaesthetic vapour, one of the rats is dropped into the jar/funnel and the time is noted (A).
The animal is observed and its respiratory and the heart rate are recorded. When the animal goes into its lateral recumbency it is removed from the jar /funnel. The time the animal goes to its lateral recumbency is recorded as (B).
After placing the animal on the table the corneal and pedal reflexes are checked at regular intervals. The time when the animal loses its pedal reflex is recorded as (C). The heart rate and respiratory rate are also recorded during the period.
The pedal reflex is gained thereafter and the time at which the animal regains its pedal reflex is recorded as (D).
The animal is observed for regaining of sternal recumbency and the time when the animal regains its sternal recmbency is recorded as (E).
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INFERENCE
Calculations:
1. Induction time: Difference in time in seconds between the time the animal is placed in the anaesthetic chamber to the time it loses its pedal reflex (C-A).
2. Anaesthetic duration:Difference in time in seconds from the time animal loses its pedal reflex to the time it regains its pedal reflex (D-C).
3. Sleeping time:Difference in the time when the animal regains its sternal recumbency to the time at which it went to its lateral recumbency (E-B).
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Ex.No.12 Date:
DEMONSTRATION OF EFFECT OF INTRAVENOUS GENERAL ANESTHETICS
Intravenous anaesthetics induce general anesthesia by CNS depression. Their potency and efficacy are compared by noting various parameters and onset and duration of general anesthesia.
Principle: Intravenous anaesthetics induce general anesthesia by CNS depression. Their potency and efficacy are compared by noting various references and onset and duration of general anesthesia. Various stages of anaesthesia produced on administration of anaesthetics can be observed by this experimentThe stages of anaesthesia are divided into distinctly, each correlating with a particular set of physiological responses or reflexes.The organisational scheme includes four stages of anaesthesia.Stage I This stage is also known as the stage of analgesia or stage of voluntary excitement or stage of analgesia and amnesia.This period begins from the beginning of induction to the loss of consciousness.Stage II This stage is also known as the stage of delirium or stage of involuntary excitement or stage of uninhibited action.This period begins from the loss of consciousness to onset of automatic respiration. Stage III This stage is also known as the stage of surgical anaesthesia. It is divided into four planes as Plane 1, Plane 2, Plane 3 and Plane 4.This period begins from the onset of automatic respiration to respiratory arrest.Plane 1 – Light surgical anaesthesia – rolling eye balls and plane ends with eyes becoming fixed.Plane 2 – Moderate surgical anaesthesia – loss of corneal and laryngeal reflex.Plane 3 – Deep surgical anaesthesia – Pupil starts dilating and light reflex is lost.Plane 4 – Excessive surgical anaesthesia – intercostal paralysis, shallow abdominal breathing.Stage IV This stage is also known as the stage of medullary paralysis or respiratory paralysis.This period is between respiratory arrest and cardiac arrest.At these above stages can be observed during the experiment.
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Objective: To study the onset and duration of intravenous anaesthetics in rats
Animal : RatApparatus required: Dental syringe with hypodermic needleDrug: Thiopental sodium, distilled waterProcedure:
The rats are weighed, numbered and the sex of the animal noted. Respiratory rate and heart rate are recorded.
A 2.5% solution of thiopental sodium is made by dissolving 0.5 G of thiopental sodium in 20 ml of distilled water. The dose of the drug required for each rat is 40 mg/kg body weight. Accordingly the dose for each rat under experimentation is calculated and the calculated dose of the drug is taken in a dental syringe and injected intra peritoneally and the time when the drug is administered is noted (A).
The animal is observed and its respiratory and the heart rate are recorded. When the animal goes into its lateral recumbency. The time the animal goes to its lateral recumbency is recorded as (B).
After placing the animal on the table the corneal and pedal reflexes are checked at regular intervals. The time when the animal loses its pedal reflex is recorded as (C).
The heart rate and respiratory rate are also recorded during the period. The pedal reflex is gained thereafter and the time at which the animal regains its
pedal reflex is recorded as (D). The animal is observed for regaining of sternal recumbency and the time when
the animal regains its sternal recmbency is recorded as (E). By this way different stages of anaesthesia i.e., Stage 1,stage2,stage3 and stage4
of anaesthesia may be observed chronologically. The animal is placed in the cage after the experiment. The same procedure is
repeated with the other rats.
