2. Introduction
Typically, PAIN is a direct response to an untoward
event associated with tissue damage, such as injury,
inflammation or cancer.
But
Severe pain can arise independently of any obvious
predisposing cause (e.g. Trigeminal Neuralgia)
Persist long after the precipitating injury has healed
(e.g. Phantom Limb Pain).
It can also occur as a consequence of Brain Or Nerve
Injury (e.g. following a Stroke or Herpes Infection).
Painful conditions of the latter kind, not directly linked
to tissue injury, are often described as ‘Neuropathic
Pain’.
3. CLASSIFICATION OF PAIN
Based on source/ location/ referral & duration
ACUTE PAIN /
TRAUMATIC PAIN
CHRONIC PAIN
VISCERAL
/SPLANCNIC
PAIN
SOMATIC PAIN MALIGNANT PAIN
OR
CANCER PAIN
NON – MALIGNANT
PAIN
OR
BENIGN PAIN
SUPERFICIAL PAIN
OR
CUTANEOUS PAIN
DEEP SOMATIC
PAIN
MUSCULOSKELETAL
PAIN
NEUROPATHIC PAIN
4. NEURAL MECHANISMS OF PAIN
Nociceptive Afferent Neurons
Modulation In The Nociceptive Pathway
Hyperalgesia And Allodynia
Transmission Of Pain To Higher Centres.
Descending Inhibitory Controls
Neuropathic Pain
Chemical Signalling In The Nociceptive Pathway
• Chemosensitivity Of Nociceptive Nerve Endings
Transmitters And Modulators In The Nociceptive
Pathway
5. A FIBRES C FIBRES
A – BETA
FIBRES
A – DELTA FIBRES
Large
Myelinated
Fast conducting
Low stimulation
threshold
Respond to light
touch
Small
Lightly Myelinated
Slow conducting
Respond to heat,
pressure, cooling &
chemicals
sharp sensation of
pain
Small & unmyelinated
Very slow conducting
Respond to all types of
noxious stimuli
Transmit prolonged dull
pain
Require high intensity
stimuli to trigger a
response
Nociceptive Afferent Neurons
7. Various stimuli (physical and chemical) can initiate or enhance the rate of
action potential firing in nociceptive primary afferent neurons (i.e. induce pain).
These afferent fibres project to the dorsal horn of the spinal cord where they
synapse on neurons projecting to higher centres.
Spinal nociceptive afferent
neurones end in Grey Matter of
dorsal horn.( lamina 1 and 2 mostly)
nociceptive afferent neurones
Release Glutamate and ATP at their
central synapse in DRG.
SG is rich in both endogenous
opioid peptides and Opioid
receptors.
Substance P and CGRP Present in
dorsal horn are mediators both at
Central and Peripheral Terminals.
8. Modulation In The Nociceptive Pathway
Acute pain is generally well accounted for in
terms of nociception—an excessive noxious
stimulus giving rise to an intense and
unpleasant sensation.
Chronic Pain states are associated with
aberrations of the normal physiological
pathway, giving rise to hyperalgesia and
allodynia or spontaneous pain without any
precipitating stimulus.
9. Hyperalgesia And Allodynia
Involves both sensitisation of peripheral nociceptive nerve terminals
and central facilitation of transmission at the level of the dorsal horn
and thalamus—changes defined by the term neuroplasticity.
• Peripheral component-bradykinin and prostaglandins acting on the
nerve terminals.
• central component- facilitation of synaptic transmission in the dorsal
horn of the spinal cord.
‘WIND-UP’—i.e. the synaptic potentials steadily increase in amplitude
with each stimulus—when repeated stimuli are delivered at
physiological frequencies. (long term potentiation in the hippocampus.)
In the dorsal horn, the facilitation is blocked by NMDA receptor
antagonists and also in part by antagonists of substance P and by
inhibitors of nitric oxide (NO) synthesis.
10. Transmission Of Pain To Higher Centres
Dorsal Horn
Contra Lateral ST Tract
Ventral and Medial Part of Thalamus
SST
11. Descending Inhibitory Controls
Cortex
Thalamus
PAG
Rostro Ventral Medulla
Nucleus Raphe
Magnus
Dorsal Horn Periphery
, 5HT,ENK
PONS
Locus Ceruleus
, NE
PAN
NSAID
NRPG
+
PAIN TRANSMISSION Through
STT Is gated by
Descending Inhibitory
pathways
Aβ fibres.
