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 α chloralose (active form) and β chloralose (inactive form) are formed. Chloralose is the active form (α 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): 7

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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: 8

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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. Mg+2 ions : responsible for contraction of smooth muselec.

2. 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 10

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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

No. 1. 2. 3. 4. 5.

Nacl KCl CaCl2 NaHCO3 Mgcl2.

6.

6H2O MgSO4

7. 8. 9. 10.

7H2O NaH2PO4 KH2PO4 Glucose Areation

Ringer

Frog's

Tyrode's

Deialon's

Kreb's - Mc

solution

ringer

solution

solution

Henseleit Ewen's

9.00 0.42 0.24 0.50 -

solution 6.50 0.14 0.12 0.20 -

8.00 0.20 0.20 1.00 0.10

9.00 0.42 0.06 0.50 -

solution 6.9 0.35 0.28 2.10 -

solution 7.60 0.42 0.24 2.10 -

-

-

-

-

0.29

-

1.0 O2

0.01 1 or 2 air

0.05 1.00 O2 / air

0.50 O2+5%

0.16 2.00 O2+5%

0.14 2.00 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. Drug Dilutions: In pharmacological experiments drugs are used in very minute quantities i.e. can be fractions of mg or µ g. These small amounts can't be weighed

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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 1% 10% 100% 0.1% 0.01% 0.001% 0.0001%

Strength 1:100 1:10 1:1 1:1000 1:10,000 1:100,000 1:10,00,000

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mg/ µ g 100mg/10ml or 10mg/ml 100mg/ml. 1000mg/ml or 1g/ml . 1 mg/ml 0.1mg/ml or 100µg/ml. 0.01mg/ml or 10 µ g/ml. 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) 2.

The entire assembly is mounted on tripod stand. 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.

LEVERSIt is the device by virtue of which response of isolated tissue can be recorded and magnified.

Principle: 14

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(i) Fulcrum- the point around which the lever moves on the lever holder is the fulcrum. (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 15

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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.

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Principle: works on displacement of air. 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 ENTEROGRAPHIt 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.

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o

The open end of the metallic tube is connected to the tracheal camula with the help of polythene tubing.

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.

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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. 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 CANNULAIt 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.

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

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.

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11.

TRACHEAL CANNULAIt 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.

21

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

This instrument has been replaced by polythene sets with similar design and function in disposable IV perfusion sets. 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 ASTHESIOMETERIt 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. 22

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

These BD glass syringes are largely replaced by plastic disposable syringe and needle.

23

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(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.

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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. 25

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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. 26

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•

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 Pilocarpine 2%

Size of Pupil(mm) RE B A

LE B

5

5

Atropine 1%

5

Ephedrine 4%

5

Light Reflex

Corneal Reflex

A

RE B A

LE B A

RE B A

LE B A

5

2

P

P

P

P

P

P

P

P

5

5

10

P

P

P

Ab

P

P

P

p

5

10

P

P

P

P

P

P

P

P

P

B- Before drug instillation

-

P-

Present

A- After the drug instillation

-

Ab-

Absent

Remarks (inference) Miotic response is seen causing constriction of pupil, no effect on LR/ CR. could be a Parasympathomimatic drug. Mydriatic effect is seen i.e. (dilatation of pupil) no change in CR LR is lost. So possibility of parasympatholytic drug Mydriatic effect is seen. CR and LR both remain unaffected. Hence, possibility of a sympathomimetic drug.

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).

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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.

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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.

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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 α 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 α - adrenergic antagonist. Adrenergeric neuron blockers

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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 β - blocker 2. Miotics – pilocarpine 3. Hypertonic mannitol 4. Acetazolamide In chronic angle glaucoma

1. β - Blocker – Timolol 2. Miotics 3. α - 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. β –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. α - 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 β blockers. Betaxolol β 1 selective antagonist, less effective and few adverse effects. Q. 14. What are the advantages and disadvantages of β glaucoma? Ans.

