Experimental Pharmacology

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EXPERIMENTAL PHARMACOLOGY
INTRODUCTION
TO EXPERIMENTAL PHARMACOLOGY
AND DIFFERENT LABORATORY ANIMALS
Pharmacology: It is the science which deals with the study of drugs which is desired
from a greek word "Pharmacon" which means drugs & "logos" means study.
Experimental Pharmacology: It is that branch of pharmacology which deals with the
effect of drugs on living system. It can be studied under heads:
a) Preclinical Pharmacology: dealing with effects of drugs on animals.
b) Clinical Pharmacology: dealing with effect of drugs on human livings.
Thus it helps in under studying the nature of drug action and the un-alterability of the
living systems to the attract by chemicals that serves as the basis on which:
a) New therapeutic agents are developed &
b) Toxic consequences of chemical exposure may be activated.
The experiment can be carried out in whole animal (in vivo) or in isolated organs (in
vitro).
Olyectives for experimental pharmacology:
1. To provide scientific weighting of a drug for its use.
2. To add new & better drug in therapeutics.
3. To ...... & discover new, simple & better techniques for experimental.
4. For bioassay and standardization of drug.
Bioassay :
Estimation of the potency of an active principle in a unit quantity of prep. or defect &
using biological methods is known as 'biological assay or Bio assay.
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Different Laboratory Animals And Their Applications In Experimental

Pharmacology :
Laboratory Animals :
These are those animals which can be used and reared (maintain) in the laboratory
under suitable conditions.
The common laboratory animals are rat, mice, guinea pig, rabbit frog & hamster. Other
animals used for experimental purpose are cat, dog, monkey, pigeon etc.
Since long animal experiments have been a mile stone in advanced medical research.
Any or every animal is not suitable for experimental work. There selection is based on
the following criteria.
1) Size: Smaller animals are prefered because they are easy to handle & less
quantity of drug is required.
2) Availability: Animals which are commonly available should be selected e.g.
frogs, rats, rabbits & dogs.
3) Sensitivity: Animals which are sensitive to drugs under trial e.g. guinea pig is
sensitive to effect of histamine.
4) Species: In rabbits intracerebro ventricular injection of 5-HT induces a lowering
of temperature, but in cats, it induces fever.
 Ethacrynic acid is almost inactive in rats, except at high doses, but quite active
in the dog.
 Following the same dose of hexobarbitone per unit body weights, the average
sleeping time of rats is about seven times that of mice, and in the dog its
effects lasts for hours.
 Guinea pig and humans are 500 more times sensitive to histamine than are rats
& mice. Histamine powerfully contracts the uterus of guinea pig while relaxes
that of rat. In rodents it produces stronger arteriolar constriction in cat slight
constriction, while in dog, monkey and man arteriolar dilation.
 The rat heart is known to be very resistant to cardiac glycosides.
 Alloxan produces diabeties mellitus in no. of species but not in guinea pigs.
 Rabbits generally show symptoms following insulin when the blood sugar
level is 45 mg percent while dogs show symptoms only when the level is
about 18 mg. percent.
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Isolation or group housing:

Dose of pentoharbitone or phenobarbitone, which produce full hypnosis in isolated
mice, produce marked stimulation when the animals are grouped.
The CVS becomes more responsive to isoprenaline in solitary housed (6-8 weeks) rats
compared to group housed rats.
CHARACTERSTICS & ADVANTAGES OF DIFFERENT ANIMALS
(i) RAT (Rattus norvegicus)
Albino rat is one of the commonest laboratory animals suitable for experimental work
because of its because of its small size & greater sensitivity to most drugs. It is also the
most standardized of all laboratory animals. It can be used to obtain pure and uniform
strains and is found to be very sturdy to withstand long periods of experimentation
under anaesthesia.
It is small in size compared to other animals so drugs are required in small quantity.
Vomitting centre is absent and so drug can be administered orally. Gall bladder and
tonsils are absent.
Because of the absence of gall bladder in rat there is continuous flow of bile into the
intestine. This facilitates the study of drugs acting on bile, cholesterol reabsorption.
Pancreas is diffused, therefore difficult to produce pancreactomy. In stomach, fundus &
pyloric parts have clear lining between them. The gastric acid secretion is continuous.
EXPERIMENTAL USES:
(Adult wt. 180-200 gm., age suitable for most of the experiment 1.5 months)
1. Psychopharmacological studies.
2. Study of analysis of anticonvulsants.
3. Bioassay of various hormones, such as insulin oxytocin, vasopressin etc.
4. Study of oestrus cycle, mating behaviour and lactation.
5. Studies on isolated tissue preparation like uterus, stomach, vasdeferens,
anococcygeus fundus strip, heart, etc.
6. Chronic study on blood pressure.
7. Gastric acid secretion studies.
8. Study of hepatotoxic and antihepatotoxic compound.
9. Acute & chronic toxicity studies.
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B. GUINEA PIG (Cavia porcellus):

It is docile animal, highly susceptible to TB & anaphylaxis and also highly sensitive to
histamine and penicillin. It requires exogenous ascorbic acid in diet.
Experimental use (Adult Wt. 400-600 gms., age suitable for experiments- 3 months)
1. Evaluation of bronchodilators
2. Anaphylaclic and immunological studies.
3. Study of histamine and antihistamincs.
4. Bioassay of digitalis
5. Hearing experiments because of sensitive cochlea.
6. Studies on isolated tissues specially, ileum tracheal chain, vas deferens, etc.
7. Study of tuberculosis and ascorbic acid metabolism.
(C) MOUSE (Mus musculus)
Swiss albino mice are commonly used. They are smallest cheap & easy to handle.
EXPERIMENTAL USE:(Adult weight:20-25 gm., age suitable for experiment, 2 month)
1. Toxicological studies, specially acute & subacute toxicities. They are also used in
teratogencity (foetal abnormalities)
2. Bio assay of insulin.
3. Screening of analgesics & anti convulsants
4. Screening of chemotherapeutic agents.
5. Studies related to genetics & cancer research.
6. Study of drugs acts on CNS
(D) RABBIT (Oryctolagus cuniculus): Rabbit are also docile animals with large ears.
Usually Newzealand white rabbits are used. It has huge caccum and large appendix.
The enzyme atropine esterase is present in rabbit liver and plasma, so it can tolerate
large does of belladona (atropine). Cardioaortic nerve forms a separate depressive
nerve. Vasodilator nerves are absent and so vasomotor reversal phenomenon can not be
demonstrated. Histamine causes increase in blood pressure. Ovulation is related to the
release of luteinizing hormone and occurs 10 hours after coitus.
EXPERIMENTAL USE- Adult weight 1.5-3.0 Kg. age suitable for experiment 5-6 months.
1. Pyrogen testing
2. Bio assay of antidiabetics, curareform drugs and sex hormones.
3. Screening of agents affecting capillary permeability.
4. Irritancy tests.
5. Screening of antitoxic agents and teratogens.
6. Studies related to reproduction (antifertility agents).
7. Isolated preparation like heart, dudenum, ileum, Finkleman preparation.
8. Study of local anesthetics (surface anaesthesia)
9. Study of miotics & mydriatics
(E) HAMSTER (Mesocriceitus auratus and Cricetulus griseus)
They have short body with short legs & tail. The skin is loose and covered with dense
short soft fur. The cheeks pouches are prominent & extend up to the shoulder region.
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EXPERIMENTAL USE: adult weight 80-90 gm average age suitable for experiments
1 month)
1. Chinese hamsters have low chromosome number making it useful for cytological
genetics tissue culture & radiation research.
2. Research on diabetes mellitus
3. Research related to virology, immunology and implantation studies.
4. Bioassay of prostaglandins.
(F) FROG (Rana tigrina)
One of the most commonly used experimental animals in physiology, pharmacology
and toxicology. It has been used in experiment for 200 yrs. It is easily available during
rainy reason. It is an amphibian animal and safe to handle. It can not be used in
laboratory. Adrenaline is neuro transmittor in the sympathetic system.
EXPERIMENTAL USES:-
1. Study of isolated tissue such as rectus abdominis muscle and heart preparation.
2. Study of drugs acting on CNS
3. Study of retinal toxicity of drugs, light bleaches rhodopsin in eye within one
hour and is regenerated within one hour in dark.
4. Study of drugs acting on neuromuscular junctions (using gastronemius, sciatic
muscle nerve preparation.)
(G) CAT:-It is carnivorous animal relatively easy to obtain and to use for experimental
purpose. The physiology of circulatory & neuromuscler system is very much similar to
that of man. It has highly developed nictating membrane which is contracted by
sympathetic nerves. Morphine produces excitation of central neruous system in cat.
EXPERIMENTAL USES:-
1. Acute experiments for the drugs affecting BP.
2. Bioassay of NA (using spinal cat)
3. Studies on ganglions blockers (using nictitating membrane in vivo)
4. Studies on neuromuscular system (using gastronemius, sciatic muscle nerve
preparation.)
5. Toxicity studies of compound like acetanilide.
(H) DOG- Commonly Mongrel or Beagle dogs are used. It is easily available & large
sized animal. Dogs can be easily tamed as well as trained. It has a small stomach and
short intestinal tract resembling those of human beings. It can be conditioned to carry a
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stomach cannula. The cervical sympathetic and vagus nerve run together inseparably in
the trunk
EXPERIMENTAL USE-
 Gastric acid secretion studies (Pavlov pouch.)
 Acute experiments for drug affecting BP and intestinal movements etc.
 Studies on antidiabetic agents.
(I) MONKEY AND APES- These are the primates belonging to the highest order of
the mammals. The anatomy & physiology of these animals are closely related to that of
man. The studies done in monkeys are directly translated to man. Considering the
human respects, tests in primates should be done only in last stage of evaluation of
drugs before clinical trials. They are used in the fields of psychopharmacology,
urology, immunology, nutrition, reproduction, parasitology, etc.
(J) LEECH:-The dorsal muscle of beech is used for bioassay of acetylcholine.
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LABORATORY ANAESTHETIC AGENTS
Object: Choice of laboratory anaesthetic agents.

General anaesthetic agents bring about loss of all sensation particularly pain along with
reversible loss of consciousness. Consciousness is regained after the agent is
metabolized or excreted.
A general anaesthetic fulfill the following requirements:
a) Induction of anaesthesia should be quick & pleasant.
b) It must have longer duration of action.
c) There should be adequate musele relaxation.
d) It should not interfere with effect of drug under study.
e) It should be cheap & non-inflammable.
G. ANAESTHESIA OF TWO TYPES:
1). Volatile general anaesthetics:
These could be liquid like ether, halothane, ethyl chloride, trichlorethylene,
gases as N2 O, ethylene & cyclopropane. They are not used commonly in
experimental animals, because they require constant monitoring and costly
instrument set up. Ethyl chloride is rarely used in experiments of rats & cats.
2). Non-volatile anaesthetics:
These agents are more commonly used for producing anaesthesia in various
animals because it is easy to administer these agents and no complicated
technique is required. Commonly used agents are-chloralose, urethane,
barbiturates, MgSo4, paraldehyde etc.
(i) Chloralose:
It is the compound of chloral and glucose prepared by heating equal parts of
anhydrous glucose and chloral when both  chloralose (active form) and 
chloralose (inactive form) are formed. Chloralose is the active form (-
chloralose) freely soluble in hot water in alcohol & in ether & slightly solutle in
cold water.
Dose: 80-100 mg/kg body wt. 1% solut used given 1.V routes.
Advantages:
1. It produces surgical anaesthesia lasting for 3-4 hrs.
2. Respiration and BP are not depressed so can be used in conditions where they have
to be recorded.
3. Reflexes are not depresed.
Disadvantage:
(1.) Poor water solubility so solution is prepared in luke warm water or 10% propylene
glycol or ether. (2) Large volume is needed. (3) Jerky movement are seen.
(4.) Unsuitable for rabbits where it produces narcosis instead of anaesthesia.
2) URETHANE (ETHYL CARBAMATE):
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It is readily soluble in water giving a neutral solution. Usually 25% solution is

