PHARMACOLOGY – I (PRACTICAL)[PHL 313]

Dr. Mohd Nazam Ansari

Lab 4: Study of Some Basic Instruments

Used for Isolated Tissue Experiments

Isolated tissue preparations are commonly used to study the effects of drugs on specific type of receptors. These preparations are also used: For bioassay of drugs, Characterization of specific receptor or its

subtypes, To determine concentration response curve of

an agonist, To study antagonism of drug and in new drug

discovery.

The in vitro isolated preparation represents an isolated organ or a piece of living tissue from a freshly killed animal. Optimum ionic environment Adequate supply of nutrition and oxygen And a stable temperature

These basic requirements should be provided if one has to maintain the isolated tissue in living state.

Basic Requirements

Physiological Salt Solutions Instrumentation Procedures and drugs – to render the

animal unconscious Tissue: isolated or whole

Physiological Salt Solutions

Optimal pH 7.0 – 7.2 Commonly used PSS are:

Frog Ringer (for heart, rectus abdominis and other preparations of frog)

Physiological Salt Solutions

Tyrode (for guinea pig ileum, rat ileum, rabbit ileum etc.)

De Jaon (for rat uterus)

Kreb’s solution (for rat fundus strip, tracheal preparations, vas deferens) etc.

Composition of PSSIngredients

Concentration on gms/l

Frog ringer

Tyrode Kreb’s bicarbonate

De jalon

NaClKClCaCl2NaH2PO4

KH2PO4

MgSO4

MgCl2GlucoseNaHCO3

6.50.140.120.05

---

1.50.4

8.00.20.180.1--

0.11.01.0

5.90.350.28

-0.160.11

-2.02.1

9.00.350.003

----

0.50.5

NaCl: To maintain isoosmolarity, isotonicity, excitability and contractility KCl: To maintain ionic balanceCaCl2 : To maintain contractility NaH2PO4 & KH2PO4 : Act as a bufferMgSO4 & MgCl2: To stabilize the preparation and hence reduce the spontaneous activityGlucose: To provide energyNaHCO3 :To provide alkaline pH

Experimental setup

Isolated organ bath The apparatus providing the basic

requirements of life to the tissue and facilitating the record of response.

Outer water bath of perspex or glass

Inner organ bath (single or multiple)-glass (15-100 ml): Organ tube

Tissue holder cum Aeration (oxygen) tube

Glass coil connected to organ bath, Marriott's bottle: Reservoir for PSS

Electric heater Preheating coil

Aeration

Oxygen or carbogen (5% CO2 + 95% Oxygen)

The purpose of aeration are: Supply of oxygen Uniform distribution of drug due to bubbling Maintenance of pH specially in Kreb’s solution

where amount of NaHCO3 used is more

Sherrington Recording Drum and Drum Cylinder

It is an instrument on which physiological responses are recorded.

Heavy base Base hoofs (legs) with adjustable

leveling screws Gear rod

Vertical shaft Drum Cylinder

Brass or ironIt has 8 variable speeds. The speed is measured in mm per second. Eight variable speeds are 0. 12, 0. 25, 1. 25, 2. 5, 12. 5, 25, 320 and 640 mm per second.

Levers

Isometric contractions: change in tension (force). E.g. spring lever

Isotonic contractions: change in length at uniform tension. E.g. isotonic frontal writing lever (light aluminum or stainless steel rod)

Smoking of Drum

Glazed paper A mixture of benzene and kerosene (1:9)

Fixing the graph (Varnishing of the graph)

A solution of resin (colophony) in methylated spirit. This solution is prepared by dissolving 150 gm of resin in 2 Lit of spirit.

Recording of responses on drum cylinder without smoking (Sketch-Pen Tip)

Contact time

For slowly contracting tissue e.g. rectus abdominis muscle it is 60-90 sec.

For fast contracting tissue e.g. Guinea pig ileum it is 30 sec.

Useful Hints For Students Step by step Setup the assembly first Assembly should be on right hand side and drum

should be on left hand side The drum should move from right to left or away from

the writing point The speed of drum should be minimum The writing lever should be horizontal and the

writing point should touch the drum. All tracing should begin after the overlap of the

paper. Always start with the minimum concentration of drug. Write the name & amount of drug. Name of

experiment and student with student ID and signature of teacher before varnishing.

Thanq