Calculations:1. Induction time: Difference in time in seconds between the time the animal is injected to the time it loses its pedal reflex (C-A).2. Anaesthetic duration:Difference in time in seconds from the time animal loses its pedal reflex to the time it regains its pedal reflex (D-C).3. Sleeping time:Difference in the time when the animal regains its sternal recumbency to the time at which it went to its lateral recumbency (E-B)
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Ex.No.13 Date:
DEMONSTRATION OF DRUGS AT THE NEURO EFFECTOR MUSCULAR JUNCTIONS USING FROG RECTUS ABDOMINIS MUSCLE
Aim: To demonstrate nicotinic cholinergic response at neuromuscular junction of
skeletal muscle and to demonstrate anticholinesterase activity of Physostigmine/neostigmine
potentiating Ach response. To demonstrate d-tubocurarine competitive blockade of Ach at neuromuscular
junction.Apparatus: Organ bath, kymograph or Chart recorder or digital polygraphDrugs: Acetylcholine, Physostigmine, d-tubocurarine.Procedure
The frog rectus preparation is usually made from Rana temporaria, Rana pipiens or Rana esculenta.
Frog weighing 20 g will do for the experiment. These can be kept for many weeks at 4 degree celcius without feeding.
The frog is stunned, decapitated and the spinal cord is destroyed. The frog is pinned down on the dissecting board and the skin of the abdomen
but not the abdomen itself is picked with the forceps and cut from the above the sternum down to the fork. It is then cut laterally to expose whole of the abdomen.
Two rectus muscle will be plainly seen, running each side of the midline from the base of the sternum to the fork. The sternum is cut through just above the xyphisternum at its base, and the pair of muscles attached to it are dissected out and transferred to a dish containing frog-ringer solution at room temperature.
The muscles are divided, unless they are very small and threads are attached to the top and bottom of each piece. One end is attached to the pin in the organ bath and the other end to the lever.
The load on the lever should be 0.5 g to 1.0 g and adjust it so that it is just balanced and then add the load, a small piece of plasticine is suitable. The preparation is kept at room temperature in frog ringer solution through which air is blown.
The capacity of the organ bath is 5-10 ml. A simple side way writing lever is suitable though a gimbal is better but is expensive. A vibrator may be attached to avoid sticking on to the writing paper.
After the muscle has been made to contract, it will not relax rapidly when the drug has been washed out, and it must be stretched gently by increasing the load.
The experiment is then carried with the drugs and the effects are recorded on the kymograph.
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Ex.No.14 Date:
COMPUTER ASSISTED LEARNING IN EXPERIMENTAL PHARMACOLOGY Introduction:
All the experiments in experimental pharmacology have been designed based on animal use. They usually involve scarification of the laboratory and/or domestic animals to carry out some invasive procedure. However, the restrictions in animal use, of late, have made it difficult to understand the effects of drugs since the effects cannot be demonstrated. To overcome this problem, we have readily available software, which have been designed in such a way that the effects of drugs can be visualized on the screen. These packages are user-friendly, interactive and full of animated sequences.
The program that is being used now is the ExPharm developed for educating graduate students. The programme is aimed to make the student understand, remember and recall drug actions. The current version that is being used can be used in both DOS and Windows mode. The basic experiments that are simulated are:
1. Effects of drugs on rabbit eye2. Effects of drugs on frog heart3. Bioassay of histamine on the guinea-pig ileum4. Effects of drugs on the frog oesophagus ciliary body movement 5. Effects of drugs on blood pressure in dogs
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Ex.No.15 Date:
COMPUTER ASSISTED LEARNING IN EXPERIMENTAL PHARMACOLOGY
EFFECT OF DRUGS ON FROG HEART
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Ex.No.16 Date:
COMPUTER ASSISTED LEARNING IN EXPERIMENTAL PHARMACOLOGY
EFFECT OF DRUGS ON RABBIT EYE
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Ex.No.16 Date: COMPUTER ASSISTED LEARNING IN EXPERIMENTAL PHARMACOLOGY
EFFECT OF DRUGS ON FROG OESOPHAGEAL CILIARY BODY MOVEMENT
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Ex.No.16 Date: COMPUTER ASSISTED LEARNING IN EXPERIMENTAL PHARMACOLOGY
EFFECT OF DRUGS ON DOG BLOOD PRESSURE AND HEART RATE
Introduction:Actions of many drugs are not confined to a single system of the body. A drug
may act on a single organ or multiple organs of the same or different systems. It may act at one site but produce an effect at another site. The present experiment which simulates a live anaesthetized dog demonstrates the effects of a few drugs on the cardiovascular system. These drugs act on different receptors and organs to produce similar or opposite effects.
