ANALGESIC MODELS

THE DEPT. OF
DRAVYAGUNA
WELCOMES YOU
ALL…

ANALGESIC
MODELS
Presented By
Dr. Puneshwar Keshari,
Ist Year PG Scholar,
Dept. of Dravya Guna,
Guided By
Dr. Prakash L. Hegde
Prof. Dept. of Dravya Guna

CONTENTS
• Definition
• Functions of Pain
• Pain classification
• Analgesics
• Classification of analgesics
• Analgesic models
• Experimental models
• Discussion
• Conclusion
• References

Definition of Pain
An unpleasant sensory or emotional
experience associated with actual or
potential tissue damage, or described
in terms of such damage. (IASP)

Nociception
Sherington, 1910
introduced the term nociception
nocere – ‘to harm’

Concept of Pain in Ayurveda
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Types of Human Pain
Various types of pain are seen in humans: eg.
– Somatic pain (arising from the skin, Muscles,
joints, ligaments and bones)
– Visceral pain
– Referred pain
– Neuropathic pain
– Cancer pain etc.

Classification of Pain
Acute Pain:
• Source is soft tissue damage, infection or
inflammation
• Short duration
• “Symptom of pain”

Chronic pain:
• Lasts for six months or longer,
• “The disease of pain”
• Common causes are cancer, neuropathic and
arthritic.

ANALGESICS
• The drugs which possess significant pain
relieving properties by acting in the central
nervous system , or on the peripheral pain
receptors without significantly affecting
consciousness.

Classification of analgesics:
NARCOTIC ANALGESICS NON NARCOTIC ANALGESICS:
Act centrally Act peripherally
Cause addiction Do not cause addiction
Produce CNS depression Do not produce CNS depression
Do not produce gastric
irritation
Produce gastric irritation
Show no anti inflammatory
effect
Show anti inflammatory effect
eg. Morphine, Tramadol,
Pethidine etc.
Diclofenac, Ibuprofen, Aspirin
etc.

Analgesics in Ayurveda
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ANALGESIC MODELS
• The animal models employed for screening of
analgesic agents are analgesic models.
• For animal models of pain, the word “model”
refers to three entirely separate entities-
– subject,
– assay and
– measure.

EXPERIMENTAL MODELS:
1. Models using thermal stimuli:
– The tail flick model using radiant heat/immersion of the
tail in hot water
– Pain withdrawal test
– Hot plate test
– Pain state models using cold stimuli
• Cold tail flick test
• Cold ethanol tail flick test (CET)
2. Models using mechanical stimuli:
– Haffner’s tail clip method
– Randall Selitto Test
– Strain gauges
– Von Frey filaments
– Inclined plane test

3. Models using electrical stimuli:
– Electrical stimulation of the tail
– Grid shock test
– Stimulation of the tooth pulp
– Monkey shock titration test
– Stimulation of the limbs.
4. Models using chemical stimuli:
– Formalin test
– Acetic acid induced writhing test
– Stimulation of hollow organs
• Rat sigmoid colon model
• Inflammatory uterine pain model

MODELS FOR CHRONIC PAIN
• Neuropathic pain models
• Vincristine induced neuropathy model
• Diabetic neuropathy model
• Persistent post thoracotomy pain model
• Rat model of incisional pain
• Rat model of bone cancer pain

IN VITRO METHODS
• H – Naloxone binding Assay
• Micro opiate receptor binding assay
• Assay to study cannabinoids activity
• Receptor binding of nociception
• Bioassays for nociception
• Vanilloid receptor binding

USE OF
SHORT DURATION STIMULI (PHASIC
PAIN) OR
MODELS FOR ACUTE PAIN

A. PAIN STATE MODELS USING
THERMAL STIMULI
1. The Tail Flick models:
• Commonly accepted model for
quantifying analgesic response in
animals.
• In this model, radiant heat is applied to
a small surface of the tail or the tail is
immersed in hot water.

a. Tail flick model using radiant heat:
• Hardy et al. 1940 used on human
• Then used in Rat (1953)
• The tail flick test with radiant heat is a
simplified method the application of
thermal radiation to the tail of an animal
provokes the withdrawal of the tail
• The morphine like drugs are capable of
prolonging the reaction time.

Procedure
–The tail of Wistar rat (170-
210gms) is placed on the
radiant source and time
taken for the rat to withdraw
its tail is recorded.
Withdrawal of the tail from
heat source is referred to as ‘
tail flick latency’. Exposure of
the tail with radiant heat is
done for 10-20 seconds.