Discussed in mechanism of action.

31

blockers used in

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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.

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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.

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The presence of corneal reflex is graded as +, ++, +++ ……after every five minutes. 10min, 15min, 20min, 25min, and 30min and tabulated. Drug I CONTROL (RE) II XYELOCAINE (LE)

5min

10min

15min

20min

25min

30min

1 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 C T

10min C T

15min C T

20min C T

25min C T

30min 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.

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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 5min I CONTORL II XYLOCAINE

REACTION TIME 10min 15min 20min

25min

30min

1 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 10min

Tone of muscles Sensation

35

After Xylocaine 20min 30min

40min

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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. 36

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-

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.

37

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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

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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)

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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 40

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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.

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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

1

ACh

0.1 ml

Contraction of muscle Potentiatioin of response

2

ACh + physostigmine

0.1ml+0.5ml

3

ACh + d-TC

0.1ml+0.5ml

Inhibition or blocade blockage of response

4

ACh

0.1ml

Contraction of muscle

INFERENCE (Remark) Nicotinic action at NMJ Because of anti cholinesterase action of physostigmine it decrease metabolism of increase Ach at NMJ hence, increase in response d-TC is having SMR action so there is positive response of ACh (Reversible action). Nictonic action reappears. There 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.” 42

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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.

43

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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?

44

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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. Uses of PSMRs :

45

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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.

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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.

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Observation Drug and Dose

Effect of Drugs Rate Force

Adrenaline

Increase

Infeunce of

contration Increase

Adr

has

ionotropic

+

chronotropic

+

Kcl Cacl2

Decrease Decrease

decrease Increase

dromotropic action (B- receptors ) Direct depresant action/ Force of contraction increased with bradycardia

ACh

Decrease

Decrease

(Has ionotropic action only) Has a inhibitory action on heart (Muscarinic

Atropine no effect

No effect

action) Atropine has blocked the muscarinic effect of

inject

After 2 min inject same

amount

ACh.

of

ACh

Difference: 1

ACh KCl Inhibitory effect on the heart is blocked by Effect not affected by Atropine

1.

Atropine Adr CaCl2 Both systdic and diastalic component are only systolic component is seen.

2. 3.

seen Heart increases Beta - blocker abolishes the effect

Brady cardia B blocker does not affect the effect

48

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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. 49

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*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 area. Indirectly acting e.g. adrenaline, atropin. b. Directly acting e.g. papaverine, nitrates. Significance : 1. To know whether drug is spasmogenic or spasmolytic 50

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2. To differentiate between directly or indirectly acting drugs. 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. 1. 2. 3.

Drugs ACh Adrenaline BaCl2

Tone increases decreases increases

Amplitude decreases decreases little or

4. 5.

Atropine Atropine + ACh

decreases no response seen

change decreases spasmolytic drug no response Spasmogenic action of seen

Inference spasmogenic drug spasmolytic drug no spasmogenic drug

ACh

is

atropine 6.

Atropine + BaCl2

increases

little change

or

blocked

by blocking

muscarinic receptors. no Spasmogenic action is not blocked as it is directly acting.

51

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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.

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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.

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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 A) Drugs acting through receptors : 1. ACh- Cholinergic receptor and their agonists. 2. Histamine- Via histaminergic receptors 3. 5HT or serotonin via serotonergic receptors. 4. Nicotine in low dose through nicotinic receptors but at high doses it blocks its own receptors. 5. Morphine via opium receptors B. Drug acting directly : a) BaCl2 b) Post pituitary extract or oxytocin. c) Vasopressin, substance - P, Bradykinin

Spasmolytics a). Adrenaline - via both alfa & beta receptors. b). Amphetamine c). Nicotine at high doses. d). Atropine by blocking muscarinic receptors. e) B2 receptor agonist.

a). Aminophyllin b). Papaverin c). Nitrates & Nitrites d) Chloroform Uses : Dysmenorrhora Spastic constipation. renal and brilliary colics etc.