used I.M. This is also suitable for acute non recovery type of experiments in
dog, cat, rabbit & rats. Duration of action is 3-4 hrs. Frogs can be anaesthetized
by keeping them covered in beaker containing 5-10% solution.
Advantages:
1. It does not affect reflexs, CVS, & respiration.
2. It is readily soluble in water.
3. Anaesthesia remains for 3-4 hrs.
Disadvantage:
1. Induction is slow so, injection of morphine 2mg/kg body wt. is given, IM 30
min before giving urethane as pre anaesthetic agent so induction is quick and
smooth.
2. It has irritant nature so animal feels pain.
3. Amount of solution injected to be large so has to be given at various sites.
4. It has delayed toxic effect on liver & may also cause agranoulocytosis &
pulmonary adenomas. Mice develop high incidence of lung tumors regardless of
route of administration.
(3) BARBITURATES:
These are most commonly used agents.
Advantage:
1. Induction is very rapid and smooth with minimal excitation.
2. No preanaesthetic medicat ion is required.
Disadvantage:
1. It produces depression of cardiovascular & spinal reflex, by interfering
with nerve impulse transmission both in CNS and ganglia.
2. Muscle relaxation is inadequate therefore, mucle relaxants have to be
given simultaneously.
3. Anaesthesia produced is of short duration, therefore small doses of these
agents are to be injected at regular intervals.
They can be classified as:
a) Long acting barbiturates:
e.g. phenobarbtone 10% aqueous solution is used. Dose 120-180 mg / kg. of
body wt. intra-peritoneally.
b) Short acting barbiturates:
e.g. thiopentone sodium 2.5% aqueous solution in dose of 15-25 mg/kg. body
wt. is given i.v. duration of action is 20-30 minutes.
PARALDEHYDE:
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It has wide margin of safety as it depresses only the cerebrum and not medullary
centres. Dose 1 mg/kg. body wt. I.P. or 2 mg/kg. body wt. I.M. in dogs & cats.
MAGNESIUM SULPHATE:
20% solution in a dose of 5 ml/kg. intravenously produces anaesthesia for on hour,
Calcium gluconate is used I.V. to counteract the depressant effect immediately. Its
principle use is in producing euthenasia (mercy killing).
Euthanasia: (Painless killing).
When animals are killed at the end of the experiment. It should be done by a human
method.
Methods of euthanasia:
1) Chemical method: It is the painless death produced by administration of
chemical poisons, Certain chemicals are injected & there are-
(1) Magnesium sulphate I.V. / intra cardiac (15gm/kg. body wt.)
(2) Chloroform. (3) A large volume of air ( /kg. body wt.)
(4) Sodium citrate in large quantity (anticoagulant) (5) Paraldehyde & MgCl 2 can
also be used. (5) In open chest operations adrenaline can be touched at the apex of
ventricles - arrythinias will be started - death.
2) Mechanical Method: The quickest & commonest method for killing mice, rats,
guinea pigs & rabbits is Stunning is carried out by striking the dorsal part of
head against the edge of table or sink.
This lead to stiffening of all muscles followed by a series of convulsions and then
gradual relaxatation of the limbs and the body. As a result of stunning animals get
sudden shock and temporarily becomes semiconscious one should have practice to stun
by a single hard stroke only. Multiple strokes does not comply the principle of
euthanasia.
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PHYSIOLOGICAL SALT SOLUTIONS
Object: To study the physiological salt solutions used in experimental pharmacology &
various drug dilutions:
As animal experiments have to be done with isolated organs, it is necessary to use a
certain no. of physiological solution of different ionic concentration which almost act
as a substitute to the tissue fluid. They provide isotonicity, nutrition and acts as a buffer
when drugs are added.
It was "Ringer" who Ist introduced the idea that tissue could be kept alive by providing
proper nutrition, O2 & temperature.
PSS can be defined as artificially prepared solution to keep isolated tissue alive under
experimental conditions. The content of these solutions carries according to tissues &
animals taken. These solution provide food material i.e. energy, O2, electrolytes as in
the same proportion as that present in tissue fluid. They exert same osmotic pressure as
that of interstitial fluid i.e. isotonic with body fluids. Any variant from the principle
will lead to shrinkage or blotting depending on hypertonicity & loss of physiological
function. For these two points should the kept in mind :
1. Solution should be prepared carefully with pure material. They can be kept for
about 24 hrs. as they are good media for the growth of micro organisms they must
be refrigerated and should be freshly prepared after 24 hrs.
Following points should be carefully noted at the time of preparation of solution :
i). Balance of cations: Absolute quantity of each ion and prepartion among each
other especially with ca+2 & K+ must be maintained. The common cations and
their significance are :
(a) Na+ions: Responsible for maintenance of excitability, contractivility
rythimicity of muscles and nerves.
(b) K+ions: responsible for increase relaxation of heart increased
neuromuscular transmission and excitability of nerves.
(c) Ca++ ions: increase force of contraction & tone of heart and decrease
excitability of nervous tissues.
2. Mg+2 ions : responsible for contraction of smooth muselec.
i). pH of solution / reaction of solution : pH of various PSS varying from
7.3-7.8 depending upon organ. At lower pH value tone of preparations
tend to decrease & effect of drug is also altered. pH affects tissue directly
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and by ionization. At higher pH ionization is less and leads to alkalinity &

thus improves cardiac & smooth muscle activity. During experiment there
can be accumulation of metabolite which may change the pH. Buffering
agents like HCO-3 & PO-4 are add in saline solution and solutions are
changed frequently.
ii). Glucose: Introduced by "Locke" and serves as an energy source, increases
contractility of tissue. It is not essential constituent for amphibians tissue
but indispensable for mammalian tissues.
iii). Distilled Water : It acts as a vehicle to dissolve various ingredients.
iv). Control of temperature: In order to get consistent effect, it is important
to maintain the temperature of PSS, particularly for mammalian tissue. For
instance, when temperature of solution is below 370C tone of intestine is
decreased, increased the contracts become smaller & contracts and
relaxation time increases. Whereas, amphibaian tissues survive for longer
time at room temperature only.
v). Areation: Air, O2 or O2 + 5% CO2 is needed for the proper functioning of
the tissues. Besides providing O2 for the tissues, the stream of gas bubbles
also stirs the solutions in the bath thereby facilitating diffusion of the
drugs.
The solution in the bath should be changed frequently because prolong
areation tends to alter pH.
Different physiological salt solutions and their uses :
i). Ringer lock's solution: It is used in isolated rabbit heart perfusion.
ii). Frog's Ringer's solut: Used in frog's rectus abdominis muscle and leech
dorsalis muscle preparation.
iii). Tyrode's solution: It is used in experiment of rabbit intestine & guinea pig
ileum.
iv). De- jalon's solution: Used in rat uterus, duodenum colon experiment.
v). Kreb's Henseleit solution : Used in guinea pig tracheal chain prep. & rabbits
aortic strip preparation.
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Components of Physiological Salt Solution:

S. Salts in g/l Ringer Frog's Tyrode's Deialon's Kreb's - Mc
No. solution ringer solution solution Henseleit Ewen's
solution solution solution
1. Nacl 9.00 6.50 8.00 9.00 6.9 7.60
2. KCl 0.42 0.14 0.20 0.42 0.35 0.42
3. CaCl2 0.24 0.12 0.20 0.06 0.28 0.24
4. NaHCO3 0.50 0.20 1.00 0.50 2.10 2.10
5. Mgcl2. - - 0.10 - - -
6H2O
6. MgSO4 - - - - 0.29 -
7H2O
7. NaH2PO4 - 0.01 0.05 - - 0.14
8. KH2PO4 - - - - 0.16 -
9. Glucose 1.0 1 or 2 1.00 0.50 2.00 2.00
10. Areation O2 air O2 / air O2+5% O2+5% O2
CO2 CO2
Smoking of Drum:
Kymograph paper is wrapped smoothly round the drum with adhesive. Either coal gas
bubbling through benzene or a mixture of benzene and Kerosen (1:9 Ratio) can be used
to get a black soot. The drum should be rotated at a reasonable speed to obtain thin and
uniform black soot.
Fixing of the tracing on smoked papers:
Fixing is done by dipping in any of the following solution & allowing to dry:-
a) 150 gm powered amber resin or colophony is dissolved in 1 ltr. of ethyl alcohol,
10 ml. of glycerine added to it, varying amount of collodion added according to the
glossiness required.
b) Granular white shellac is dissolved in alcohol till a large amount of it remain
undissolved & the whole solution allowed to remain for a week or more. The clear
supernatant is decanted & kept in well corked bottle to prevent evaporation of
alcohol.
C) Any quick drying spirit varnish diluted 10-12 times with methylated spirit & a
very small amount of castor oil added to give shine.
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Drug Dilutions: In pharmacological experiments drugs are used in very minute

quantities i.e. can be fractions of mg or g. These small amounts can't be weighed
accurately even in analytical balance and hence substance are usually weight in larger
quantities and dissolved in solvents.
Generally stock solutions are prepared which are 1%. Solution & further diluted
according to dose required 1%. solution is prepared by dissolving 100mg. of the drug in
10ml of distilled water.
Drug Dilutions:
% of solution Strength mg/ g
1% 1:100 100mg/10ml or 10mg/ml
10% 1:10 100mg/ml.
100% 1:1 1000mg/ml or 1g/ml .
0.1% 1:1000 1 mg/ml
0.01% 1:10,000 0.1mg/ml or 100µg/ml.
0.001% 1:100,000 0.01mg/ml or 10g/ml.
0.0001% 1:10,00,000 0.001mg/lml or 1µg/1ml.
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INSTRUMENTS
1. DALE'S ORGAN BATH- or isolated organ bath
It is an apparatus used for studying the effects of drugs/ chemical substances on
isolated tissues in vitro.
The apparatus consist of
(i) An inner glass tube or organ bath containing PSS and tissue and connected to
reservior through polythene or rubber tube.
(ii) Areation cum tissue holder tube to hold tissue and supply O2 / air to perfusion
fluid.
(iii) An outer bath made up of glass/perspex filled with water the temperature of
which is checked with the help of thermometers & maintained at 37° for all
mammalian experiments.
(iv) A lever for recording the responses of the tissue on a kymograph drum.
(v) The isolated organ bath meant for research purpose also possesses an inbuilt
warming device the thermostat, a fluid warming cell and a stirrer meant for
automatic temperature control of the water of the outer bath.
(vi) The entire assembly is mounted on tripod stand.
2. SHERRINGTON'S RESEARCH KYMOGRAPH
It is the instrument on which physiological responses such as contraction and
relaxation of muscle are recorded.
It consist of a heavy base and a vertical shaft. Heavy base gives stability to
drum.
It has
(i) base loofs (legs): with adjustable leveling screw to keep drum horizontal on
the uneven surface.
(ii) side hoofs- to turn the drum on its side so that shaft become horizontal.
(iii) Gear rods- arrangement with gear & clutch to obtain desirable speed of drum.
(iv) Drum cylinder- is a brass or iron cylinder around which paper is wrapped and
smoked for recording of tracings
3. LEVERS-
It is the device by virtue of which response of isolated tissue can be recorded
and magnified.
Principle:
(i) Fulcrum- the point around which the lever moves on the lever holder is the fulcrum.
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(ii) Stylus- is the writing point which record the tracing on the smoked paper of the
drum. It is either made of celluloid parchuments paper, aluminium foil or thin
photographic or x-ray film.
Magnification- The fulcrum (F) should be so placed that there is some magnification
of the actual contraction (response). In order to achieve this, the distance between the
writing point and the fulcrum (F) should always be greater than the distance between
fulcrum and point of attchment of tissue (T). By adjustment of these relative distances
from the fulcrum any degree of magnification can be obtained.
Therefore lesser the inherent contractility of tissue higher the magnification needed or
vice-versa
Name of tissue magnification
1. Guinea pig ileum 5-10 times
2. Rat uterus 4-6 times
3. Frog rectus abdominis muscle 10 times
4. Rat fundic strip 16 times
Adjustment for the load or tension
The muscle preparation has to be properly relaxed without affecting the normal tone
and rhythimic activity so that efficient contractions are achieved when stimulated and it
also relaxes to its full length after wards. This is achieved by the following way:-
Select the proper length of longer and shorter arm after fixing magnification for
particular tissue and fix the fulcrum.
Balance the lever by putting the wt (plasticin) at the end or shorter arm and mark the
point of tissue attachment.
At equidistance i.e. the distance between the F &T from the (F) on the longer arm of
lever the desired load required for particular tissue. The tension (load) prescribed for
various commonly used tissues is as follows :
1. guinea pig ileum 1 gm
2. guinea pig trachea 0.2 gm
3. guinea pig vas deferens 0.5gm
4. rat colon 0.5 gm
5. rat uterus 1.0 gm
6. rat fundus 1.0 gm
7. frog rectus abdominis muscle 1.5 gm
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The writing levers are light in weight, rigid and are generally made up of wood
(straw), light aluminium or stainless steel. The levers are of 2 types
(i) Isotonic type- i.e. change in length due to contraction is recorded while the
tension on the muscle remains the same e.g. simple lever frontal writing lever.
(ii) Isometric type-These levers are used under special circumstances for instance,
when a twitch is produced by stimulating a muscle suspended between two
rigid points,one being a strong spring, the muscle does not shorten but only
creates a force or tension which is recorded; the twitch is also much faser in
action. e.g. Paton's auxotonic lever will serve purpose well.
Different types of lever
(i) Simple lever- (sideway writing): It is simplest type of lever made up wood
(straw), stainless steel or aluminium. A celluloid writing tip (stylus) is attached
at the end of the longer arm. The contractions are recorded as curved lines.
(ii) Frontal writing lever (writes frontally)- This 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 contraction are
recorded as straight line.
(iii) Starling's or Heart Lever:-This lever is used to record the contraction of heart.
The differences between this & other isotonic levers is that fulcrum lies at one
end beyond the point of attachment. It consist 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.
(iv) Universal lever (Brodies): It is a lever of versatile utility there is an adjustable
spring support which counters the pull on the writing arm. The spring helps in
bringing the pulled writing arm back to its original position. This lever is
mainly used for recording sudden repetative contraction of muscles/movements
of a part of the body e.g. contraction of a gastronemius muscle in response to
sciatic nerve stimulation.
4. ONCHOMETERS:-
These are used to study volume changes of oragans due to the effect of drug
(i.e. measure blood volume changes in a particular organ). These have to be
connected to Marey's tambour or piston recorder.
Principle: works on displacement of air.
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Types:-(1) Spleen on onchometer (2) Kidney onchometer

