PG Instruments (Pharmacology).pptx

Instruments Used in
Experimental Pharmacology
Presenter:-
Dr Arun Singh
Senior Resident
Department of Pharmacology
SMS Medical College, Jaipur

Rota rod Flame Photometer Physiograph Plethysmograph
Analgesiometer pH Meter
Electroconvulsiom
eter
Actophotometer
Cook’s Pole
Climbing
Apparatus
Eddy’s Hot Plate Others : HPLC

• UG batch- Pupilometer, Critical Flicker Fusion
Apparatus

• Estimation of Na+, K+ routinely is done by flame photometer method.
Detection limit is as follows:
• Detection limit of Na+= 0.0001ppm Detection limit of K+= 0.001ppm
Procedure: Stock solution of 1 gm/mEq/L of Na+ and K+ is prepared.
• Then, solutions are diluted to make different strength solution.
• Na+ std solution 110 mEq/L and 170 mEq/L and K+ std solution are 8,
6, 4 and 2 mEq/L.
• The reading of photometer are adjusted, and corresponding reading of
unknown samples are directly read from photometer.
• Observation and Results Na+ conc. of unknown = ? K+ conc. of
unknown = ?

Discussion: In the flame photometer, the solution evaporates, and
substance is first converted to atomic state.
• The electrons in the higher energy orbit are in a meta stable state and
are prone to return to lower energy orbit including ground state and
energy previously absorbed is released as quanta of light, the
wavelength of which depends upon the energy levels which the
electrons can attain and are characteristics of each substance
commonly called “emission spectrum”.

• In emission flame photometry, the relatively low energy flame excites
a few elements mainly alkali metals.
• Part of light which is emitted in all directions is collected by a reflector
and falls on a detector.
• The light intensity and hence the detector output is directly
proportional to the concentration of substance.
• Frusemide causes marked diuresis in terms of urinary volume.
• The onset of diuresis is within 1 hour.
• The peak effect occurs within the first and second hour.

• The duration of action is 6-8 hours.
• The duration of diuretic effect is approximately 2 hours.
• It also causes an increase in Na+ and K+ excretion.
• Total urine output usually occur within 6 hrs approximately 3500 ml.
K+ and Na+ excretion increases peaking at 1 hr.
• BP and HR is not usually affected. Volunteers may complain of bad
taste and weakness.

Analgesiometer
(Tail Flick
Method)

Aim: To demonstrate the analgesic effect of morphine in mouse using
tail flick method Background.
The method deals on the principle of the thermal radiation heat.
This principle is used in the animal experiment for the evaluation of
the centrally acting analgesics, and hence this method found to be the
differentiating between the centrally acting opiates and non-opiates
analgesics.

Tail Flick Method:
Animal is placed into restrainer and leaving the tail exposed outside
the restrainer.
Clean the tail with the help of cotton soaked in water or ethanol.
Then, leave the tail for drying and also to settle down the rat/mouse in
the restrainer.
When rat/ mouse settled, then keep restrainer on the “tail flick
analgesiometer”.
1/3rd tail proximally left due to the thick and keratinized skin and then
keep tail on the place made for tail above hot wire (measure the height
of tail from wire) of the analgesiometer

• The time of tail flick is measured and recorded.
• The cut off time is set up 15-20 sec in case of mouse whereas in the
case of rat, cut off time is 20-30 sec to avoid any further injury to the
tail .
Materials and Methods:
Materials Animal/species : Mice/ albino Swiss Sex/Body weight : Either
sex/ 20-30 g Syringe/needle : 1ml/ preferably 24G onwards Drug :
Morphine (5-7.5 mg/kg, i.p. or s.c.)

• Other drugs : Codeine hydrochloride (30 mg/kg s.c.), Pethidine
hydrochloride (30 mg/kg s.c.) and Phenazone (400 mg/kg s.c.)
• Record the response at 30, 60 and 120 min after the saline/drug
treatment (Tail flick test).