Uses : Paralytic ilivs Retention of urine etc.

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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 drugtreated 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. 55

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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.

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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. 57

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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.

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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, Hear rate

Sex Male, / Min.

Weight -10 Kg.

Rate of Respiration 59

/min

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Blood pressure: Aneasthesia used: morphine + urethane (1.5gm1kg) Effect of various drugs on dog blood pressure : S. No. 1.

Drugs & Dose Adrenaline

Blood Pressure 1st ↑ then (biphasic

2.

(2µ g/ Kg.) Nor adrenaline-

response) ↑

3.

(3-5 µ g / kg.) Isoprenaline

↓↓

↑

Sympathomimetic drug

4.

(2µ g / kg.) Acetylcholine

↓

Bradycardia

Cholinergic drug

5.

(2µ g / kg.) Histamine

↓

↑

Naturally occuring auto

6.

2-3 µ g / kg. Ach. (2µ g / kg.) +

No effect of Ach i.e.

↑ H.R.

coid. Atropine blocks the

atropine 1mg/kg.

(↓ in bp)

muscarinic receptore, so

↑

↑

block the action of ACh. Nicotinic act & release of

↑

cate-cholamine. Tolazoline block Alpha

7.

ACh (5 mg/kg)

8.

Adrenaline +

↓

9. 10.

tolozoline (10 mg/Kg.) Adrenaline +ppnl. Histromine + no fall in BP.

Heart Rate ↑↑

Remarks Sympathomimetic drug Sympathomimetic drug

-

receptor. Beta effect also blocked 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) 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

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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. Dale's Vasomotor Reversal

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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.

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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. 1. 2. 3.

Drug and dose Ach (1 µ gm/kg.) Ach (3 µ gm/kg.) Atro 3 mg./kg. After 20 mtr. Ach 1µg./kg.

Effect on Blood Pressure Fall in B.P. muscarinine Action G reater fall in B.P. Increase Or decrease in B.P. No effect Rise in B.P. (Nicotinic action)

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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 α receptors.

3.

After 20 mts. when there is effective blockade of α 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 α and β . Due to its effect on α receptors it causes rise in B.P. and by its effect on β receptors it causes fall in B.P. when priscoline is injected it block α receptors and when Adr is repeated again it stimulates only β receptors causing fall in B.P. from the beginning.

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(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 Demonstratio 1 n 1 Discussions 7. Screening of local anaesthetic agents Demonstratio 1 n 1 Discussions 1 (Surface anaes) Gen. Class 8. Screening of antiepileptic drugs Demonstratio 1 n 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.

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S.No. 1. 2. 3. 4. 5.

6.

7.

8. 9. 10.

(A) LIST OF EXERCISES IN PHARMACY Topic Introduction of Pharmacy/Definition Dosage forms of drugs Abbrevialion, Weight & Measures used in Pharmacy. Prescription writing Mixtures in General Carminative mix Demo.  Preparation of Carminative Mixture  Purgative mix demo & Prep  Antidiarrhoeal Mixture (Demo & Prep). Powder in General & ORS Powders (Demo  Preparation of ORS Powders  Seidlitz's Powder (Demo & Prep.) Lotion in General Demo KMnO4 Lotion (Demo)  KMnO4 Lotion (Prep.)  Calamine Lotion (Demo & Prep) Turpentine Liniment (Demo & Prep.) Emulsions in General & Cod Liver Oil Emulsion (Demo) Preparation of Cod lives oil emulsion Ointments in General and Whitfield Ointment (Demo)

Probable No. of Lectures 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 19 Total hours of teaching -- 76 hrs as whole class is divided into two batches & each lectures is of 2 hrs.

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MANUAL OF PHARMACY PRACTICAL

DEPARTMENT OF PHARMACOLOGY DR. S.N. MEDICAL COLLEGE JODHPUR (RAJ)

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