(3) Heart onchometers (4) Intestine onchometer
They are made of metal according to organ shape & are likewise called spleen
kidney etc.
When an organ relaxes in the oncometer then the piston of the piston recorder
moves up, where as it moves down when the organ contracts.
5. JACKSON'S ENTEROGRAPH-
It is the device used for recording the movements of an isolated segment of
intestine "in situ" This has been named after its discoverer. It consist of:-
a) hollow metallic tube about 15 cm in length and 15 cm in diameter.
b) hook welded at its lower end
c) a pulley fixed at the lower end.
One end of an isolated segment of intestine “in situ” is hooked at its lower end
and the other end is connected to a writing lever with there of a thread which
passes over the pulley.
Thus, the contraction of longitudinal muscles of this segment of intestine are
transmitted to the recording lever.
(Since the two ends of isolated segment of intestine are tied up in length wise
fashion, it records the responses of the longitudinal muscles of intestine without
any record of the contractions of the circular muscle.)
6. MAREY'S TAMBOUR:
o It is an apparatus used for recording delicate pressure changes in a gaseous column.
o It is particularly useful for recording the respiratory excursions in anaesthetized
animals.
o It consist of -
(a) metallic hollow tube (about 3-4 mm in diameter and about 10 mm in length
connected internally to a hollow disc/cup.)
(b) a tightly fitted soft rubber diaphragm covering the entire upper opening of disc/
cup.
(c) a writing device which moves with each up & down movement of the
diaphragm and marks it on the kymograph paper.
o The open end of the metallic tube is connected to the tracheal camula with the help
of polythene tubing.
17
o The pressure changes in the hollow passage of the system are transmitted by the
diaphragm to the writing lever whose magnification can be adjusted by changing
the position of the writing tip of the lever.
o Changes in the resp. rate and depth are easily reorded by Marey's tambour.
7. PISTON RECORDER:
o It is used to record the slightest blood volume changes in the organ easily.
o It consist of an internal ground glass tube in which an aluminum piston works very
smoothly through out its entire stroke.
o The piston is further attached anteriorly to the lever assembly.
o The lower end of the piston recorder is connected to the onchometer by means of an
air tight pressure tube.
8. MERCURY MANOMETER:
o Hg manometer, originally designed by Poissullic in 1828, was modified by Carl
ludwing in 1847 to allow graphic record to be obtained from a float on the Hg so
that progressive changes in the blood pressure could be studied.
o Due to inertia of Hg column, the assembly does not register accurately the rapid
changes of pressure in the artery with each heart beat which appear as
comparatively small fluctuation.
o These fluctuation become large when the heart beat is slow because the manometer
is more nearly capable of keeping up with the slow rate.
o Thus assembly gives us only a true and valuable record of the mean arterial
pressure.
o The Hg manometer consists of a glass U. tube (5 mm) bore with two vertical limbs
about 30 cm in height which are half filled with Hg.
o Since the Hg is displaced equally up in one limb and down in other, it is obvious
that any displacement as recorded must be multiplied by two to obtain the actual
pressure in terms of Hg column.
o To obviate this, a millimeter scale with doubled value is fitted, with the manometer
so that these can be read of directly in mm up to 250 mm Hg.
o On the surface of the Hg in one limb is a cylindrical float of vulcanite from which a
stiff fine wire rises bearings on its upper end a stylus (writing paint) which writes
on the traveling surface of smoked paper.
18
o The other limb of the manometer has a side tube which is connected through
inextensible (pressure or resistance) tube made up of thick rubber of polythene to an
arterial cannula.
o The upper end of this limb is also connected with a reservoir bottle containing some
anticoagulant fluid which can be pumped into this limb and through the
interconnecting tube to the arterial cannula.
9. ARTERIAL CANNULA:-
o It is a small glass apparatus used in animal experiments to cannulate an artery
(usually common carotid or femoral) for recording the blood pressure in
anesthetized animals e.g. dog, cat, rabbit & rats.
o It consists of hollow bulb connected to three arms, open into the cavity of bulb.
o The small globular arm is bevelled at its outer end to help in its insertion into the
artery. The size (length & thickness) of this arm depends on the size of the artery to
the cannulated.
o The larger thicker arm is used for connecting the cannula with the Hg manometer.
o The small thicker arm is used for flushing out and for removing the blood clot from
the cavity of the cannula if it occurs otherwise it is clamped with the help of punch
clip.
o During the experiment the entire cannula is filled up with a solution containing
sodium citrate or heparin as anticoagulant.
10. VENOUS CANNULA-
It is small hollow glass tube about 4-5 cm in length & 3-4 mm in diameter and
is used for cannulating a vein for I.V. administration of drugs/ fluids during
experiment on dog, cat, & rabbit etc.
 At one end there is small globular projection with bevelled outer end help its insertion
into the vein.
 The base of this projection is thinner to retain the tied thread in place & prevent
slippage of the cannula out of the vein.
 A piece of rubber tube is slipped on the other thicker end of the cannula and is
clamped with a punch clip to prevent back flow of blood.
 The cannula is filled up with saline after cannulating the vein.
19
11. TRACHEAL CANNULA-

It is a small metallic tubular device meant for cannulating trachea for
 Artificial respiration
 For recording of respiration in animals
They are of following types
1. Straight
2. Z- shaped
3. "Y" shaped
 "Straight" and "Z" shaped tracheal cannula are used whenever respiration is to be
recorded.
 One end of these cannula is inserted into lumen of trachea and the another end is
connected to Marey's tambour with the help of rubber tube.
 These cannula have adjustable slit, this is used to adjust the entry of air.
 "Y" shaped tracheal cannula is used whenever artificial respiration is to be given.
Stem of this "Y" cannula is inserted into the lumen of trachea out of the remaining
two arms one is connected to the outlet of the respiration pump and another one to
the inlet of the pump with the help of rubber tubing.
 Depending on the size of trachea, small or large tracheal cannula may be used in
animals like dog, cat, rabbit guinea pig & rat.
12 MURPHY'S DRIP-
 It is small glass hollow sealed apparatus used for assessing the rate of flow of a liquid
(blood or drug solution) in a transfusion system.
 It consist of a closed cylindrical body about 4cm in length and 1 cm in diameter and
is connected to two thinner tubes at its two ends.
 The upper input tube projects into the body cavity of the Murphy's drip. Such that the
rate of input of fluid can be easily observed.
 The upper end is connected to the bottle containing blood or perfusion fluid while the
lower end is connected to needle with the help of polythene tubing. This lower end is
used to drain out the entire content of the body of Murphy's drip.
 This instrument has been replaced by polythene sets with similar design and function
in disposable IV perfusion sets.
20
13 BULL DOG CLAMP-

 As the name implies is a small clamp meant for tightly compressing a hollow
tube/structure just like the jaws of bull dog.
 Usually employed for occluding an isolated artery/vein during arterial and venous
cannulation in animal experiments.
 It is made up of stainless steel consist of 2 sturdy prongs connected with each other
at the base in a crossed fashion such that the jaws of the clamp open up with this
crossed portion is pressed with fingers.
 The inner flat surface of each prong has serration for tight grip of the vessel and also
to minimize tissue damage to the vessel wall.
 For more precautionary measure these 'jaws' of the clamp may be covered with
rubber tubing to prevent tissue damage.
14. VON FREY'S HAIR ASTHESIOMETER-
It consists of hair preferably of horse or nylon hair which is adjustable to
increase or decrease the tension.
It is used to see touch, deep touch for touch sensatation in local anaesthesia
15. Syringes-
(i) Simple Glass Syringe
 It is also known as B&D syringe. B&D stand for Beckton & Dickinson, who
manufactured it.
 It is glass syringe with hollow floating piston.
 Available in 2cc,5cc,10cc,50cc & 100 cc capacity.
 Sterilized by autocalving.
(i) Needle- The B&D needle has a bevel, body & shoulder. The needle thickness
varies. The lower the number the thicker the needle.
 Sterilized by boiling for half an hour or by autoclaving.
 In the BD syringes the needle can be locked by applying a metallic lever lock to the
nozzle.
 These BD glass syringes are largely replaced by plastic disposable syringe and
needle.
21
(ii) Tuberculin Syringe

It was used initially for the tuberculin test (PDP or pure protein derivative test)
therefore the name tuberculin was given.
Now a days it is used for many other sensitivity test e.g. lepromin test.
It is 1 ml capacity syringe and divided into:
100 parts having further sub divisions i.e. minimum.01 ml drug can be gaien
50 equal further division 0.02 ml minimum drug can be given. or
16. RAT HOLDER-
 It is a metal instrument used in experiments where rat tail is used
 To screen the
(i) Analagesic activity of drugs,
(ii) Collection of blood from rat tail.
 In consist of hollow cylindrical tube.
 At one end there is small rounded door with a hole through which the tail of the rat
comes out.
The other end of the holder is closed and perforated which provides air for respiration.
22
MYDRIATIC & MIOTIC EFFECT OF DRUGS ON RABBIT'S EYE.
Object To study the mydriatic & miotic effect of topically applied drugs on
rabbit's eye.
Experimental Animal: Rabbit - (2.5 Kg in wt.)
Experimental Requirement; - Torch, Scale (6”small), Cotton, Hair aesthesiometer,
Scissors, Dropper, Rabbit holder (cage)
Drugs : Normal saline 0.9%
Pilocarpine 1-2%
Ephedrine 4%
Atropine 0.5-1%
Phenylepherine 5%
Xylocaine 0.5%
Principle
Local action of a large number of drugs in an eye can be achieved without systematic
effect by the application of drugs as eye drops or eye ointment. Most of these drugs
belong to antimicrobial, autonomic or local anaesthetic groups. The eye is supplied
both by sympathetic & parasympathetic nerves.
The iris has two types of muscles
1.Dilator pupillae (radial muscle)
2.Sphincter pupillae (circular muscle)
Circular muscle has parasympathetic supply while radial muscle has sympathetic
supply. The cilliary muscle is also supplied by parasympathetic nerves & when it
contracts, the cilliary body move inwards & forwards. Because of this the lens bulges
forwards & eye is accommodated for near vision. The opposite effect is produced by
the relaxation of cilliary muscles resulting in paralysis of accommodation (cycloplegia).
When the pupil dilates the iris folds back near the opening of the canal of schlemn &
the drainage of aquous humour is decreased thereby increasing intraocular pressure and
constriction of pupil by opposite action that will increase drainage & reduce intra
ocular tension.
Topically applied drugs can affect the eye by changing conjunctival congestion,
pupillary size, corneal sensitivity & intra ocular pressure. However the effect of drugs
on Pupillary size, Light Reflex (L.R.) & Corneal Reflex (C.R) can be studied easily.
23
1. Pupillary size- Can be measured by placing a transparent plastic scale in front of

the eye, as close as possible.
2. LR- Elicited by directing the light of a torch towards the pupil from back. The
positive response shows constriction of pupil.
3. Corneal Reflex- Sensitivity of cornea is tested by touching cornea gently with a
fine cotton wick/hair aesthesiometer from the side & not from the front of the
eye.Positive response show blinking of the eyelids.
Physostigmine is a reversible cholenestrase inhibitor which increases the endogenous

acetylcholine, this inturn stimulates the circular muscle of iris to produce pupillary
constriction without producing loss of light reflex & corneal reflex.
Ephedrine & Phenylephrine- Stimulate the radial muscle of the iris, this produces
dilatation of pupil without any effect on light reflex & corneal reflex.
Atropine- An antimuscarinic agent, block the effect of endogenously released. ACh in

the circular muscle of the iris. This also causes stimulation of ciliary muscle. This
produces mydriasis & spasm of accommodation leading to cycloplegia but without
producing loss of corneal reflex.
Ciliary muscle is present in the eye mainly supplied by parasympathetic nerves & weak
sympathetic supply. This is responsible for accommodation. Anticholinergic drugs eg.
atropine causes paralysis of ciliary muscle, tightening of suspensory ligaments, the lens
is flattened & focal length increases. Eye is fixed for far vision known as 'paralysis of
accomodation' or cycloplegia
EXPERIMENTAL SET UP (PROCEDURE)

One rabbit is used to study the action of one drug. Different rabbits are required to
study the action of various drugs. Their eye lashes are trimmed with scissors, as they
interfere with elicitation of corneal reflex/conjunctival reflex.
 Place a rabbit in the rabbit holder box. Keeping its head outside.
 Observe size of the pupil, LR & CR in both the eyes [examine the LR by holding
the torch in front of the eye moving light beam to & fro (from back to front),
examine the corneal reflex by touching side of the cornea with horse hair
aesthesiometer or cotton wick.
24
 One eye of the rabbit is used (RE) as 'control' for the experiment.
 Instill few drops of normal saline in right eye & atropine(Drug) in left eye (test eye).
 Record the pupillary size, LR & CR after 10 mins of drug instillation & tabulate
the observation
 Repeat the test with other drugs (pilocarpine, ephedrine etc)
OBSERVATION TABLE
Drugs Size of Pupil(mm) Light Reflex Corneal Reflex Remarks (inference)
Pilocarpine 2% RE LE RE LE RE LE Miotic response is seen
B A B A B A B A B A B A causing constriction of
pupil, no effect on LR/ CR.
could be a Parasympatho-
5 5 5 2 P P P P P P P P mimatic drug.
Atropine 1% 5 5 5 10 P P P Ab P P P p Mydriatic effect is seen i.e.
(dilatation of pupil) no
change in CR LR is lost. So
possibility of parasympa-
tholytic drug
Ephedrine 4% 5 5 10 P P P P P P P P P Mydriatic effect is seen. CR
and LR both remain
unaffected. Hence,
possibility of a
sympathomimetic drug.
B- Before drug instillation - P- Present