In event of repeated
stimulation,
May lead to activity dependent
facilitation of transmission
(wind up).
NRPG
Nucleus Reticularis
ParaGigantocellularis
13. Chemical Signalling in The Nociceptive Pathway
Ligand gated ion channels- Acid Sensitive Ion Channel, ATP sensitive channels, Capsaicin
sensitive Channel.
GPCRs regulate channel function via 2nd Messenger.
Growth factor like NGF act via TrkA receptors to control ion channel function and gene
expression.
14. Chemical Signalling in The Nociceptive Pathway
Mediators Comments
•TRP Channels (Transient
Receptor Potential)
TRPV1, TRMPM8, TRPA1
AGONIST –
Capsaicin
Anandamide
•Kinins Bradikinin , Kallidin
Potent pain producing substance
Acts on B2 receptors , facilitates TRPV1
opening.
•Prostaglandins Enhance Pain Producing Effet Of 5HT,
BRADYKININ.
Analgesic NSAIDS act by inhibition of PG.
•ATP Downregulation of P2X3 reduces
inflammatory pain.
15. Transmitters And Modulators In The Nociceptive Pathway
Endogenous Opioid Peptides
CGRP
SUBSTANCE P- Antagonists at NK1 receptors shown to
be effective analgesic drugs in animal model.
Glutamate
GABA
ATP
5HT- transmitter of inhibitory neurones.
Noradrenaline - transmitter of inhibitory neurones.
Adenosine
GALANIN
16. Opioids
Opioid: any substance, whether endogenous or
synthetic, that produces morphine-like effects that
are blocked by antagonists such as Naloxone
Opiate: compounds such as morphine and codeine
that are found in the opium poppy.
Narcotic Analgesic: old term for opioids; narcotic
refers to their ability to induce sleep. Unfortunately,
the term narcotic has subsequently been used
inappropriately by some to refer generically to
drugs of abuse.
18. Opioids
Important morphine-
like agonists include
Diamorphine,
Oxycodone And
Codeine.
The main groups of
synthetic analogues are
• piperidines (e.g.
pethidine and fentanyl)
• methadone-like drugs
• benzomorphans (e.g.
pentazocine)
• thebaine derivatives
(e.g. buprenorphine).
19. Opioid Receptors
μ Receptors are responsible for most of
the analgesic effects of opioids, and for
some major unwanted effects
(e.g. respiratory depression, euphoria,
sedation and dependence). Most of the
analgesic opioids are μ-receptor
agonists.
δ Receptor activation results in analgesia
but also can be proconvulsant.
κ Receptors contribute to analgesia at
the spinal level and may elicit sedation,
dysphoria and hallucinations. Some
analgesics are mixed κ agonists/μ
antagonists.
ORL1 receptors are also members of the
opioid receptor family. Activation results
in an antiopioid effect (supraspinal),
analgesia (spinal), immobility and
impairment of learning.
20. Opioid Receptors
σ Receptors are not true opioid receptors but are the
site of action of certain psychotomimetic drugs, with
which some opioids also interact.
All opioid receptors are linked through Gi/Go-
proteins and thus open potassium channels (causing
hyperpolarisation) and inhibit the opening of calcium
channels (inhibiting transmitter release). In addition
they inhibit adenylyl cyclase and activate the MAP
kinase (ERK) pathway.
Functional heterodimers, formed by combination of
different types of opioid receptor or with other types
of G-protein-coupled receptor, may occur and give
rise to further pharmacological diversity.
21. In vitro tests
3H-Naloxone binding assay-
Opiate agonist and antagonists have ability to displace
Radiolabelled Naloxone . IC50 values for 3H-Naloxone in the
presence or absence of Na+ is calculated.
3H-Dihydromorphine binding to μ opiate receptors-
3H-Dihydromorphine is selective for μ receptor, The test is used
to detect compounds that inhibit binding of 3H-DHM in a
synaptic membrane preparation obtained from rat brain.