Discussion: Tail-flick test mimic acute thermal pain and persistent pain model
by the formalin test.
Hot-plate and tail-flick test are two different methods for evaluation of
nociception.
These experimental models of pain commonly used to tests for response
thresholds to high intensity stimuli (acute pain tests) or persistent pain models.
Tail-flick test is predominantly a spinal response and Hot-plate is mostly at
supraspinal level.
Studies have conducted to evaluate participation of nitric oxide in the agmatine
mediated potentiation of morphine-induced analgesia in mice indicate that
agmatine potentiates morphine-induced spinal but not supraspinal analgesia,
and this effect is not mediated by a nitric oxide-dependent mechanism.

Aim: To observe the effect of various drugs on the isolated heart
(Langendorff’s preparation).
 Background In 1897, Oscar Langendorff established the isolated
perfused mammalian heart preparation which was considered to be the
breakthrough in cardiovascular research.
 This is based on the principle of retrograde flow in the aorta either at
constant flow or constant pressure.
 The entire perfusate enters the coronary arteries via the ostia at the
aortic root .
 The physiological salt solution (PSS) passes through the coronary
circulation and then, perfusate drains into the right atrium via the
coronary sinus.
 Coronary flow rate is measured on volumetric determination.

Animal Required: Albino Rats (300 gm and at least of 1 yr of age), or
New Zealand Rabbits (1.5-3 kg and 3 years of age) or Guinea pig (300-
450 gm and 2-3 years of age)
Animal is housed at ambient temperature (23°C ±2°C) under a 12:12
hr light and dark cycle and with free access to tap water and food ad
libitum.
The animals are acclimatized to the laboratory conditions for at least 1
week prior to experimentation. Precautions.
Animal should be pretreated with heparin (55-110 µg/kg, i.p. or s.c.
[for rabbit] or heparin (500-1000 IU/kg, i.v. in rabbit and rat and i.p. in
guinea pig), to avoid thrombosis in heart, if mounting take > 2-3 min
after the surgical procedure) and then sacrificed after 20-30 mins

Maintain the PSS flow rate adequately (Edema may develop in cardiac
tissues, if the perfusion pressure is too high) (The expected flow rate
through the cannula differs depending on species and it ranges from 7-9
ml/min for rat to 20 ml/min for rabbit)
While attaching the heart to the cannula, there should be already some
flow through the aortic cannula, which helps to avoid air bubbles
entering the coronary arteries
Insert cannula firmly so that it should not penetrate into the aortic
valve.
Maintain the sufficient hydrostatic pressure by maintaining the
distance between reservoir and heart position.

Method: Animal is pretreated with the heparin (55-110 µg/ kg, i.p. or s.c
[for rabbit] or heparin (500–1000 IU/kg, i.v. in rabbit and rat and i.p in
guinea pig).
Animal is stunned and the thorax is opened immediately, and the heart
exposed.
Heart is dissected out and removed as rapidly as possible and
transferred immediately into cold Krebs solution (rat and guinea pig
heart) or McEwens solution (rabbit heart).
The ascending aorta is separated gently using forceps from the
pulmonary artery and dissect the heart.

Figs :Showing the coronary supply of the rat or rabbit heart; (B) Bilateral coronary supply in guinea
pig, different from the rat or rabbit.

• It has 2 right and 2 left coronary arteries and (C) Position of cannula
in the ascending aorta (above aortic valve) with at least 1 cm aorta
intact. (Usually, the aorta is cut just before it divides.)
• The heart is gently squeezed several times to remove blood from the
heart and prevent formation of thrombus.
• Trim away any excess connective and lung tissue from the heart
taking care not to damage the aorta. The glass cannula (3 mm outer
diameter for rats and guinea pig; 4 mm outer diameter for rabbit) tip is
placed 0.5 cm into the aorta and firmly kept in place using a thread.
• It is important to exclude the air entrap into the system to prevent air
emboli.

• Initially side arm is flushed to remove air bubbles from the apparatus
before the experiment is started.
• Langendorff’s preparation is a constant head reservoir system, used for
providing pressure, raise this to about 18 inches above the heart or
alternatively, if a peristaltic pump is used set the rate for about 15-20
ml/min, reducing it to 3-5 ml/min maintain perfusion chamber and
aerated Krebs solution in it at 37-38°C.
• Attachment of Heart Attach a heart clip complete with a length of
thread to the tip of the ventricles (another small clip may be bound to
the auricles).
• The thread is passed around a pulley arm vertically below the heart.
• Attach the thread to the transducer after passing it over a second pulley
horizontal to first and about 20 cm apart and attach to the transducer.