A- After the drug instillation - Ab- Absent
Definitions
Mydriatics: These are the drugs which causes dilation of the pupil.
Miotics: These are the drugs, which produce constriction of the pupil.
Cycloplegic: These are the drugs, which cause loss of the accommodation by
casing paralysis of the ciliary muscle.
Discussion (Effect of Drugs)

Pilocarpine : Is a parasympathomimetic drug. It acts directly. It causes contraction
(++) of circular muscle of the iris & leads to decrease in size of pupil (constriction of
pupil).
Physostigine (Eserine): Is indirectly acting drug it is anticholineterase agent. It
decreases the metabolism of ACh. Hence increase the concentration of ACh locally.
Thus, causes miosis. (same as parasympathomimetics).
25
Adrenaline /Ephedrine
These are sympathomimetic drugs they cause contraction of radial muscle (++)
(Ephedrine act indirectly by releasing Adr / NA & causes mydriasis. The light reflex &
corneal reflex are not affected by these drugs.
Atropine: It is a parasympatholytic drug. It causes paralysis of circular muscles by

blocking muscarinic receptors which release ACh. Thus, causing predomination of
sympthetic action. There is unopposed action on radial muscles this produces mydriasis
and there is loss of accomodation (cycloplegia) & light reflex, due to action on cilliary
ganglion.
Advantages of the Experiment

1. To study the effects of drugs on ANS (eye)
2. Also to screen the drugs for surface anaesthesia.
Clinical relevance
1. Miotic effect of physostigmine is useful in glaucoma as drainage of aquous humor
is facilitated resulting into decrease in I.O.P.
2. Short acting mydriatic effect of phenylepherine is used in fundoscopy, where
topical anticholinergic (mydiatrics) are avoided.
3. Topical effect of xylocaine is used to produce surface anaesthesia for minor local
surgery eg. Foreign body removal, symptomatic relief of corneal/ conjunctival
irritation.
4. Atropine is used for fundoscopy & to provide rest to the iris, e.g. in iritis
5. To counteract the mydriatic & cycloplegic effect miotics are used.
Miotic effect of phystostigmine is used in glaucoma as the drainage of aquous humor is

facilitated & decrease in I.O.P occurs
Phenylepherine is short acting and not causing cycloplegia. Hence, often preferred over
atropine and its substitutes specially in elderly.
26
MYDRIATIC AND MIOTICS (VIVA-VOCE QUESTIONS)

Q. 1 What are mydriatics? Name some mydriatic agents
Ans. Those drugs which cause dilatation of pupil, are called mydratics. The
commonly used mydriatics are
a. Atropine - Having slow and long lasting action.
b. Homatropine - less potent than atropine short duration of action
c. Cyclopentolate, tropicamide.
d. Sympathomimetics – ephedrine, adrenaline, phenylepherine.
Q. 2 Discuss the mechanism of mydriasis, what do you understand by “Active
mydrasis and Passive mydriasis”?
Ans. There are two types of muscles present in the iris
a) Dilator pupillae or radial muscle, is supplied by both and α and β receptors
innervated by sympathetic nerves.
b) Sphinctor pupillae or circular muscle, it is having muscarinic cholinergic
receptor innervated by parasympathetic nerves.
Topical instillation of sympathemimetic in the eye causes stimulation of radial muscle
of iris and causes mydriasis because there is constriction of muscle, it is called 'active
mydriasis' this is not associated with cycloplegia.
When anti cholinergic drugs, like atropine instilled locally in the eye it causes paralysis
of circular muscle of the iris and there is unopposed action of dilator pupillae or radial
muscles. This causes dilation of pupil this is called 'passive mydriasis' because it is
caused by paralysis of muscle. This is associated with cycloplegia.
Q. 3 What is cycloplegia?
Ans : Mydriasis caused by atropine is associated with paralysis of circular muscle. This
produces tightening of the suspensory ligament, resulting in flattening of lens
with a consequent increase in the focal length of the lens. Thus, the individual is
able to see things clearly only at a long distance due to fixing of lens for far
vision. He is unable to constrict the pupil due to paralysis of circular muscle for
viewing of near objects or in response to bright light (photophobia) this
phenomenon is known as loss of accomodation or cycloplegia.
27
Q.4 Name some drugs which produces mydriasis with cycloplegia?

Ans. Sympathomimetics, viz. adrenaline, phenylepherine, cyclopentolete (less cycloplegia).
Q. 5 Name mydriatic agent which preferred in children?
Ans. Atropine and homatropine.
Q.6 Name mydriate agent in elderly
Ans Phenylepherine, tropicamide, sympathomimetics.
Q. 7 Why atropine is not preferred in elderly?
Ans. Because of aging process elderly have shallow anterior chamber of eye. So, when
we use atropine it causes mydriasis with cycloplegia it may precipitate glaucoma.
Also it may cause systemic adverse effects, e.g retention of urine.
Q. 8 Why atropine substitue are preferred over atropine?
Ans. Mydriasis caused by atropine is long lasting. Also it causes cycloplegia. Some
times it may cause severe adverse effects if absorbs systemically specially, in
children. So, atropine substitutes are preferred over atropine.
Q. 9 Can cocaine is used for opthalmic purpose?
Ans. Cocaine is a local anaesthetic agent causes mydriasis due to  receptor action. It
is not used for this action purpose because it causes corneal necrosis. Also it is a
drug of abuse and leads to strong psychological dependence. It also stimulate
CNS Other systemic effects are seen like rise in BP, tachycardia, etc.
Q. 10 Enumerate the therapeutic uses of mydriatics ?
Ans (a) For the refraction and others ophthalmic examinations like retinoscopy fundoscopy.
(b) To break adhesion between iris and cornea or iris and lens, used alternatively
with miotics.
(c) In inflammatory conditions of eye to relieve pain.
C/I of mydriatics – Glaucoma
Q. 11 What are miotics? Name some miotic agents.
Ans. Those drugs which cause constriction of pupil are called miotics. Commonly used
miotics are :
Pilocarpine – occusert
Phystostigmine, neostingmine
Carbachol
- adrenergic antagonist.
Adrenergeric neuron blockers
28
Demcarium is the longest acting miotic but not used clinically because it
accelerate the development of cataract. Pilocarpene (occusert) is available now
as (long DDS).
Q. 12 What is the mechanism of action of miotics?
Ans. Topical instillation of parasympathomimetic drug in the eye stimulates circular
Muscle of the iris and causes miosis.
Q. 13 What is glaucoma? Name drugs used in glaucoma
Ans. Increase in I.O.T. is known as glaucoma (>19mm Hg). In Acute narrow angle.
Glaucoma.
1. Timolol – topical  - blocker
2. Miotics – pilocarpine
3. Hypertonic mannitol
4. Acetazolamide
In chronic angle glaucoma
1. - Blocker – Timolol
2. Miotics
3.  - adrenergic agonist Dipivefrine
4. Acetazolamide
Mechanism of Action of drugs used in glaucoma

Miotics – Increase the tone of cilliary muscle and sphinctor pupilae thus facilitate the
flow of aq. humour. But short acting so fluctuation in I.O.T is common.
–blockers–no fluctuation/ no change in size of pupil, no induced myopia, no headache
but causes systemic adverse effects e.g. bronchospasm; bradycardia, CHF specially, in
elderly.
 - adr agonist increase– aq outflow, increase aq formation e.g. Dipivefrine is a
prodrug of adrenaline, longer and consistent action used as adjuvant to miotics or 
blockers.
Betaxolol  selective antagonist, less effective and few adverse effects.
Q. 14. What are the advantages and disadvantages of  blockers used in

glaucoma?
Ans. Discussed in mechanism of action.
29
Q. 15 Name the drug which produces miosis by central action?

Ans: Morphine
Q. 16 Will morphine produce miosis when applied locally in the eye?
Ans. No, because morphine causes miosis due to its action on E.P nucleus of 3rd
cranial nerve which is its central action. Morphine does not have local miotic
action.
Q. 17 Enumerate therapeutic uses of miotics?
 Miotics are used in the treatment of glaucoma
 Alternatively with mydratics, they are used to prevent adhesions or to break
adhesion in iritis.
 To counteract the mydriatic effect of atropine and others.
30
SCREENING OF THE LOCAL ANAESTHETIC
Object: Screening of the local anaesthetic activity of drugs.

Methods of screening and evaluation of local anaesthetic activity of drugs.
Apparatus: Cotton hair-aesthisiometer, dropper, toothed forceps, pins, tuberculin,
syringe with needle, stop watch.
Animals: Rabbit, guinea pig, Rat, frog
Drugs: Lignocaine 2%; Normal saline 0.9%.
Drugs are often applied topically to produce loss of sensation locally. Thus local
anaesthetic activity of drugs can be utilized clinically by surface application or by
infiltration method. Several experimental methods are available to study the local
anaesthetic property of drugs.
The basic principle involved in these methods is to apply the drug locally on a sensitive
area and test for the sensation locally. Since corneal surface and skin are easily
approachable, local anaesthetic effect is often tested on these tissues. For infiltration
anaesthsia the drug solution may be infiltrated either into the subcutaneous tissue or in
close vicinity of a sensory nerve and the loss of sensation in the area of distribution of
the nerve is tested. For spinal anaesthesia the drug solution has to be injected into the
subarachnoid space to produce its effect on the cauda equina.
METHODS OF PRODUCING LOCAL ANAESTHESIAS
1. Surface anaesthesia – a) Corneal anaes in rabbits
2. Infilteration anaesthesia- b) Intsadermal wheal method in guinea pig.
3. Conduction or nerve block- c) Rat's tail pinch method
4. Spinal anaesthesia
(A) SURFACE ANAES
The surface anaes effect (topical anaes) is studied in rabbit's cornea. A rabbit is placed
in a rabbit box with its head protruding out. By gently touching the cornea with the tip
of a cotton swab (hair aesthisiometer), the corneal reflex is elicited to detect the
sensitiveness of cornea. The lower lid of one eye is pulled and few drops of the solution
of the test drug (2% xylocaine) is instilled into its pocket. The lids are then closed for
2min. The corneal sensitivity is then tested every five mins till it is fully recovered. The
other eye (RT eye) of the same rabbit serve as control for the comparison. In the RE
0.9% NS is instilled.
31
The presence of corneal reflex is graded as +, ++, +++ ……after every five minutes.
10min, 15min, 20min, 25min, and 30min and tabulated.
Drug 5min 10min 15min 20min 25min 30min

I CONTROL (RE) 1
II XYELOCAINE (LE) 1
Time of instillation of drug – x sec

On set of action (-ve blinking response) - x1 sec
Disappearance of anaesthesia (+ve blinking response) - x2 sec
Duration of anesthesia x2 – x1,
INFILTRATION ANEASTHESIA
The local anesthesia activity of a drug after infiltration into the S.C. tissue or
intradermally can be studied in guinea pig or rabbit. An area of skin about 4cm in
diameter on the back of the animal on either side of the vertebral column is shaved
carefully without damaging the epidermis. The solution of the test drug (0.5ml of 0.5%
xylocaine) is injected intradermally on one patch while other patch serves as a contol.
The pain reactions in response to prick with a sharp needle is recorded at interval of
five min each. For each set of observation 5 pricks are made in each spot at regular time
interval and progressive disappearence and reappearance to the sensation are recorded
and tabulated as follow.
Failure to squeak
Drug 5min 10min 15min 20min 25min 30min
C T C T C T C T C T C T
I CONTORL (RE) 1
II XYLOCAINE (LE) 1
Total no of prick stimulate are 25-30. The criteria of production of anaesthesia is failure
of the animal to squeak The no of times the animal failed to squeak is taken as the
anasthetic score.
32
NERVE BLOCK ANAESTHESIA

The effect of local anaesthetics on conduction of impulse along the peripheral nerves
can be studied in frogs. The brain and the upper 1/8 of the spinal cord of a frog is pithed
and the lower part of the ventral sac is opened exposing the right sciatic N Hold the
frog in a clamp and dip the foot of the frog in an N/10 solution of HCL. Note the time
taken by the frog to withdraw its foot ie reflex time which is an indication of impulse
transmission along the nerve. Rinse the foot of the frog in 0.1% saline after each
observation so that tissue damage due to contact of the acid with the skin of the foot is
minimized. Soak a small cotton plug in 1% xylocaine and carefully place it around the
sciatic nerve for 2-3 min. Repeat the test at regular interval and note the reaction time.
Blockade of nerve conduction after application of xylocaine is manifested by marked
increase in reaction time. The data obtained in the expt should be recorded in the table.
DRUG REACTION TIME

5min 10min 15min 20min 25min 30min
I CONTORL 1
II XYLOCAINE 1
SPINAL ANAESTHESIA
The local anaesthetic effect of the drugs on spinal cord is studied in dogs. A dog about
10kg in weight is taken. The animal is placed on the table in the lateral portion with
spine maximally flexed. A long hypodermic needle or a LP needle is inserted between
L3 and L4 Vertibrae in an horizontal direction till its tip pierce the spinal theca or
reaches sub arachnoid space indicated by the appearance of CSF through the needle.
Inject 2ml of 5% xylocaine within the space and remove the needle. Keep the head of
the animal above the level of body of the animal. After every 10 min, observe the
animal regarding tone of skeletal muscle and the sensations in the lower limbs of the
dog and record the results.
Parameters Control After Xylocaine