3H-Bremazocine binding to κ opiate receptors –
Bremazocine is potent agonist of Κ receptor ,binding to μ and δ
receptors is prevented by inclusion of the peptide DAGO (Tyr-
D-Ala- Gly-N-Me-Phe-Gly-ol) to mask the μ receptor and of [D-
Pen2,5]-enkephalin (Tyr-D-Pen-Gly-Phe-D-Pen) to mask the δ
receptor.
22. In vitro tests
Inhibition of Enkephalinase-
Enkephalins are endogenouus opioid peptide, enkephalinase inhibitor Triptophan
shows antinociceptive activity in mice. The inhibitory potencies of test
compounds are compared with the standard.
Receptor binding of Nociceptin-
Nociceptin/OFQ acts on Nociceptin Opioid Receptor(NOP/ORL1). Nociceptin
induces analgesia when administered intrathecally.
Vasoactive intestinal polypeptide (VIP) and pituitary adenylate
cyclase-activating peptide(PACAP) binding assay-
Important In Somatosensory Processing Of Pain. Altered transmission of sensory
information in neuropathic pain.
23. In vitro tests
Cannabinoid receptor binding assay.
Cannabinoids have been shown to produce analgesia without the respiratory
problems associated with opioid analgesics. CB1 (CENTRAL) , CB2
(PERIPHERAL) receptors for cannabiniod. Anandamide produces Anti
nociception.
Vanilloid Activity-
Capsaicin and related vanilloids are unique in that the initial stimulation by
vanilloids is followed by a long lasting refractory state.
The endogenous ligand of CB1 cannabinoid receptors, anandamide, is also a
full agonist at vanilloid VR1 receptors.
A detailed pharmacological characterization was conducted using the Ca2+-
sensitive dye, Fluo3AM in a fluorimetric imaging plate reader (FLIPR).
Several Vanilloid receptor antagonists were described, such as Capsazepine or
Iodo-resiniferatoxin.
Inhibition of rapid heat responses in nociceptive primary sensory neurons of
rats by Vanilloid receptor antagonists has been shown.
24.
25. In-vivo tests for Central Analgesic activity
Model using mechanical stimulus-
1. Haffner’s Tail Clip Method In Mice.
Model using thermal stimulus-
1. Tail Flick Or Other Radiant Heat Methods,
2. Tail Immersion Tests.
3. Hot Plate Methods In Mice Or Rats,
Electrical Stimulation
1. (Grid Shock, Stimulation Of Tooth Pulp Or Tail).
2. Monkey Shock Titration.
Chemical stimulus-
1. Formalin Test In Rats.
26. Peripheral Analgesic Activity
Writhing Tests.
Pain in Inflamed Tissue-RANDALL-SELITTO-Test
Mechanical Visceral Pain Model in the Rat.
Antagonism Against Local Effects of
Bradykinin.
Effect of Analgesics on Spinal Neurons.
Antagonism to Nerve Growth Factor.
27. Chronic Pain Models
Neuropathic Pain
1. Chronic Nerve Constriction Injury
2. Peripheral Nerve Injury Model
3. Spared Nerve Injury Model
4. Spinal Cord Injury
Chemotherapy-Induced Pain
Trigeminal Neuropathic Pain Model.
Migraine Model in Cats
28. Post Operative Pain Model
1. Persistant Post Thoracotomy Pain Model.
2. Rat Model of Incisional pain.
29. Mechanical stimulus models
• Tail-clip method:
1. Noxious stimulus by using artery clip placed at the root of tail.
2. Response- biting the clip/tail.
3. Reaction time is noted.
4. Cut off time- average reaction time plus 3 times the sd of the combined
latencies of the control mice at all time period.
30. Tail flick test
• Use of light beam exerting radiant heat, focused to proximal 1/3rd of the
tail.
• Nociceptive spinal reflex response- flicking tail away from heat source.
• Escape reaction- turning the head away.(more reliable)
• Latency period is compared.
• Minimal inter animal variation.
31. Tail immersion test
• Young female Wister rats(170-210 g).
• Placed in cages with tail hanging out freely.