A dog is weighed and anaethetized using intravenous chloralose (100 mg/kg of body weight). It is fixed on a dog table in supine position. Right femoral vein is cannulated with a catheter to inject drugs. Its neck is dissected to expose carotid arteries and vagus nerves. Left common carotid is inserted with an arterial cannula connected to a mercury manometer or to a pressure transducer, which is connected to a polyrite / physiograph. A chart recorder or a kymograph is set up to receive signals from the polyrite/physiograph to produce graphical representation of changes in blood pressure. The left side vagus is cut into central and peripheral ends for applying electrical stimulation later in the experiment. The right common carotid is identified and exposed for later use.
Experimental setup:A dog is weighed and anaethetized using intravenous chloralose (100 mg/kg of body weight). It is fixed on a dog table in supine position. Right femoral vein is cannulated with a catheter to inject drugs. Its neck is dissected to expose carotid arteries and vagus nerves. Left common carotid is inserted with an arterial cannula connected to a mercury manometer or to a pressure transducer, which is connected to a polyrite / physiograph. A chart recorder or a kymograph is set up to receive signals from the polyrite/physiograph to produce graphical representation of changes in blood pressure. The left side vagus is cut into central and peripheral ends for applying electrical stimulation later in the experiment. The right common carotid is identified and exposed for later use.Drugs are injected (one by one) into the femoral vein and BP is recorded on the chart. The heart rate (beats/min) is counted and noted on the chart. Drug administration is also marked.
Limitations:Realistic simulation of real life events on a computer is not easy - especially the
biological processes which are so complex. Many factors come into play and control an
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organ or a system (say CVS), and attempting to simulate each one (factor) will make the coding a highly complex affair which the author (of the software) is not capable of handling. Hence, the simulated results obtained with the software may not be very accurate, although care has been taken to eliminate gross errors. The minor inaccuracies, may not come in the way of learning, because the aim of the software is to teach the students about major facts that have practical utility.
Although the program may be used for self-learning, a demonstration by a teacher would be of great help to students to understand the experimental procedure and the theory behind it.
Aim:To demonstrate the actions of a few drugs on the blood pressure (BP) and heart
rate (HR) of dog.
Procedure:Inject the drugs one by one. Observe the following parameters before and after
drug administration: (a) BP (mm of Hg)(b) HR (beats per minute)Parameters (a) and (b) are assessed by observing the recording. The BP
recodings are indicated in black and that of the HR in red. Two scales for measuring BP and HR are provided one at each side of the chart. The one in which the measurements are marked in red indicates HR (beats/min) and the other with black marking indicates BP(mm Hg). Moving the mouse pointer over the chart will display a crosshair that helps you measure the correct BP and HR. Apart from drugs, a few procedures such as carotid occlusion and electrical stimulation of cut ends of vagus can be performed to see the changes that these procedures would bring about on the BP and the HR.
1.The drug can be selected by clicking the mouse on the drug list. On selection, the selected drug and the recommended dose to be injected are displayed. The list includes carotid and vagal procedures also.
2. Always use the appropriate dose. The conventional dose range and the recommended dose for different drugs will be displayed. The dose can be altered by the user and the drug is injected by clicking the "Syringe" icon. Double clicking the drug name (on the drug list) will also inject the recommended dose of drug.
3.Be careful while selecting the dose. Low doses may not produce any effect. Whereas higher doses might ‘kill the dog’. Do not repeat the same blocker often. It might kill the dog.
4.The effect of blockers can be removed by pressing 'Remove Blocker' button. Names of the blockers in action and their amount in the body will be displayed. You can remove
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any or all blockers. The level of blockers (concentration) will automatically decrease with time.
5.For 'Epinephrine' you can give doses as low as 0.1 mg/kg to see the beta receptor action, and for 'ACh' doses as high as 150 to 200 mg/kg can be given to see the nicotinic action.6. Start with carotid and vagal procedures and continue with agonists - This is called 'Normal Response Brackets or Normal Panel'. Then administer a blocker followed by the corresponding agonist to see the 'Agonist - Antagonist' interaction.