10min 20min 30min 40min
Tone of muscles
Sensation
33
CLINICAL RELEVANCE
Topical application of local anaesthetic is often practiced not only for producing local
anaesthesia for minor local surgical procedures but also for their local analgesic effect
in certain painful condition. Surface applications & infiltration in the tissue produces
anaesthetic effect at the site of application. Often the solution of adrenaline is mixed
with LA agent to retard the absorption & therefore increase the duration of the LA
agent. Infiltration of the anaesthetic agent around a nerve produces anaesthetic effect in
the area of the distribution of the nerve permitting more extensive surgical interference.
However, adequate precaution must be taken to avoid nerve damage.
The use of LA for spinal anaesthesia though extensively useful, but require more
caution while administrative. hyper boric soln. (heavier than CSF) of a local
anaesthetic, head end of the patient should be raised so that the solutions stay in the
lower most part of spinal theca without producing any resp. insufficiency. On the
contrary, while adminitring hypoboric solution (Lighter than CSF) of a L.A., the head
end of the patient should be lowered so that the LA solution stays only in lower part
(placed above the lower of heart) of the spinal theca.
Lastly precautions viz prevention of infection, hemorrhage, nerve damage, etc. must be
taken care of while adm. spinal anaes.
LOCAL ANAESTHESIA
LA drugs block nerve conduction, when applied locally to nerve tissue. They act by
producing stabilization of neuronal membrane thus preventing initiation & propagation
of action potential. Pain sensation is blocked first (rapidly) followed by other
modalities e.g. temp; pressure, touch. Non myelinated fibres are blocked quickly than
myclinated fibres. Fibres of small nerves blocked first than thick fibres. Nerves of the
A.N.S. are also blocked by L.A. adrenergic nerves blockage produce arteriolar
dilatation & hypotension. Cholinergic blockade may produce atony of rectum & urinary
bladder. When absorbed they produce adverse effects or C.N.S. symptoms.
- Local anaesthetic agents with high lipid solubility & water solubility produces
rapid onset of action. Also useful in surface anaes e.g. Amethocaine &
xyelocaine.
- LA like procaine with low lipid solubility are not useful as S.A.
- Some LA like xyelocaine & procaine produces membrane stabilizing effect on
myocarduim also & hence they are used in the treatment of cardiac arrhythmias.
34
- Adrenaline in the conc of 1:10,000 to 1 in 50,000 is mixed with local anaesthetic

to reduce systemic absorption, retard the rate of metabolism & prolong the
duration of action.
To study the LA action of drugs on lumber plexus / planus, of frog is a good example
for bioassay of drugs.
- Planus / plexus anaes. is one of the imp. experiment employed to screen &
estimate the action of the local anaes.
- Other methods include - guinea pig intradermal wheal method.
- Block anaes. in rabbit.
35
VIVA-VOCE QUESTIONS
Q. 1. Define the term local anaesthetic agent?
Ans. Local anaesthetics are drugs which upon topical application or local injection
cause reversible loss of sensory perception of pain in a restricted area of the
body. They block the generation & conduction of nerve impulse at all parts of
neurons.
Q. 2. What are the various methods of producing local anaesthesea mention one
example of the drug used for each type?
Ans. Various methods of producing LA are:
(i) Surface anaesthesia - Tetracaine, lignocaine, Benzocaine
(ii) Infiltration anaes. - Lignocaine, Bupivacaine
(iii) Conduction block - Lignocaine
(iv) Spinal anaesthesia - Lignocaine, bupivacaine, tetracaine
(v) Epidural anaesthesia - Lignocaine, bupivacaine
(vi) Intravenuous regional anaesthesia- Lignocaine
Q. 3 Mention three uses of xyelocauine as surface areasthetic ?
Ans. Eye- Tonometery,
Mouth & throat - stomatitis,
Anal canal and rectum- urethra cathetrization,
Fissure, painful pile urethra, for dilatations, catheterization.
Q.4 Mention three common side effects of spinal anaesthesia?
Ans. Headache, hypotension, resp. paralysis, cauda equina syndrome, septic
meningitis, etc.
Q.5 Name local anaesthetic used in cardiac arrhythmia?
Ans. Lignocaine is used in ventricular arrhythmia.
Q.6 How can you prolong the duration of action of LA ?
Ans. We can prolong the duration of action of LA by using it with varoconstrictor.
Most commonly used vasoconstictor is adrenaline.
Q.7 What is the order of loss of sensation by LA?
Ans. Autonomice fibres are more susceptible than somatic nerves, order of blockade
pain, temp., sense, touch, deep pressure. As for as for tongue- bitter taste is lost
first sweet, sour, salty taste, last of all.
Q. 8 Differences between GA & LA
36
Q. 9 What is the mechanism of LA?

Ans. They decrease the sodium permeability & having membrane stabilizing action.
Q.10 Most cardiotoxic LA?
Ans. Cocaine & bupivacaine
Q. 11 Local anesthetic which is having only surface anesthetic activity?
Ans. Cocaine
Q.12 LA does not have surface anaesthetic activity is?
Ans. Procaine
Q.13 Is Local anaesthesia drug action can be seen by other drugs? Name the
drug groups?
Ans. Anticholinegic, Antihistaminic, propranolol, quinine chlorpromazine.
Q.14 Which LA does not required adrenaline for prolongation of its action. ?
Ans. Bucricaine.
Q.15 Most potent & most toxic LA agent is?
Ans. Cocaine
Q.16 Name local anaesthetic agent causes Mydriasis?
Ans. Cocaine causes mydriasis by blocking the reuptake of adrenaline from the nerve
ending & hence increases the local conc of adrenaline (sympathomimetics)
which caused dilatation of pupil.
Q.17 Name drug which block Na+ channels?
Ans. (i) Quinine (ii) Cardiac glycosides (iii) Antiarrhythmic drug.
Q.18 Name LA which causes methameloglobinaemia?
Ans. Prilocaine (its metabolites)
37
EFFECT OF DRUGS ON FROG'S RECTUS ABDOMINIS MUSCLE
Object: To study the effect of drugs on Frog's Rectus Abdominis muscle.
Apparatus: Recording drum, kymograph, frog dissecting board, frog rectus bath,
simple straw lever, stand, X-blocks, tuberculin syringe, pipette, stop watch, various
dissecting instruments, plasticin, thread.
EXPERIMENTAL ANIMAL – FROG

Physiological solutions – Frog's ringer solution
Drugs Concentration Dose
Acetylcholine 1:10000 (100µg/ml) 0.1-0.2 ml
Physostigmine 1: 10000 (100µg/ml) 0.5-2.0ml
d- tubocurarine 1: 10000 (100µg/ml) 0.5-2.0 ml
Frog rectus abdominus muscle is a voluntary skeletal muscle producing slow

contraction in response to ACh. Isolated frog rectus preparation is a simple isolated
tissue preparation widely used for the study of nicotinic effect of ACh Since, frog
rectus is a skeletal muscle, nicotinic mechanism operates at NMJ. Cholinergic drugs
stimulate these nicotinic receptors and produce contraction of skeletal muscle. The
response of these drugs on nicotine receptors is blocked by neuromuscular blocking
drug (d-TC guanethidine, Sch).
Experimental SET UP
Dissection: A frog is pithed and laid out on the frog dissection board. The skin of the
anterior abdominal wall is cut by a midline incision which is extended laterally onto the
anteior aspects of the limbs. This exposes the flat whitish muscle of the anterior
abdominal wall from their pubic origin to their sternal insertion. Cuts are placed
separately on each rectus abdominus from the surrounding musculature and from its
fellow.
The two recti are removed and placed in Frog's Ringer solution in a shallow dish. They
are carefully cleaned and one of them is trimmed to the desired size and mounted in an
organ bath in Frog's Ringers solution at room temp aerated with oxygen. A very small
38
bath of 5ml capacity is sufficient but in this case solution of drugs must be so made that
when a dose is added, a change of more than 15% in bath fluid volume should not
occur. For recording purpose an isotonic lever with a sideway writing point is used
tangential to the smoked drum balanced for a tension of 2.5gm with an extra load of
1gm on the long arm. The later serves to bring back the lever to baseline since when the
rectus has contracted it does not relax rapidly even on washing out of the drug.
The muscle is relaxed for 30-45 min by applying load with the help of plasticin (as
mention above) on the long arm of the lever that is equidistant form fulcrum after
loading new, the lever is balanced.
EXPERIMENTAL PROCEDURE
2. The base line is taken for approximately 2-3 cm at the lower one third of the drum.
Switch off the drum.
3. Start the drum and add 0.1 ml of ACh then record the effect of the drug for 90sec.
Stop the drum and wash the tissue with Frog's ringer solution 2-3 times. There must
be atleast 30 sec to 60 sec, gap between each washing (total pd 6-7 min)
4. After 5 min repeat the same cycle (add ACh). Two successive tracing having the
same height of contraction with same drugs in same concentration of the drugs for
the same period (90sec) obtained, these tracings are known as 'control' tracings.
 Give the rest pd of 5min.
 Add physostigmine (0.3 to5ml), wait for 5 min.
 Start the drum and add 0.1 ml of ACh in the same organ bath in the presence of
physostigmine.
 Record the tracing for 90sec
 Stop the drum and wash the tissue in the same manner as mentioned earlier.
 Again take two control tracing with ACh.
 Now, add 0.5 ml of dTC and wait for 5 min.
 Start the drum and add 0.1 ml and ACh in presence of d-TC.
 Record tracing for 90 sec. Stop the drum and wash the tissue 2-3 times
 Again take two control tracing with ACh.
39
PRECAUTIONS
1. The level of ringer solution in the organ bath should remain constant throughout the
expt. by marking the level with elastic ring/marker.
2. The thread of the rectus muscle should not come in contact with the wall of the
rectus bath
3. A steady and constant stream of air/O2 should be passed in the bath.
4. 6-7 min cycle should be followed rigidly
5. Speed of the drum should be slow and the lever should be placed tangential to the
drum.
6. The no. of washing and the interval between them should be kept constant.
7. If the desired response is not obtained with 0.1 ml of ACh, then the dose should be
increased.
OBSERVATION TABLE
S.No. DRUGS DOSE EFFECT INFERENCE (Remark)
1 ACh 0.1 ml Contraction of Nicotinic action at NMJ
muscle
2 ACh + physostigmine 0.1ml+0.5ml Potentiatioin of Because of anti cholinesterase
response action of physostigmine it
decrease metabolism of increase
Ach at NMJ hence, increase in
response
3 ACh + d-TC 0.1ml+0.5ml Inhibition or blocade d-TC is having SMR action so
blockage of response there is positive response of ACh
(Reversible action).
4 ACh 0.1ml Contraction of Nictonic action reappears. There
muscle is blockade action of d-TC and not
the muscle is fatigued.
DISCUSSION
Nicotine receptors are present in skeletal muscle, atropine does not produce any direct
effect in this preparation. ACh produces contraction of the muscle through the
stimulation of nicotinie receptors. We take response/contraction for 90 sec only because
to get submaximal response (occupation of receptors) so that we can observe the
potentiation of action with cholinomimetic/ anti cholinergic drugs, when used with
ACh.
Physostigmine is a reversible antiche agent, it prevents the metabolism of ACh by

inhibiting the enzyme ChE. Thus, when physostigmine present, more ACh is available
for the effect and the height of contraction of ACh is increased. This effect of
plysostigmine is known as “potentiation.”
40
d-TC is nicotine receptor blocking agent. In presence of d-TC, the response to ACh is
reduced. This effect of d-TC is known as “Inhibition”.
We take two control tracing after d-TC to observe that the inhibition of response is due
to the effect of d-TC and not due to muscle fatigue.
Nicotinie receptors are present at :
(a) NMJ (b) Autonomic ganglias.
The nicotinic receptors of both the sites are of different nature. ACh can excute both
these receptors but d-TC cannot block both these receptors.
Drugs like DMPP (dimethyl phenyl piperazinium) stimulate, specially the nicotinic
receptors of autonomic ganglia; whereas the durgs like PTMA (Phenyl trimethyl
ammonium) stimulate specially the nicotine receptors of skeletal muscles. Drugs like
tetraethylammonium and hexamethonium block the receptors of autonomic ganglia and
termed as ganglionic blocking agents. The drugs like gallamine, d-TC, decamethonium
blocks the receptors of skeletal muscle and are known as skeletal muscle relaxants.
Clinical relevance
Physostigmine is reversible anti chE having both central as well as peripheral action so
that it can be used in atropine belladona poisoning. Although though it also increases
muscle tone by accumulating ACh at NMJ but it cannot be used / preferred in the
treatment of myasthenia gravis. In this condition neostigmine is preferred because it is
devoid of central adverse effects of physostigmine and also it has some cholinomimetic
action (partial agonist).
d-TC is SMR competitive antagonist to ACh at NMJ. It competes with the ACh for the
nicotinie receptors at motor end plate. After dTC the response of the exogenous ACh is
decreased because it fails to occupy the sufficient numbers of receptors to cause
stimulation and contraction. We can obtain response with large doses of ACh (effect
can be reversed by neostigmine). This relaxant effect of d-TC and other SMR is used to
relax the muscles during surgery.
41
FROG’S RECTUS ABDOMINIS MUSCLE PREPARATION

(VIVA-QUESTIONS)
Q. 1 What type of muscle rectus abdominis is?