Evaluation:
• The tail flick latency in the test, standard, and
control animals are compared
• A lengthening of the reaction time is
interpreted as an analgesic action.
• Using various doses, ED-50 values can be
calculated.

Merits
• Effective for screening Morphine like
analgesics
• Simple technique, which does not
require any special skill
• Results of experiments are quite
accurate and less time consuming.

Demerits:
• Tail flick response is prone to habituation
• Efficient only for revealing the activity of
opioid analgesics
• Prolonging exposure to radiant heat
beyond 20 seconds may cause burning of
tail.

Modification
• USG induced tail flick procedure
• Ear withdrawal by applying radiant
heat
• Tail flick formalin test in rodents:
changes in skin temperature

b. Tail flick model using immersion of the tail:
• Distal 5 cm of the tail of wistar rat
is marked and immersed in a cup
of warm water ( 55°Cto56°C) for
15 seconds.
• The reaction time is determined
periodically after administration
of test drug (0.5, 1, 2, 3, 4, and 6
hours)

Tail flick model using immersion of
the tail
Mouse Tail-Flick, Allegheny College.mp4

• Response and Evaluation: - Withdrawal of tail or
attempt to escape or abrupt movement of tail
and sometimes the recoiling of whole body is
seen.
• Centrally acting analgesics are capable of
causing prolongation of tail withdrawal
reflex( more than 6 sec.).
• Modification:
–Cold tail Flick test
–Cold ethanol Tail flick test

2. Hot Plate Model
• Introduced by Woolfe and
Mc Donald in 1944
• Mice are put on the
hotplate(temp. 55° to56
°C)and then latency is
noted following
administration of test drug.
• Response – jumping or paw
licking.

EVALUATION
• The prolongation of latency time between the
test, standard and control animals are
compared (by using t-test)
• Using various doses ED-50 values can be
calculated.

B: PAIN STATE MODELS USING
MECHANICAL STIMULI:
1. Haffner’s Tail Clip Method:
• Preferred sites for applying nociceptive
mechanical stimuli are the hind paw and the tail
• Highly sensitive for centrally acting drugs
• Tests using constant pressure have been
abandoned progressively for those applying
gradually increasing pressures

PROCEDURE
• An artery clip is placed at the root of tail of mice.
• A quick response is seen as biting the clip or tail,
where clip has been placed. The reaction time is
noted by a stopwatch.
• The test compounds are administered
subcutaneously or orally.
• Then after 15, 30, and 60 minutes, the same
procedure is repeated and the reaction time is
measured.

EVALUATION
• Reaction time of the test animals, greater
than the cut off time denotes a positive
response, indicating analgesic activity.

PAIN STATE MODELS USING
ELECTRICAL STIMULI: Grid shock
method
-Blake et al (1963)
-Used for central and peripheral analgesics
-Stimulus is given in the form of square wave pulses
to male mice . The output of the stimulator is
connected to the wires of grid with fixed
resistance which is kept parallel to oscilloscope
-Pain thresholds are determined in each individual
mouse twice before and after administration of the
test drug.
-Response- on oscilloscope , a starling reaction ,
increase locomotion or attempt to jump.

EVALUATION
• Current in milli-ampere is
recorded in each mouse before
and after administration of drug
is noted.
• The average pain threshold
values for each group at each
time interval are calculated and
statistically compared with the
control values.

D. PAIN STATE MODELS USING
CHEMICAL STIMULI
• Slow, progressive, and
irreversible form of
stimulation
• Closest in nature to
clinical pain

1. Formalin Test
• 10% formalin solution is used as noxious stimulus
which is injected into the paw of rat or mouse and
inflammation is created as a model of persistent
or chronic pain.
• Two phases reactions are shown – Early phase
starts immediately after injection, and lasts for 3-5
minutes
• The second phase starts after 10—15 mins. of
injection and lasts for 20-40 mins.
• Opioid analgesics are effective in both phases,
while NSAIDs are effective in the second phase.

• Male wistar rats ( 180-300gms) are administered
subcutaneously with 0.05 ml of 10% formalin in
dorsum of the front paw.
• Each animal is placed into separate cages for
observation of pain responses in early and late
phases.
• Responses are as elevation or favoring of paw, or
excessive licking and biting of the paw
PROCEDURE

• Scoring is done according to the pain scale
• Then test drug is administered and again scoring is
done after 30, 60, minutes for comparison
• If both paws of animal are allowed to rest on the
floor with no obvious favouring of the injected paw,
this is taken as positive analgesic response.
Contd..

b. Writhing Test
• Model of visceral or peritoneal
pain in animals
• Test is issued to detect
peripheral analgesic activity
• Pain is induced by intra
peritoneal administration of
chemicals, which irritate serous
membrane and provoke a
stereotype behaviour in the
mouse or rat known as writhing.
• Chemicals used are acetic acid,
acetylcholine, bradykinin,
aconitine, 4% NaCL, etc.