Note: Two or more drugs can not be administered simultaneously; the drugs have to be given one after another only. The response to agonists can be tested while the antagonists are still present and acting on the body.The recordings can be saved, viewed, or deleted using the options(buttons) under 'Session'.
Nature Drug/ Code ( dose / Range in g/kg)Agonist Epinephrine Code : Epi Dose : 2 Range : 1 - 3
It stimulates the alpha and beta adrenergic receptors. Conventional doses will increase the BP followed by a short fall before reaching the basal level (biphasic response due to alpha and beta receptor responses). To see the beta action alone, use a low dose such as 0.1 mg per kg of body weight. The heart rate decreases due to vagal reflex.
Agonist Norepinephrine Code : Nepi. Dose : 3 Range : 2 - 5 It stimulates mainly the alpha and beta1 receptors. The heart rate is generally reduced due to vagal reflex in response to increased BP. Hence if the dog is pretreated with a muscarinic blocker (atropine), norepinephrine will show an increase in the heart rate
Agonist Isoprenaline Code : Iso. Dose : 3 Range : 2 – 5 It is a potent, non-selective beta adrenergic stimulant. It increases the systolic BP (sometimes it remains unchanged) but decreases the diastolic BP. Because the decrease is more pronounced than the increase, the mean arterial pressure typically falls.
Agonist Acetylcholine Code :Ach. Dose : 5 Range : 2-10Intravenous administration of Acetylcholine (ACh) leads to a sharp a fall in BP which returns to basal level quickly. Though ACh generally reduces heart rate, small doses may generally lead to reflex tachycardia. After complete atropinization (this might require a higher dose of atropine), a large dose of ACh (150 - 200 mg/kg) may be tried to demonstrate the 'Nicotinic or Ganglionic' action.
Agonist Histamine Code : Hist. Dose : 3 Range : 2 – 5Acts on H1 and H2 receptors to produce a fall in BP which may be completely blocked when H1 and H2 antagonists are acting concurrently. Either of the antagonists, if given alone will produce only a partial block.
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Stimulation of H1 produces a rapid onset short lived decrease in BP whereas H2 stimulation leads to a fall characterized by slower onset and longer duration.
Agonist Ephedrine Code : Ephe. Dose : 100 Range : 100 – 200 It acts on both alpha and beta receptors and in addition enhances the release of norepinephrine from sympathetic neurons. It increases the BP and heart rate. Repeated administration within a short time will lead to a gradual decrease in response. This is known as tachyphylaxis. To demonstrate this phenomenon, repeat a conventional dose 4 to 5 times without allowing a large interval between doses
Antagonist Phentolamine Code : Phen. Dose : 1000This drug, an alpha blocker, reduces BP and also affects the alpha components of other drugs.
Antagonist Propranolol Code : Prop. Dose : 1000It is a beta blocker which reduces BP and heart rate. It affects the beta components of other drugs.
Antagonist Atropine Code:Atro. Dose : 750 Range : 500 – 1000This drug is a muscarinic cholinergic antagonist. It competitively antagonizes ACh. Atropine in conventional doses may not completely block muscarinic receptors. It may require a higher dose ranging from 1000 to 1500 mg/kg. To demonstrate the nicotinic action of ACh, complete atropinization is a must.
Antagonist Mepyramine Code : Mep. Dose : 5000Mepyramine is a H1 blocker. It does not have any action on the CVS but when given alone it blocks partially the fall in BP by histamine.
Antagonist Cimetidine Code : Cime. Dose : 5000It is a H2 blocker, which also partially blocks the effect of histamine on BP.
Not Known (to be determined)
Unknown Code:Unkn. Dose: 0.1 ml Range : 0.1 - 0.2 mlUnknown solution contains one of the drugs listed above. It is randomly chosen by the software and varies each time you run the experiment (i.e. new experiment). Unknown solution is given only when the software is run in Examination mode. It contains only one drug.
Neutral Saline Code:Sal. Dose :1ml Range : 1 - 2 mlWhile conducting the live experiment, a small volume of saline is administered following every drug injection, so that the drug remaining in the catheter can be flushed completely into the system.
Exercise: 1. Interpret the various graphs relatively mentioning the receptors involved in the agent’ s action
2. Schematically represent the pharmacological actions of epinephrine, norepinephrine, Isoproterenol, histamine, atropine sulfate on various body systems
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