• Cardiac Muscle Preparation : Langendorff apparatus set-up for the
assessment of activity of different drugs which further attach to the coupler
of student physiograph.
• After the experiment animals are disposed in the yellow polythene bags for
the incineration.
Instrument: The recording is done by attaching the thread to a strain gauge
transducer, an attachment with the student physiograph. The paper speed can
be adjusted as required.
The sensitivity of the recording apparatus to the heart contractions can be
adjusted based on the sample tracings and the baseline is adjusted so that the
tracing is in middle of the paper.
Standardized speed is maintained. Drugs Drug additions by attaching a
syringe to the injection port (or into a rubber tubing) preferably keeping the
volumes < 0.2 ml.

• Recording: Reading of the rate of heating and of the coronary flow can
most commonly be taken over a period of 30 seconds.
• If the movements are too fast to count, the kymograph can be run at a
high speed for 15- 30 seconds before the drug reaches the heart and, in
most instances, the effects passes in a few minutes as the drugs are
washed through.
• Recording of the rate of heating of flow should be taken once every 2
minutes.
• Further, doses should not be given until the preparation has recovered
completely and the rates are steady or until they have settled down
steadily at new control levels.

• Note: Important to follow the step: Initially the experiment is started
with the stimulant drugs (epinephrine, norepinephrine, isoprenaline
and calcium in increasing doses), followed with depressant
(acetylcholine, potassium) drugs and finally antagonists (propranolol
and atropine) are added as the effects of the antagonists take a long
time to wear off.
• Observations: The preparation is observed for the inotropic (by
measuring the amplitude of contractions) effect, chronotropic (by
measuring the rate of heartbeat) and effect on coronary vascular
resistance (by measuring the rate of collection of drops from the
preparation).

• Aim: To demonstrate muscle relaxant property of diazepam in mouse
using rotarod apparatus.
• Background: Rota rod test is used to evaluate fore and hind limb motor
coordination of rodents.
The apparatus consists of a horizontal metal rod (coated with rubber) of
3 cm diameter attached to a motor with the speed adjusted to 2
rotations/minute to 6 rotation/min.
The rod is 75 cm in length and is divided into 6 sections by plastic
discs, thereby allowing the simultaneous testing of 6 mice.

The rod is at a height of about 50 cm above the tabletop in order to
discourage the animals to escape from the instrument.
The cut off time for the test is 2 min.
The retention time (sec) for each mouse/rat is recorded.

• Materials and Methods: Materials- Animal/species : Mice/ Swiss
albino Sex/Body weight : Either sex/ 20-30 g Syringe/needle : 1ml/
preferably 24G onwards Drug : Diazepam (3-5mg/kg, i.p.)
• Precautions before Experimentation :Laboratory should be dim lighted
and noise free
Animal should be marked properly, to avoid mixing in two groups
Handle the animals with care (minimize the stress and pain to animal).
Precondition the mouse with the procedure.

• Methods
Step 1
Weigh the animals and mark properly to distinguish from one another
Divide animals into two groups (n = 6 in each group)
Step 2
Group 1: Control group (n = 6); mice are given saline at the equivalent
dose of drug
Group 2: Treatment group (n = 6); mice are given diazepam at the dose of 3
mg/kg, ip
Step 3
Observe the animals for 2 min on the rota rod apparatus
Observe the animals, to fall down and note the time of falling down

• Note: For mouse: Mouse is placed on a 2.5 cm diameter rod rotating at
3 rev/min. The animal’s muscle coordination is considered to be
impaired, if it falls from the rota rod within 120 sec observation
period.
• Dimensions of Rota Rod Mouse: Rod is 75 cm in length, 30 mm
diameter and is divided into 6 sections by plastic discs and of about 50
cm above the tabletop.
• Rat: Rod is 75 cm in length, 60 mm diameter and is divided into 6
sections by plastic discs and of about 50 cm above the tabletop.