Ans. It is a skeletal muscle having cholinergic nicotinie receptors.
Q.2 What are the different sites of nicotinic receptors?
Ans.Various sites of nicotine actions are NMJ, autonomic ganglia and adrenal medulla.
Q. 3 Why we choose rectus muscle?
Ans Many reasons are there :
 It is a ribbon like muscle and fibres are monopinnette, so easy to record better
contractions.
 It is a slow contracting muscle, so it is very convenient to observe its contractile
response.
 It is easily available or approachable.
Q. 4 What is the purpose of Airaction?
Ans. * For proper mixing of drugs. * To supply O2 to the tissue.
Q. 5 Why temperature is not maintained?
Ans. Because frog is cold blooded amphibian, can live on land and water and easily
acclamatized or resistant to change in temperature so temperature regulation is not
required.
Q. 6 Which type of action ACh exhibit on rectus?
Ans. ACh causes contraction of skeletal muscle by acting or to change in temperature
or stimulating nicotinie receptors.
Q. 7 What is a control tracing?
Ans. 'Control' is defined as the pharamacological response of drug obtained after
administration of the same dose, under same condition. In other words these are
two alike successive responses of the same magnitude by administering the same
dose of the drug under same conditions.
Q. 8 Why do we record control tracing at the commence of experiment?
Ans. It is done to confirm that with a fixed dose of ACh for fix duration of time, muscle
contrats to the same extent i.e. to show muscle / tissue stability so that, we can
compare the effects of other drugs.
Q. 9 Why is the contraction recorded for 90sec only?
Ans. We are recording the submaximal response of the muscle and not the maximum
response so that, we can see or compare the effect of drugs, which are potentiating
the action of ACh.
Q. 10 Why we apply additional weight on the long arm of lever?
42
Ans. Because of trauma during dissection, the muscle goes into a state of spasm.
Additional weight of (0.5-1gm) is applied so that, the muscle relaxed properly
and regain its original length so better conctactile responses are recorded.
Q.11 Name the drug which are potentiating the action of ACh?
Ans. Drugs potentiating the action of ACh are antichE Two groups of antichE drugs:
(a) Reversible – eg neostimine and physiostigmine.
(b) Irreversible – organophosphanus compound.
Q. 12 Which is most suitable lever for the expt?
Ans. Gimbal liver is the best but we use the simple straw lever because it is easily
available.
Q. 13 Which is the most sensitive tissue for ACh bioassy?
Ans. Leech dorsalis muscle.
Q. 14 Enumerate the therapeutic uses of physostigmine and neostigmine
Ans. a) Physostigmine 1. atropine poisoning 2. Glaucoma
b) Neostigmine 1. Myasthenia gravis 2. Post operative paralyitc ileus
3. Urinary retention (Post Operate) 4. Cobra bite.
Q.15 Why neostigmine is preferred over physostigmine in the treatment of
myasthenia gravis?
Ans. Neostigmine is a potent drug and having dual action (act as partial agonist) Also
it doesn’t cross BBB so, No central action or adverse effects occur and having
peripheral action only.
Q. 16 Name the drug used in atropine poisoning?
Ans. Physostigmine (0.5-2mg) IV, repeatedly used.
Q. 17 Why ACh is not used clinically?
Ans. a) Because ACh metabolized rapidly by plasma pseudo cholinesterase and
true ChE at the site of action (NMJ).
(b) Also non selectivity of action
(c) Having various systemic adverse effects.
Q.18 What is the antidote of different types of SMRs?
Ans. i) d-TC – Increases the concentration of ACh (agonist) use
anticholinestrase.
ii) Succinyl choline – blood transfusion is the only treatment because
succnylcholine is metabolized by psedochE only, which is present in
plasma. Their is no specific antidote.
Q.19 Name various drugs producing neuromuscular blockade? Give three
therapentic uses of peripherally acting SMRs?
Ans. (a) Directly acting – Dantrolene, quinine.
(b) Competitive blockade – Gallamine, dTC, pancuronium, etc.
(c) Persistent depolarization- succinylcholine.
43
Uses of PSMRs :
1. Used as adjuvant to GA.
2. During ECT, convulsion and trauma are avoided by using muscle relaxant.
3. SMR also used in severe cases of tetanus and status epilepticus which are not
responding to diazepam.
Q.20 Can you name any tissue possessing both skeletal muscle and smooth
muscles?
Ans. Get of trench fish having outer layer of skeletal muscle and inner layer of
smooth muscles.
Q. 21 Give important uses of centrally acting SMR?
Ans. (1) Acute muscle spasm (2) Torticolis, neuralgia (3) Anxiety and tremors
(4) Spastic neurological diseases (5) Tetanus (6) During ECT.
Q. 22 By seeing the tracing can you predict whether the unknown drug is
physostigmine or neostigmine?
Ans. Yes, because neostigmi has dual action so, it cause only one response with
presence of twitching or fasciculation, while the response of physostigmine is
smooth.
Q. 23 How will you treat post operative atonic bladder?
Ans. Neostigmine is drug of choice 0.5 to1mg SC.
Q. 24 Why dTC is not used now a-days?
Ans. Because it releases histamine which causes wide spread adverse drug effects
and at the same time better drugs are available i.e. never SMRs.
Q.25 How will you differentiate action of dTC and Sch?
Ans. a) dTC 1. the action of d-TC can be reduced by increasing the dose of ACh.
2. Its action is slow in onset, long duration of action and cause
histamine realse.
b) Sch 1. It waves off its own, since it is metabolize by pseudo ChE
2. Action is rapid and short duration of action gradual.
Q.26 What is the purpose of this experiment?
Ans. To know the action of ACh on skeletal muscle and to know the action of
various drugs which have potentiating or blocking action at various sites so that
we can use them accordingly.
Q. 27 Why controls are recorded at the end of experiment ?
Ans. To show that decrease in response after dTC is due to relaxant (inhibiting) action
of dTC and not due to the muscle fatigue.
44
PERFUSED HEART OF FROG
Object:- To study the effects of drugs on perfused heart of frog.
Setting up of the experiment:- A big sized frog is taken. It is stopped and pithed and
is placed on a frog board on its dorsal surface. A mid line incision is given and the skin
over the anterior abdominal wall is removed. Heart is exposed by cutting the bones of
the girdles with the help of a bone cutter. Heart with its great vessels is seperated from
the connective tissues and pericardium is removed. Rt. branch of aorta is ligated with
the help of thread, Lt branch is cut and opened for perfusion fluid to come out. A small
thread is passed behind the inferior vena cava then a small cut is given over the inferior
venacava and a venous cannula is inserted through this cut into the luman of vena cava.
The other end of cannula is connected through a rubber tubing to a bottle containing
perfusion fluid. The rate of perfusion is kept 30-04 drops/min.
It is adjusted with the help of muphy's drip. A pin hook is passed through the apex of
verticle and connected to a brodies heart liver, which writes on a smoked drum.
Drug under study:-

Drug Concenrtation dose
1. Adrenalin (1:100,000 solution) 0.05 ml
2. KCl (1% solution) 0.05 ml
3. CaCl2; (1% solution) 0.05 ml
4. ACh (1:100,000 solution) 0.05 ml
5. Atropine (1:10,000 solution) 0.05 ml
Procedures and recording of observation :- First of all a column of normal tracing is

recorded for at least two min. Drug to be tested is injected in the rubber tubing close
to the perfusion cannula and observe the effect on force of contraction and heart rate.
After testing each drug wait, till the heart returns to normal and record a column of
normal tracing before and after testing each drug.
45
Observation
Drug and Dose Effect of Drugs Infeunce
Rate Force of
contration
Adrenaline Increase Increase Adr has ionotropic + chronotropic +
dromotropic action (B- receptors )
Kcl Decrease decrease Direct depresant action/
Cacl2 Decrease Increase Force of contraction increased with bradycardia
(Has ionotropic action only)
ACh Decrease Decrease Has a inhibitory action on heart (Muscarinic
action)
inject Atropine no effect No effect Atropine has blocked the muscarinic effect of
After 2 min inject ACh.
same amount of
ACh
Difference:
ACh KCl
1 Inhibitory effect on the heart is blocked by Effect not affected by Atropine
Atropine
Adr CaCl2
1. Both systdic and diastalic component are only systolic component is seen.
seen
2. Heart increases Brady cardia
3. Beta - blocker abolishes the effect B blocker does not affect the effect
46
ISOLATED RABBIT ILEUM PREPARATION
Object : To study the effects of various drugs on isolated rabbit ileum preparation.
Apparatus : Dales organ bath, tuberculin syringe, thermometer, stop watch dissecting
instruments & beaker, pipettes etc.
Animals Used : rabbits
Drugs Drug Conc Dose
1. Ach. 1:10,000 0.1-0.3 ml.
2. Adrenaline 1:10,000 0.1-0.3 ml.
3. Bacl2 2% 0.1-0.3 ml.
4. Atropine 1:10,000 0.1-0.3 ml.
Parameters to be studied :
1. Amplitude : It is recorded as height of contraction.
2. Tone : Ability of tissue to maintain its size and shape. It is the partial state of
contraction at rest.
Procedure : Divided into three steps :-
1. Prep. of organ bath.
2. Fixing of ileum'
3. Recording of observations.
1. PREPARAT OF ORGAN BATH :
The outer chamber of organ bath is cleared and filled with warm water and
temperature is maintained at 37°C throughout the experiment. The inner tissue
bath is filled with Tyrode sol. The solution is aerated by passing the stream of
air. The frontal pointing lever is fixed on metal rod of organ bath.
2. Fixing of ileum:
A healthy rabbit is kept fasting for 24 hours. Then it is stunned by blow on the
head & allowed to bleed by cutting throat. Abdomen is opened and ileum is
identified, feces removed and placed in Petri dish containing tyrode solution.
The intestine is cleaned by passing tyrode solution in lumen with help of
pipette. Gently cut the pieces (2.5 cm) of lumen. The ends of piece of intestine
are tied with thread and is mounted in Dale's organ bath by tying its one end to
curved and of O2 delivery tube & the other and is tied to the end of frontal
writing point lever. Inner bath is filed with tyrode solution which is aerated by
O2 delivery and the temperature is maintained at 37°C help of warm water.
47
*the ileum of rabbit is selected because this portion gives maximum contraction.
(3) Recording of observation

In intestines two types of mussels are found :-
1. Circular
2. Longitudinal
Normally intestines have three types of movement :-
1. Pendular
2. Segmental
3. Peristalsis
4. Antiperistalsis
In this experiment only pendular movement are recorded. Stablize the tissue for 20-30
minutes and than lever should be balanced. Bring writing point of lever closed to
smoked surface of drum. Drum is started at 1 rev/64 minutes. A small control tracing is
recorded. Then 0.1ml of drug is added to solution of inner chamber of Dale's organ
bath. Effect of drug is noted for 30 sec. and after recording drum is stopped. & tissue is
given wash 3-4 times by tyrode solution then it is allowed to recover for 3-4 minutes.
Start drum again & take control tracing. Then add next drug & observe its effect.
Modification of effect of ACh & BaCl2 by atropine is recorded by prior atropinizing the
tissue.
Two types of drugs acting on intenstine are studied :-
1. Spasmogenic drugs which increase spasm of intestine by increasing its tone &
amplitude. Types of drugs are -
a. indirectly acting drugs - these act through receptor e.g.-ACh, histamine, 5
HT.
b. Directly acting drugs - e.g. BaCl2, morphine & extract of post pit. gland.
2. Spasmolytic drugs - which decreases spasm of intestine by decreasing tone &
amplitude. Types are-
a. Indirectly acting e.g. adrenaline, atropin.
b. Directly acting e.g. papaverine, nitrates.
Significance :
1. To know whether drug is spasmogenic or spasmolytic
2. To differentiate between directly or indirectly acting drugs.
48
Clinical Importance
1. Spasmogenic drugs are used in paralytic ileus & retention of urine.
2. Spasmolytic drugs are used in various colics and dysmenorrhoea.
Precautions :
1. Tissue should be held gently, not be over stretched.
2. Constant 02 / Air supply should be there.
3. Temp. of bath should be maintained at 370 C.
4. Speed of drum should not be changed during experiment.
5. Pipette should be washed before adding drug.
6. Lever should be balanced before starting experiment.
7. Next drug should be added only when normal tracing is recorded.
Observation Table :
S.No. Drugs Tone Amplitude Inference
1. ACh increases decreases spasmogenic drug
2. Adrenaline decreases decreases spasmolytic drug
3. BaCl2 increases little or no spasmogenic drug
change
4. Atropine decreases decreases spasmolytic drug
5. Atropine + ACh no response seen no response Spasmogenic action of
seen ACh is blocked by
atropine by blocking
muscarinic receptors.
6. Atropine + BaCl2 increases little or no Spasmogenic action is
change not blocked as it is
directly acting.
49
DISCUSSION OF RABBIT ILEUM PREPARATION
Q. 1 What is the aim of experiment on rabbit's ileum ?