PROCEDURE
• Acetic acid solution is
administered to the mice(20-
25gm.) and placed individually
in the glass jar.
• The test and standard drug is
administered 15 minutes prior
to the acetic acid administered.
• Responses are noted.

Writhing Test
writhing test.mp4

EVALUATION
• The writhing period is recorded for 10 minutes
and compared with the control group.
• Writhing response in the drug treated must be
less when compared to the acetic acid treated
control.

IN VITRO MODELS
BIOASSAYS FOR NOCICEPTION
Purpose and rationale:
Nociception receptors in the periphery can be
characterized by studies in isolated
organs(Guerrini et al.1998,Bigoni et al.1999) .
The guinea pig ileum , according to Paten 1957,
the mouse vas deferens according Hughes etal.
1975, the rabbit vas deferens according to Oka
et al. 1980, the guinea pig renal pelvis according
to Giuliani and magi 1996.

PROCEDURE
• Tissues are taken from male Swiss mice, guinea
pigs, Sprague Dawley rats, and New Zealand
albino rabbits.
• Suspended in 10 ml organ baths containing
Krebs solution oxygenated with 95% oxygen
and 5% carbon-dioxide.
• Temperature is set around 33-35 degrees and a
resting tension of 0.3-1 gm is applied
• The tissues are stimulated through two
platinum ring electrodes

• The electrically evoked contractions are
measured isotonically with a strain gauge
transducer and recorded on a multichannel chart
recorder.
• After equilibration period about 60 mins, the
contractions induced by electrical field
stimulation are stable. At this time, cumulative
concentration response curves to nociception or
opioid peptides are performed.
Contd..

• Four electrical field stimulation are performed
with each tissue at 30 min intervals.
• Agonists and antagonists are added to the
bath
• Contractile response to electrical field
stimulation are expressed as % increment to
the spontaneous activity of the tissue.
• The biological effects of the application of
agonists or antagonists are expressed as %
inhibition of electrical field stimulation
induced contraction.
Contd..

EVALUATION
• The agonists and antagonists potencies are
recorded and expressed as means of +- SEM.

MODELS OF CHRONIC PAIN:
• Rat Model of Incisional Pain:
• This model helps to understand mechanisms of
sensitization caused by surgery and investigate new
therapies for post operative pain
• A longitudinal incision of 1 cm is given through the skin,
fascia and muscle of the plantar surface of the hind
paw in halothane anesthetized rats.
• Withdrawal responses are measured using Von Frey
filaments at different areas around the wound before
surgery and for the next 6 days.
• The results of tests for withdrawal responses suggest
that surgical incision of the rat foot causes mechanical
hyper-algesia lasting for several days after surgery.

RESEARCH ARTICLES
• The necessity of animal models in pain research
• Jeffrey S. Mogil a,*, Karen D. Davis b,c, Stuart W. Derbyshire
• IASP PAIN 151 (2010) 12–17
• Abstract-There exists currently a fair degree of introspection in
the pain research community about the value of animal research.
This review represents a defense of animal research in pain. We
discuss the inherent advantage of animal models over human
research as well as the crucial complementary roles animal
studies play vis-à-vis human imaging and genetic studies. Finally,
we discuss recent developments in animal models of pain that
should improve the relevance and translatability of findings using
laboratory animals. We believe that pain research using animal
models is a continuing necessity–to understand fundamental
mechanisms, identify new analgesic targets, and inform, guide
and follow up human studies–if novel analgesics are to be
developed for the treatment of chronic pain.

Animal models of pain : progress and challenges
Jeffrey S. Mogil
Nature Review, Neuroscience; Vol. 10, April 2009;
pg 283-94
Abstract:
Many are frustrated with the lack of translational progress in the pain field, in
which
huge gains in basic science knowledge obtained using animal models have not
led to the development of many new clinically effective compounds. A
careful re-examination of animal models of pain is therefore warranted.
Pain researchers now have at their disposal a much wider range of mutant
animals to study, assays that more closely resemble clinical pain states, and
dependent measures beyond simple reflexive withdrawal. However, the
complexity of the phenomenon of pain has made it difficult to assess the true
value of these advances. In addition, pain studies are importantly affected by a
wide range of modulatory factors, including sex, genotype and social
communication, all of which must be taken into account when using an animal
model.