Discussion:
Motor coordination is a physiological process to achieve movement
which is an essential interaction between neural processes involved in
moving a limb, and the actual limb in movement.
This is mainly processed through peripheral nervous system (PNS).
It is responsible for both the transmission of the efferent to the CNS
and the movement of the limb.
CNS also plays a vital role in integrating the afferent feedback and
efferent signals.
There are several experimental models which are established to study
the motor co-ordination in the rodents such as rotarod method,
chimney test, grip strength, treadmill performance etc.

• In the either tests, rodents (mouse or rat) are tested for their ability to
retain the muscle co-ordination such as in rotarod test.
• Mouse/rat should remain on the revolving rod or in chimney test; it
should climb backward to show the muscle coordination.
• The drugs which can be screened through these tests are anesthetics
like Phenobarbital, etc. centrally active skeletal muscle relaxants such
as benzodiazepam, chlordiazepoxide or diazepam, zolpidem,
zopiclone, etc.

• A pH meter is used to determine the acidity or alkalinity of the
solution.
• pH is the concentration of hydrogen ions in the solution.
• A solution containing more H+ ions remains acidic while the solution
containing more OH- ions remains alkaline.
• pH value of solutions ranges from 1 to 14.

Cook’s Pole Climbing Response Apparatus

Aim: Effect of psychotropic drugs on condition avoidance response
Background: Psychotropic drugs influence the learning/memory process
by modulating central neurotransmitter systems.
Cook’s pole climbing apparatus was used to determine the antipsychotic
activity and is based on principle of ability of psychotropic drugs to
avoid conditioned response.
This apparatus was used for inducing stable baseline behavior.
The aim of the study is to evaluate the antipsychotic activity of the given
drugs on condition avoidance response.

• Requirements: Animals: Albino rats of either sex weighing 200-250
gms
Drugs: Saline-1ml, Chlorpromazine- 4 mg/kg, Phenobarbitone
sodium- 15 mg/kg.
• Instrument: Cook’s pole climbing apparatus
The Cook’s pole climbing apparatus comprises of a transparent
compartment with an electrified floor and a lid to which is attached to
the pole.
The lid can be moved after the rat has mounted the pole.
The entire chamber is enclosed by a wooden box and there is an
arrangement for beeper.

• Procedure: Select animals of both sexes and divide them into three
groups consisting of 3 animals in each group.
Group receiving saline serves as control.
The other two groups receives freshly prepared chlorpromazine and
phenobarbitone respectively through intraperitoneal route, 2 hours prior
to the experiment.
Learn the rats to jump on a pole to avoid foot shock.
Use a tone of 50 Hz as the conditioned stimulus and a foot shock of 1.0
mA as the unconditioned stimuli.
Train the animals to climb the pole to escape shock by placing in the
apparatus.

• In the training procedure, initially allow the animal to adapt in the
chamber for 1 min then train them how to avoid electric shock by
responding promptly to danger signal (buzzer sound) for a period of 15
sec each.
• End the trial either after the animal respond by jumping on the pole or
after 30 secs, whichever was earlier.
• Give such trial for all selected animals every day for 10 days.
• Test the animals for learning process before and after drug treatment for
retention of memory of painful stimuli developed in the learning
process.

• Response of an animal to a signal is termed as conditioned response
(CR) while the response to the noxious stimuli referred as
unconditional response (UR).
• Inject 1 ml of saline to control group and place the animal in pole
climbing apparatus and give buzzer sound followed by electric shock
and note the response.
• The same procedure is to be done for the other two groups receiving
chlorpromazine and phenobarbitone respectively and the data of
avoidance/escape latency in seconds should be taken for 3 days and
the readings are compared.

• Conclusion- Animals that received saline shows response to both CR
and UR and confirms normal responsiveness in animal.
• Animals treated with chlorpromazine blocks CR effectively by
blocking CR, so that animals forget what they learned but do not
interfere in UR.
• Animals treated with phenobarbitone blocks both CR and UR.
• From these observations it is evident that chlorpromazine acts as
tranquilizer and phenobarbitone as hypnosedative.

• Body plethysmograph is an instrument for measuring changes in
volume of whole body in which signal represents the sum of two flows,
one is nasal flow (movement of air into and out of the nose) and
another is thoracic flow (rise and fall of chest cavity).
• The animal makes respiratory efforts against the closed shutter, causing
their chest volume to expand and decompressing the air in their lungs.
• This is based on the principle of changes of chest volume which alter
the internal box volume (increase in chest volume slightly reduces the
box volume) and thus slightly increases the pressure in the box.
• The body plethysmograph is a two chambered acrylic box.