Ans To see the various effects of different drugs on the isolated rabbit ileum i.e. we
record the contraction known as spasmogenic response and relaxation is
spasmolytic response and their use in different conditions. e.g. spasmogenic -
paralytic ileus, spasmolytic - in different types of colics.
Q. 2. What is the nature of this muscle & write different types of intestinal
movements. ?
Ans. It is a smooth muscle and 4 types of intestinal movements are recorded.
a) Segmental - due to circular muscle contraction.
b) Peristaltic - Helping mixing and propagation of food.
c) Antiperistaltic - Helping mixing and propagation of food.
d) Pendular - are controlled by longitudinal muscles and are
important because they produce mixing of food and also help in
adjustment of intestine over abdomen. In this experiment pendular
movements are recorded.
Q.3. Why ileum is selected ?

Ans. The advantage of selecting ileum are :
- Easily available.
- Maximum contraction
- By simply sacrifying one animal we set up 20-40 experimental setup at a time.
Q.4 Why frontal point writing lever used. ?

Ans. - Because it is very light as it is made up of aluminium.
- Write smoothly
- It magnifies the contraction in appropriate manner.
50
Q.5 Which are the various drugs used as spasmogens ?

Ans. Categories :
i) Which act through receptors like cholinergic, serotonergic, etc. their
effects are blocked by their blockers.
ii) Directly acting drugs are - BaCl2
Oxytocin
Vasopressin
Angiotensin
Bradykinin
Substance P.
Cromium, magnesium
Q. 6 In which clinical condition we use spasmogenic drugs ?

Ans. Paralytic ileus - Neostigmine. In retention of urine - carbachol, bethanechol.
Q.7 Give e.g. of drugs which are smooth muscle relaxants ?

Ans. 1. Adrenaline Nicotine Amphetamine - Acts on alfa & beta Receptors
2. beta2 Stimulants - Isoxsuprine - threatened abortion, preterm labour.
3. Muscarinic blockers - atropine.
4. Directly acting - Aminophylline, papaverine, various nitrates.
Q.8 Name the PSS used in this experiment ?

Ans. Tyrode solution.
Q.9 Why temperature is maintained at 370 C.?

Ans. Because rabbit is warm blood animal and its body temp is about 370 C.
Q. 10 Name commonly used antimotility drug ?

Ans. Dicyclomine atropine, paparverine, pirenzepine.
Q. 11 Causes of spontaneous movements of intestine even when all nerves are cut. ?
Ans. Due to presence of Auerbach's plaxus these muscles have inherent contractility.
51
Q. 12 How will you identify 2 responses of two different types of spasmogenic drug.?
Ans. We would atropinize the tissue and in the presence of atropine, we give
spasmogenic drug (un known) . Before adding the "Spasmogenic drug" allow
atropine to come in contact with tissue for 5 min, without washing. Add the
drug (A) and see the response, no contraction that means drug may be
cholinergic, since at atropine is muscarinic blocker blocks the action of
cholinergic like drugs. Add unknown drug (B) in presence of atropine shows
contraction (increase tone & amplitude). The amplitude increases in ladder
pattern. It may be a directly acting drug.
Q.13. How will you confirm that the drug is smooth muscle relaxants ?
Ans. For a known against for L+B blocker Ach- Atropine.
Q.14 Write difference Spasmogenics & Spasmolytics
Spasmogenics Spasmolytics
A) Drugs acting through receptors :
1. ACh- Cholinergic receptor and their agonists. a). Adrenaline - via both alfa & beta receptors.
2. Histamine- Via histaminergic receptors b). Amphetamine
3. 5HT or serotonin via serotonergic receptors. c). Nicotine at high doses.
4. Nicotine in low dose through nicotinic d). Atropine by blocking muscarinic receptors.
receptors but at high doses it blocks its own
receptors.
5. Morphine via opium receptors e) B2 receptor agonist.
B. Drug acting directly :

a) BaCl2 a). Aminophyllin
b) Post pituitary extract or oxytocin. b). Papaverin
c) Vasopressin, substance - P, Bradykinin c). Nitrates & Nitrites
d) Chloroform
Uses : Uses :
Paralytic ilivs Dysmenorrhora
Retention of urine etc. Spastic constipation.
renal and brilliary colics etc.
52
SCREENING METHODS OF ANALGESICS

Object: Screening methods of Analgesics.
Introduction: Analgesics are those drugs, which relieve pain as a symptom without
causing loss of consiousness. The analgesics can be classified as :-
1. Narcotic Analgesics : Are those analgesics, while relieving pain, also cause
marked depression of C.N.S. e.g. morphine and pethidine.
2. Non-Narcotic Analgesics: While relieving pain, those do not produce
depression of C.N.S. e.g. Aspirin.
Screening of Analgesics :
Most of the reflex activity initiated by a potentially noxious stimulus is of an ecape
nature. The tail flick, skin twitching and limb-flexion are all movement which would
tend to remove the animal or the affected part away from the stimulus. Such reflexes
have formed the basis for determining the pain threshhold in animals and analgesic
drugs are screened by testing their ability to raise this threshhold.
Methods : Following are the methods of screening of analgesics:-
I. Thermal methods :
a). Hot wire method: The instrument used for this purpose is known as
analgesiometer. It is fitted with a nichrome wire which can be heated to a
required temperature and it can be maintained with the help of a regulator
(usually electric current of 6 volts is passed). A jacket surrounds the nichrome
wire, which helps the platform cool. The animal (Rat) is kept inside the rat
holder, so that the tail projects out. A portion of the tail, 3 cms. away from the
tip is placed over the platform and the reaction time is noted (appearance of a
characterstric tail flick). Control group of animals are compared with the drug-
treated ones. Delayed reaction time is suggestive of analgesic activity.
b). Hot Plate Method: In this method a copper or zinc plate is treated of 550 C.
and this temperature is kept constant. The experimental animal (Rat or Mouse)
is kept over the plate and observed carefully for the signs of discomfort.
Reaction time is noted before and after the drug treatment.
c). Radiant Heat Method: Light of 6-8 volts, kept at a distance of 6" is thrown
over the tip of rat's tail. Pain induced will manifest in form of sudden
functioning of the tail. This procedure is done before and after the drug
treatment. Increase in reaction time will be suggestion of analgesic activity of
the drug.
53
II. Mechanical Methods :

a). Mechanical Compression Method : A constant pressure of gas from a
cylinder is used to drive a plunger on the rat tail which is fixed.
An artery forceps or bull dog clamp may be applied to the tail of the rat.
Untreated animals try to remove the clamp or foceps. After an
appropriate dose of analgesic, animal dis-regards the clip.
III. Electrical Method: In this a mouse cage is used floor of which is mode of
metal strips, receiving an electric current of known voltage.
A mouse is kept in this cage and the animal electrical current is determined.
Which causes the animal to cry.
The test drug is then injected. A drug having analgesic property will raise
the pain threshold and higher voltage of current will be required to induce
pain.
IV. Chemical Method: I.P. injection of aqueous solution of phenylquinone in
mice elicits a response which is antagonized by weak analgesics (Non -
narcotic analgescis).
V. Methods used in Men :
a). Walf -Hardies Method: Light from a bulb is focused with the help of a
convex lens on a particular part of fore head. Person feels pain on that
particular point. After administration of analgesics, light of more
intensity will be required to induce pain.
b). Electrical Stimulation of pulp of the tooth.
c). Nail bed pressure method.
54
SCREENING OF "ANTI-CONVULSANTS"
Object : Screening of "Anti-Convulsants".

Anti-Convulsants :
These are the drugs which are capable of controlling convulsions. In medical
practice anti-convultsnts are used to control convulsions occurring in epilepsy,
tetanus, eclampsia and posioning with convulsants. The greatest use is in the
treatment of epilepsy. According to neurological theory "epilepsy is a self limiting,
paroxysmal cerebral dysrhythmia in which there is excessive E.E.G. discharges,
tonic and clonic convulsions and some time loss of consciousness. According to
seizures pattern epilepsy is categorized as grandmal, petitmal, psychomotor.
Most of the experimental methods for directing anticonvulsant acitivity involve the
artificial induction of convlusions and their inhibition by organic compounds.
Screening methods:
1. Physical Methods -
A. Electro shock method.
B. Sound induced seizures.
2. Chemical Methods
A. Metrogol (80 mg./kg. s.c.) induced conclusions.
B. Picrotoxin test (2 mg./kg. s.c.)
C. Strychnine test (0.3 mg./kg. s.c.)
A. Electro Shock Method:
Expt. animals are rats, animals are divided in two groups. First group serves as
control and is not given any drug and the other group is treated with the test drug.
Maximal Electro Shock seizures are induced with a stimulus of 0.2 sec. Duration
and a current of 120 m. amps. Firstly electro shock is given to the animals of
control group.
Animals exhibit a seizures pattern. The tonic flexor component of the hind limbs is
seen first of all, following this is the tonic extensor component, the phase of
intermittent, whole body clonus is seen in the last. Now the electro shock of same
intensity is given to the animals of drug treated group. Absence or diminisition in
the intensity of convulsions indicate that the drug has protected the animals from
the conculsions and has got anti-consvulsant activity.
55
Sound induced seizures in rats :

The time used to induce seizures was a 60 sec. exposure to a door bell ringing with
a intensity of 90 decibles in a sound proof box. The seizures evoked were a rapid
and wild running about and then tonic and clonic convulsions. Animals treated with
anticonvulsant drugs do not respond to this stimulus.
Metrozol induced convulsions: (Pentylene tetrazol)

Rats are divided in two groups, One group is not given test drug and serves as
control and the animals of another group are given the test drug. After 30 min. of
administration of drug, animals of both the groups are given metrozol (80 mg. / kg
S.C.). Animals of control group will show convulsions. If the animals of the group
treated with test durg are protected from the metrozol induced convulsions then this
will be suggested of anticonvulsant acuity of test drug.
In place of metrozol, strychinine (0.3 mg. / kg S.C.) and picrotoxin (2 mg. / kg S.C.)
can also be used to give chemo stick.
Significance of this experiment:

By these methods anticonvulsant activity of a new drug can be found out and these
drugs may prove of value in the treatment of various convulsive disorders. Drugs
protecting animals from electroshock convulsions may prove effective in grandmal
and drugs protecting animals from chemoshock may prove of value in petitmal
epilepsy and produces very little depression of vital medullary centers.
56
EFFECT OF VARIOUS DRUGS ON DOG'S BLOOD PRESSURE

Object: To study the effect of various drugs on dog's blood pressure.
Apparatus: Research Kymograph, operation table, U shaped mercury manometer
tuberculin syringe. Burette arterial venous & tracheal cannula, connecting rubber
tubing dissecting instruments catton & thread.
Drugs & solutions
Sodium Citrate : 8.5% (Anticoagulant)
Heparin : 500 I.U. (Anticoagulant)
Normal Saline : 0.9 % NaCl
Morphine : 1µg 1Kg.
Urethane : 1.5 gm/Kg.
Adrenaline : 100µg/ml
Acetylcholine : 100µg/ml
Histamine : 100µg/ml
Noradrenaline : 100µg/ml
Isoprenaline : 100µg/ml
Tolazoline : 1 mg/ml
Atropine : 1 mg/ml
Propranolol : 1µg/ml
mepyramine : 1 mg/ml
Procedure: A healthy adult dog is weighed and anesthetized with morphine (1mg /
1kg) + urethane (1.5gm/kg). The animal is led dorsally on the operation table by tying
the limbs to the four corners of the operation table. After that femoral vein is exposed
in the thigh region and venous cannula is inserted in the vein. Venous cannula is
connected to a burette with the help of rubber tubing which contains normal saline. The
route used for injecting the drug is I.V. After that a midline incision is given in the
neck skin and fascia are reflected, muscles are separated and and trachea is exposed.
After exposing trachea search for the carotid artery, lying in the deeper tissue on the
side of trachea. An arterial cannula (heparinized) is inserted in the carotid artery and
this cannula is connected to a mercury manometer with the help of pressure rubber
tubing. This set is used for recording of B.P. A partial transverse cut is given to the
trachea and a Y shaped tracheal cannula is inserted in the lumen of trachea and
connected to a respiratory pump.
Observation:
Animal: Dog, Sex Male, Weight -10 Kg.
Hear rate / Min. Rate of Respiration /min
57
Blood pressure:
Aneasthesia used: morphine + urethane (1.5gm1kg)
Effect of various drugs on dog blood pressure :
S. No. Drugs & Dose Blood Pressure Heart Rate Remarks
1. Adrenaline 1st ↑ then (biphasic ↑↑ Sympathomimetic drug
(2g/ Kg.) response)
2. Nor adrenaline- ↑ Sympathomimetic drug
(3-5 g / kg.)
3. Isoprenaline ↓↓ ↑ Sympathomimetic drug
(2g / kg.)
4. Acetylcholine ↓ Bradycardia Cholinergic drug
(2g / kg.)
5. Histamine ↓ ↑ Naturally occuring auto
2-3 g / kg. coid.
6. Ach. (2g / kg.) + No effect of Ach i.e. ↑ H.R. Atropine blocks the
atropine 1mg/kg. (↓ in bp) muscarinic receptore, so
block the action of ACh.
7. ACh (5 mg/kg) ↑ ↑ Nicotinic act & release of
cate-cholamine.
8. Adrenaline + ↓ ↑ Tolazoline block Alpha
tolozoline (10 mg/Kg.) receptor.
9. Adrenaline +ppnl. - - Beta effect also blocked
10. Histromine + no fall in BP. H1 receptor antogonist.
mepayramine
Noradrenaline (NA)
It is a sympathomimetic catecholamine having predominet alpha receptor action. The
effect of NA differs from that of adrenaline that the heart rate is not increased. On the
contrary it may be decreased slightly. Only rise in blood pressure is seen and this is due
to the vasoconstriction specially those which supply blood to skin and mucosa. Since
beta action of NA is not seen, the effect of rise in B.P. is more than adrenaline only.
Isoprenaline (ISP)
58
It is a sympathomimetic amine which produces specific beta1 and beta2 receptor