Evaluation of the Analgesic and Anti inflammatory
Activity of Kadamba (Anthocephalus cadamba miq.) –
an experimental study
Thesis dissertation by Dr. Prakash L Hegde, submitted to
the RGUHS
• Used model – Hot Plate Method
• Conclusion - Alcoholic extract of Kadamba has potent
analgesic and anti-inflammatory activity almost like
standard drug.
• Thus kadamba is a freely available safe drug showing
efficacy in single use both in extract and kashaya form on
rats. Same has to be reconfirmed on human beings.

EXPERIMENTAL MODELS IN
AYURVEDA

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How the Vaidya examines and certifies
the King’s food:
First, he should test it by fire, then, by
feeding it to a peacock to determine whether
it is poisonous or not. Then, he should chant
holy hymns and chants to ward of evil, and
should ensure that the king has donned and
sacred stones and medicaments as

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There are four stages in the case of quadrupeds, and
three stages in that of birds. In the animal, in the first
stage, there will be asthenia and whirling; in the
second stage the animal quivers, and in the third,
passes into stupor and takes no food. In the fourth
stage respiration becomes hard and it dies. The bird in
the first stage of poisoning feels depression and in the
second stage whirling in the third stage its limbs get
paralysed and death ensues.

DISCUSSION
• Pain –
– Common unpleasant sensory and emotional
experiences.
– Acute, chronic, somatic, visceral, Neuropathic etc are
different ways of experiences of pain.
– In Ayurveda pain is described as Vedana, Ruja, Shoola,
Angamarda, Arti etc.
– Nociception – Pain due to noxisious stimuli

• Analgesics –
– Various types
– New drugs are introducing continuously due to
limitations and adverse effects of existing one.
– Bright future for Ayurvedic drugs in field of Analgesics
– Ayurveda system seeking potent analgesic drugs for
different types of pain
• Ayurveda and Analgesic
– Acharyas mentioned different drugs in Vedana-
Sthapaka, Shoolprashaman, Angamardaprashaman
Gana.
– These drugs should be tested by analgesic models and
Contd..

• Analgesic models
– Analgesic models are equally applicable for phasic or
acute pain as well as tonic or chronic pain.
– Centrally acting and peripherally acting analgesics. both
can be evaluated by analgesic models.
– Different thermal, mechanical electrical and chemical
stimuli are used.
– In-vitro models are also used.

– Haffner’s tail clip method, hot plate method, tail
immersion method, grid shock test, formalin test in rats
etc. are used mainly for central analgesic activity.
– Responses given by the subject of the analgesic models
helps to determine analgesic effect.
– Writhing test, Randall Selitto test, mechanical/visceral
pain models in the rat etc. Are used mainly for
peripheral analgesic activity.
– Animal models are necessary for pain research, having
various challenges.
– In Ayurveda, birds and other animals were used as
experimental model in few extent.
Contd..

CONCLUSION
• Analgesics have to be tested not only for their in
vitro activity, but also for their in vivo analgesic
potential
• The most commonly used methods for
measuring peripheral analgesics activity are the
writhing test in mice.
• Tail flick method, Hot plate method and
Haffner’s tail clip method are common methods
for measuring central acting analgesic effects.

• Herbs described as vedanastahpaka, vedanahara,
shothahara,
shoolprashaman,Angamardaprashaman could be
tested for their respective analgesic effects by
using analgesic models.
• Limitations of different models should be tackled
with modification in subject, assays and
measures.

References
• Charak Samhita
• Astanga Sangraha
• K. D. Tripathi. Essential of Medical Pharmacology
• Drug Screening Methods : SK Gupta
• Dr. Prakash L. Hegde; Evaluation of the Analgesic and Antiinflammatory Activity
of Kadamba (Anthocephalus codamba Miq.): An Experimental Study
• DANIEL LE BARS,MANUELAGOZRIU, AND SAMUELW.CADDEN-Animal Models of
Nociception,The American society for pharmacology and experimental
therapeutics : vol 53 no.4 ; 597- 612
• Parle Milind, Yadav Monu. Laboratory Models for Screening Analgesics.
International research Journal of Pharmacy. 2013;4 (1): p-15-19
• Jeffery, S. Mogil, Karen D. Davis, Sturt W. Derbyshire The necessity of animal
models in pain research – comprehensive review, PAIN 151 (2010); p.12-17
• Jeffery, S. Mogil – Animal models of pain: progress and challenges, Nature
Reviews / Neuroscience; 2009 April: (10); p-283-294

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