• The animal chamber holds the animal in position and is made up of
four parts: Posterior cylinder is designed to fit the various sizes of
rodents with slanted anterior end.
• Its house rodents comfortably for the experiment and also fixes the
animal for unwanted movements.
• An anterior cylinder fits tightly over the posterior cylinder.
• Its anterior end is slanting downwards and has a conical opening in the
middle.
• When the anterior chamber is pushed over the posterior chamber, the
snout of the animal enters the cone, and the head gets fixed.

• Neck plate has two plates with adjustable hole to fit neck of the
different rodents.
• Upper plate is removable and adjusted with the screw whereas the
lower plate is fixed permanently.
• A nares cap is screwed over the protruding end of the anterior piece.
This cap has a hole on front to hold the pneumotachograph and another
on its top for air or aerosol.
• This animal chamber is placed inside an outer box of about 15-liter
capacity (size may vary).
• This box contains six openings, three for transmitting airflow and box
pressure signals, one for passing air or aerosol, one for exhaust and one
for pressure leakage.

• Equipment: It consists of six built in photo sensor and 4-digit digital
counter to indicate the loco meter activity.
It measures then spontaneous and indicated activity with digital
totalize.
It also incorporates electric shock of up to 100 volts for activating rats.
The stimulus is variable from 0 to 100v & indicating on meter.

• Principle:
Most of the CNS acting drugs influence the locomotor activities in
man & animals.
The CNS depressant drugs such as barbiturates & alcohol reduce the
motor activity while the stimulants such as caffeine & amphetamines
increase the activity.
In other words, the locomotor activity can be an index of wakefulness
(alertness) of mental activity.
The locomotor activity (horizontal activity) can be easily measured
using an actophotometer which operates on photoelectric cells which
are connected in circuit with a counter.

• When the beam of light falling on the photocell is cut off by the
animal, a count is recorded.
• An actophotometer could have either circular or square arena in which
the animal moves. Both rats & mice may be used for testing in this
equipment.
• Procedure:
1. Weigh the animals (20-25 g mice) & number them.

2. Turn on the equipment (check & make sure that all the photocells are
working for accurate recording) and placed individually each mouse in
the activity cage for 10 minutes.
Note- the basal activity score of all the animals (6).
3.Inject the drug chlorpromazine hydrochloride (Dose: 3 mg/kg, ip;
make a stock solution containing 0.3 mg/ml of the drug & inject 1
ml/100 g body wt. of mouse), and after 30 mins re-test each mouse for
activity scores for 10 mins. Note the difference in the activity, before &
after chlorpromazine.
4. Calculate percent decrease in motor activity.

• Commonly Used Drugs:
CNS depressants: Chlorpromazine hydrochloride (3 mg/kg, ip in case of
both rat & mice), Fluoxetine (10 mg/kg, ip in case of rat), Imipramine
(10-20 mg/kg, ip in case both mice & rat), Phenobarbitone sodium (10
mg/kg, ip in case of both rat & mice), Alcohol (0.5-2 g, ip, po in case of
both mouse & rat)
CNS stimulants:
Caffeine (8-10 mg/kg, ip in case of mice & 30 mg/kg, ip in case of rat)
Amphetamine (1.5 mg/kg, ip in case of mice & 3-5 mg/kg, sc, ip in case
of rat)

Interference:
Reduction in the motor activity indicates CNS depressant property of
the drug.
Increase in the motor activity indicates CNS stimulant property of the
drug.

Eddy’s Hot
Plate
Analgesiometer

• To demonstrate the analgesic effect of morphine in mouse using hot
plate method.
• Background: The method deals on the principle of the thermal
radiation heat. This principle is used in the animal experiment for the
evaluation of the centrally acting analgesics, and hence this method
found to be the differentiating between the centrally acting opiates and
non-opiates analgesic.
• Tail-flick test is predominantly a spinal response and Hot-plate is
mostly at supraspinal level.