agonistic action. Beta1 receptors present in heart are excited by isoprenaline as a result
of which there is increase in blood pressure and heart rate. When drug reaches
periphery there occurs odilation of blood vessels specially which are supplied to
skeletal muscle. As a result of this there is fall in blood pressure.
Isoprenaline is not the substrate for uptake, hence, the termination of action
takes time and there is delayed recovery.
Acetylcholine:
It is a parasympathomimetic agent which can stimulate both muscarinic as well

as nicotinic receptors. However, in low doses it produces only muscarinic receptor
action and hence, there is fall in blood pressure due to dilatation of blood vessels.
The action of Ach is rapidly terminated by cholinesterase enzyme and hence,

the response of Ach gets quickly recovered. Heart rate dose not change in response to
Ach.
Histamine
It is a naturally occurring autacoid which causes generalized vasodilatation

through H1 and H2 receptors. When histamine is administered in the dog there is
marked fall in blood pressure. The recovery from histamine is rapid as compared to
isoprenaline but slower as compared to acetylcholine.
Adrenaline
It is a sympathomimetic catecholamine which produces effect through alpha

beta1 and beta2 receptors. As a result of beta1 receptor stimulation there is sudden
increase in heart rate and force of contraction & increase B.P. Immediately due to
reflex inhibition there is slight decrease in B.P., but drug reaches to the periphery where
alpha1 receptor stimulation produces vasoconstriction and hence, further rise in blood
pressure is observed. A notch is seen due to sudden changes in the response.
Adrenaline action is slowly terminated by reuptake & by monoamine oxidase
(MAO) and catechol-o-methyl transfers (COMT) enzymes. When the concentration of
adrenaline is reduced beta2 receptor action predominates and as a result slight fall in
blood pressure (secondary fall) is seen.
59
Dale's Vasomotor Reversal
Dale (1914) found that when adrenaline is given after administration of ergot
alkaloid, it produces fall in blood pressure instead of rise. This unusual phenomenon
was described as vasomotor reversal.
Adrenaline produce rise in blood pressure through alpha adrenoceptors. When

alpha-adrenoceptors are blocked by any alpha-blockers (like phentolamine, tolaxoiine,
phenoxybenzamine or ergot alkaloids), the beta2-adrenoceptor action of adrenaline
predominates and fall in blood pressure is seen. This fall in blood pressure is known as
Dale's vasomotor reversal.
Nicotinic receptor action of acetylcholine
The nicotinic receptor action of ACh can be demonstrated by blocking all

muscarinic receptor using atropine like drugs and by using high dose of ACh.
Nicotinic receptors are found in (1) skeletal muscles and (2) autonomic ganglia.
Since the muscarinic receptors are blocked, autonomic ganglia are stimulated by ACh
as a result of which there is rise in blood pressure. This is nicotinic receptor action of
ACh.
The response of isoprenaline can be blocked by beta blocker propranolol.
The response of noradrenaline is blocked by phentolamine- the alpha- blocker.

The response of histamine is blocked by mepyramine and metiamide, the H1 and H2
receptor antagonists respectively.
60
61
d). Object : To study the muscarinic and nicotinic actions ..................... of

Ach on B.P. of an anaesthetized dog.
Set-up of the experiment: After anaesthetizing a healthy........... dog, femoral vein is
exposed in the thigh region and a venous cannula is inserted in the vein. Venous canula
is connected to a burette with the help of rubber tubing. Burette contains normal saline.
The route is used for injecting the drugs intravenously.
A mid line incision is given in the neck, skin and fascia are reflected, muscles are
separated and the trachea is exposed. After exposing trachea, search for the the carotid
artery, lying in the deeper tissue on the side of trachea. An arterial cannula is inserted in
the carotid artery and this cannula is connected to a mercurymeter with the help of
pressure rubber tubing. This set is used for the recording of B.P.
A partial transverse cut is given on the trachea and a "Y" shaped trachea cannula is
inserted in the lumen of trachea "Y" shaped tracheal annula is connected to an
respiratory pump.
Producer: Record a column of control tracing of normal B.P. Now inject Ach in a dose
of 1µgm./ kg of body wt. in the femoral vein through the tubing observe the effect on
B.P. and wait till the blood pressure returns to the normal level. When the effect passes
off again record with normal tracing and try another dose of 'Ach (3µgm/kg.) and see
the effect. When the recovery takes place, inject atropine in a dose of 3 mg./ kg very
slowly through i.v. rout. Wait for 20 mins. Test the blocked by injecting Ach1/µgm/kg
which should not produce any fall in B.P. Now inject a large amount of ach (1mg./kg )
and observe the effect on B.P.
Precautions :
1. After injecting the drug flush it wt. 4-5 c.c. of normal saline every time.
2. Inject the drug very close to the glass cannula, into rubber tubing.
3. Syringe should be rinsed thorougly before and after injecting each drug.
4. Take control tracing before and after injecting each drug.
OBSERVATIONS:
S.No. Drug and dose Effect on Blood Pressure
1. Ach (1gm/kg.) Fall in B.P. muscarinine Action
2. Ach (3gm/kg.) G reater fall in B.P.
3. Atro 3 mg./kg. Increase Or decrease in B.P.
After 20 mtr. No effect
Ach 1µg./kg. Rise in B.P. (Nicotinic action)
62
Inference:
Ach produces a short fasting fall in B.P. Fall in B.P. with Ach is due to the inhibitory
effect on heart and peripheral vasodilation (Muscarinic actions). Administration of
atropine when a large amount of ach is injected a rise in B.P. is observed. It is due to
few Nicotive action of Ach, which we are able to see after the blockade of mucarinic
action by atropine.
Object : Demonstration of vasomotor leversal phenomnon of dale.
Procedure:
1. Adrenoline is insected in the quantity of 3µgm./kg. of body wt. through femoral
vein. effect is observed wait till the blood pressure returns to normal again.
2. Priscoline in a dose of 5 mg./kg. body wt. is injected to block  receptors.
3. After 20 mts. when there is effective blockade of  receptors Adr. is injected in
two same also and see the effect.
Observation and inference:-
When Adr is injected a typical respose is observed on B.P.
1. Steep rise : This is due to b1 reeptor action increase inheart rate increase in force
of contraction and periphral vesoconstriction
2. Notch: At the peak of graph effect there is a little uready carelia (due to vagal
stimulang) which menfests it self inform of notch.
3. Secondary Rise: This is due to the stimulation of V.M.C.
4. Fall of B.P. below vase line is due to lea effect of actr. or B receptors.
Adr. act on two types of receptors these are  and . Due to its effect on 
receptors it causes rise in B.P. and by its effect on  receptors it causes fall in B.P.
when priscoline is injected it block  receptors and when Adr is repeated again it
stimulates only  receptors causing fall in B.P. from the beginning.
63
(A) LIST OF EXERCISES IN EXPERIMENTAL PHARMACOLOGY (E.P.)

S.No. Topic Probable No.
of Lectures
1. Introduction of E.P. 1
2. Choice of Laboratory animals 1
3. Anesthetic agents used in E.P. 1
4. Physiological salt solution & drug dilution 1
5. Various instruments used in E.P. 2
6. Screening of analgesic drugs
1
Demonstration 1
Discussions
7. Screening of local anaesthetic agents
1
Demonstration 1
Discussions 1
(Surface anaes) Gen. Class
8. Screening of antiepileptic drugs
1
Demonstration 1
Discussions
9. Effect of drugs on isolated Frog's rectus abdominis muscle 2
Discussion & Q/A
10. Effect of drugs on isolated frog's heart perfusion Discussion 1
Q/A 1
11. Effect of drugs on Rabbit's intestine Discussion 1
Q/A 1
12. Effect of drugs on Rabbit's eye (mediates & Miotics)
Pilocarpine, atropine 1
Demonstration 1
Discussion 1
Gen. Class
13. Effect of drugs on Dog's B.P. Discussion 1
Q/A 1
Total Classes 25
Total hours of teaching -- 100 hrs as whole class is divided into two batches & each
lectures is of 2 hrs.
64
(A) LIST OF EXERCISES IN PHARMACY

S.No. Topic Probable No.
of Lectures
1. Introduction of Pharmacy/Definition 1
2. Dosage forms of drugs 2
3. Abbrevialion, Weight & Measures used in Pharmacy. 1
4. Prescription writing 1
5. Mixtures in General Carminative mix Demo. 1
 Preparation of Carminative Mixture 1
 Purgative mix demo & Prep 1
 Antidiarrhoeal Mixture (Demo & Prep). 1
6. Powder in General & ORS Powders (Demo 1
 Preparation of ORS Powders 1
 Seidlitz's Powder (Demo & Prep.) 1
7. Lotion in General Demo KMnO4 Lotion (Demo) 1
 KMnO4 Lotion (Prep.) 1
 Calamine Lotion (Demo & Prep) 1
8. Turpentine Liniment (Demo & Prep.) 1
9. Emulsions in General & Cod Liver Oil Emulsion (Demo) 1
 Preparation of Cod lives oil emulsion 1
10. Ointments in General and Whitfield Ointment (Demo) 1
19
Total hours of teaching -- 76 hrs as whole class is divided into two batches & each
lectures is of 2 hrs.
65
MANUAL OF PHARMACY PRACTICAL
DEPARTMENT OF PHARMACOLOGY
DR. S.N. MEDICAL COLLEGE
JODHPUR (RAJ)
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Experimental Pharmacology

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Different Laboratory Animals And Their Applications In Experimental

Isolation or group housing:

B. GUINEA PIG (Cavia porcellus):

LABORATORY ANAESTHETIC AGENTS

Object: Choice of laboratory anaesthetic agents.

It is readily soluble in water giving a neutral solution. Usually 25% solution is

PHYSIOLOGICAL SALT SOLUTIONS

and by ionization. At higher pH ionization is less and leads to alkalinity &

Components of Physiological Salt Solution:

Drug Dilutions: In pharmacological experiments drugs are used in very minute

Types:-(1) Spleen on onchometer (2) Kidney onchometer

11. TRACHEAL CANNULA-

13 BULL DOG CLAMP-

(ii) Tuberculin Syringe

MYDRIATIC & MIOTIC EFFECT OF DRUGS ON RABBIT'S EYE.

1. Pupillary size- Can be measured by placing a transparent plastic scale in front of

Physostigmine is a reversible cholenestrase inhibitor which increases the endogenous

Atropine- An antimuscarinic agent, block the effect of endogenously released. ACh in

EXPERIMENTAL SET UP (PROCEDURE)

B- Before drug instillation - P- Present

Discussion (Effect of Drugs)

Atropine: It is a parasympatholytic drug. It causes paralysis of circular muscles by

Advantages of the Experiment

Miotic effect of phystostigmine is used in glaucoma as the drainage of aquous humor is

MYDRIATIC AND MIOTICS (VIVA-VOCE QUESTIONS)

Q.4 Name some drugs which produces mydriasis with cycloplegia?

Mechanism of Action of drugs used in glaucoma

Q. 14. What are the advantages and disadvantages of  blockers used in

Q. 15 Name the drug which produces miosis by central action?

SCREENING OF THE LOCAL ANAESTHETIC

Object: Screening of the local anaesthetic activity of drugs.

Drug 5min 10min 15min 20min 25min 30min

Time of instillation of drug – x sec

NERVE BLOCK ANAESTHESIA

DRUG REACTION TIME

Parameters Control After Xylocaine

- Adrenaline in the conc of 1:10,000 to 1 in 50,000 is mixed with local anaesthetic

Q. 9 What is the mechanism of LA?

EFFECT OF DRUGS ON FROG'S RECTUS ABDOMINIS MUSCLE

Object: To study the effect of drugs on Frog's Rectus Abdominis muscle.

EXPERIMENTAL ANIMAL – FROG

Frog rectus abdominus muscle is a voluntary skeletal muscle producing slow

Physostigmine is a reversible antiche agent, it prevents the metabolism of ACh by

FROG’S RECTUS ABDOMINIS MUSCLE PREPARATION

Q. 1 What type of muscle rectus abdominis is?

PERFUSED HEART OF FROG

Object:- To study the effects of drugs on perfused heart of frog.

Drug under study:-

Procedures and recording of observation :- First of all a column of normal tracing is

ISOLATED RABBIT ILEUM PREPARATION

(3) Recording of observation

DISCUSSION OF RABBIT ILEUM PREPARATION

Q. 1 What is the aim of experiment on rabbit's ileum ?

Q.3. Why ileum is selected ?

Q.4 Why frontal point writing lever used. ?

Q.5 Which are the various drugs used as spasmogens ?

Q. 6 In which clinical condition we use spasmogenic drugs ?

Q.7 Give e.g. of drugs which are smooth muscle relaxants ?

Q.8 Name the PSS used in this experiment ?

Q.9 Why temperature is maintained at 370 C.?

Q. 10 Name commonly used antimotility drug ?

Q.14 Write difference Spasmogenics & Spasmolytics

B. Drug acting directly :

SCREENING METHODS OF ANALGESICS