• Observations and Results:
Centrally acting analgesics can be evaluated by the hot plate method
whereas peripherally acting analgesics are not effective. e.g: Aspirin
showed no effect in hot plate method even at very high doses False
positive results: sedatives and muscle relaxants (Woolfe and
MacDonald, 1944) or psychotomimetic (Knoll, 1967) may give the
increase reaction time.

• Aims and Objectives: The study was aimed to evaluate the anticonvulsant
effect of hydro-alcoholic seed extract of croton tiglium in rats and mice.
• Materials and Methods: Forty -eight each of rats and mice of either sex were
randomised into four groups and subjected to seizures induced by
electroconvulsiometer and pentylenetetrazol.
The hydroalcoholic seed extract of croton tiglium (250 and 500mg/kg) was
studied for its anticonvulsant effect using sodium valproate (200mg/kg) as
standard and distilled water as control.
The parameters observed were time for onset of HLE (Hind Limb
Extension) and duration of HLE in electrically induced seizures, and time for
onset of convulsions and duration of convulsions in chemically induced
seizures.
Mortality of the animals over 24 hours was observed in both the models.

• Oil free air compressors play a pivotal role in manufacturing medicine
and related products.
• With growing FDA scrutiny, strict warnings over toxic impurities, and
delicate pharmaceutical manufacturing processes, the quality of
compressed air meeting the highest standards is of utmost importance.
• In addition, disruption in continuous air supply can lead to an immense
loss of production and negatively impact the manufacturer’s reputation
due to product contamination.

• Pupillary distance measurement- In the most common sense,
a Pupilometer is a tool for measuring pupillary distance (PD).
It is used for fitting eyeglasses so that the lenses are centered in the
visual axis. This is the most common nomenclature.
It may also be used to verify a PD measurement taken from a
millimeter ruler placed across the bridge of a patient's nose for distance
and near focus.
• Pupil Response:
Alternatively, a pupilometer is a type of pupil response monitor -- a
monocular device measuring the amount of dilation of the pupil in
response to a visual stimulus.

In ophthalmology, a pupillary response to light is differentiated from a
pupillary response to focus (i.e.. pupils may constrict on near focus, as
with the Argyll Robertson pupil) in the diagnosis of tertiary syphilis.
Although a pupillometer can be used, the diagnosis is often made with
a penlight & near-point card.
The extent of dilation of the pupil in the eye could be an indicator of
interest and attention.
Methods of reliable measurement of cognitive load, such as the
dilation or constriction of the pupils, are used in marketing research to
assess the attractiveness of TV commercials.
Dilation of the pupils reflects an increase in mental processes, whether
it be attentiveness, or psychomotor responsiveness.

• The CFF apparatus consists of a flickering light source against a dark
background.
• A dial is provided in front to adjust the number of flicker per second.
The volunteer is asked to see the flickering light for a min at 5 Hz.
• The dial is then slowly moved at the rate of 2 Hz/sec.
• The volunteer is then asked when the light appears fused.
• Volunteer is asked to look at this frequency for a minute.
• Then the rate is decreased at 2 Hz/sec till the light appears as
flickering.
• This frequency is then noted. The average of 3 such reading is noted.

• Critical Flicker-Fusion Frequency (CFF), the frequency at which a
flickering light is perceived as continuous, is widely used for
evaluating visual temporal processing.
• However, substantial variability in the psychophysical paradigms,
used for measuring CFF, leads to substantial variability in the reported
results.
• Here, we report on a comprehensive comparison of CFF
measurements through three different psychophysical paradigms:
methods of limits; method of constant stimuli, and staircase method.
• Our results demonstrate that the CFF can be reliably measured with
high repeatability by all three psychophysics methods.

High
Performance
Liquid
Chromatography

• The main purpose of the HPLC technique is to identify, quantify and
purify a particular analyte or compound.
• Both quantitative and qualitative analysis can be done.
• HPLCs can be used in the following applications:
i. Water purification
ii. Detection of impurities in pharmaceutical industries
iii. Pre-concentration of trace components
iv. Ligand-exchange chromatography
v. Ion-exchange chromatography of proteins
vi. High-pH anion-exchange chromatography of carbohydrates and
oligosaccharides

PG Instruments (Pharmacology).pptx

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