• Distal 5 cm tail is immersed (max 15 sec)
in a cup of warm water(55°C)
• Tail withdrawal reflex is seen
• Recording done after ½ , 1,2,3,4 and 6 hours.
• latency period ˃6s is taken as positive.
• Modifications- cold mixture of water & ethylene glycol at -10°C./ cold ethanol
at -20°C.
32. Hot plate method
• Purpose- to evaluate central analgesics.
• Methods-
1. Mice weighing 18-22g are used.
2. Standard or test drug given orally/ sc
3. Animals placed on hot plate(55-56° C.)
4. Response- jumping/paw withdrawal/ licking of paws.
5. Latency period is measured after 20, 60 & 90 min.
6. Those increasing the latency period at least 50% are taken positive.
7. ED-50 values are calculated.
• Drawback-
• False positive results with sedative/ muscle relaxants/ psychotomimetic.
33. Electric stimulus models
Grid - shock test
Drugs like Morphine, Acetylsalicylic acid can be measured by the Flinch – jump
response.
• Male mice (18-20g)
• The floor of the box is wired with stainless steel wire
• The stimulus in the form of
square wave pulses ( 30 cps).
• The output of stimulator is connected
to alternate wires of grid.
• The fixed resistance is placed with
the grid & parallel to an oscilloscope
to allow calibration in mill amperes.
34. Contd…..
• With increase in shock intensities the mice flinch, exhibit startling reaction &
increase locomotion or attempt to jump.
• The behavior is accurately reflected on the oscilloscope by marked
fluctuations of the displayed pulse.
• Pain thresholds are determined in each individual mouse twice before & after
the administration of the test drug.
• The average pain threshold values for each group at each time interval are
calculated and statistically compared with the control values.
35. Electric stimulus models
• Tooth pulp test-
1. Rabbits(2-3kg) are anaesthetized with thiopental at 15 mg/kg i.v.
2. Clamping electrodes are placed into tooth pulp chamber through drilled
holes.
3. After 30 min, stimulus given by rectangular current(50 Hz) upto 1 sec
4. Animals starts licking- current threshold is measured.
• Modification: stimulus given via subcutaneous electrodes at the tail
• Monkey shock titration test: shock given via Coulbourn Instrument
Programmable shocker at tail
36. Chemical stimulus models
• An irritant/ algogenic chemical agent given as nociceptive stimulus
• Slower mode of stimulation.- progressive & persisting for longer duration
• Both for central & peripheral analgesics
• Formalin test-
o 10% formalin injected at paw.
o Biphasic response
Early: immediately due to chemical stimulation of nociceptors causing C-
fibre activation
Late: after 10-15 min due to combination of an inflammatory reaction and
functional changes at dorsal horn of spinal cord.
Opioid effective in both phase, but NSAIDs are effective in only second
phase.
37.
38. Peripheral Analgesic Activity
Writhing test
• Model for visceral/peritoneal pain
• Used to detect peripheral analgesic activity of a compound.
• Mice(20-25g) are given i.p injection an aliquot of 0.25 ml of
phenylquinone(0.02%) suspended in 1% suspension of
carboxy methylcellulose
• A characteristic stereotyped behavior known as writhing is
seen.
• The writhing phenomenon can also be observed in rats
(Fukawa et al. 1980).
39. Writhing test
• Series of contraction along abdominal wall, turning movements of the
body and extension of hind limbs.
• Number of writhes ( stretching of the abdomen with at least one hind
limb) are recorded for 10 min.
• Other chemicals- acetic acid, acetylcholine, bradykinin, PGE1, 4% NaCl,
ethacrynic acid.
41. Peripheral Analgesic Activity
Randal Selitto test:
• Inflammation increases the sensitivity to pain and that this sensitivity is
susceptible to modification by Analgesics.
• Inflammation decreases the pain reaction threshold and this low pain
reaction threshold is readily elevated by non-narcotic analgesics of the
salicylate type as well as by the narcotic analgesics.
42. Peripheral Analgesic Activity
• Induction of hyperalgesia by producing inflammation via sc
injection of Brewar’s yeast at hind paw, followed by application
of pressure.
• Brewers yeast has been used as an inducer for inflammation
which increases pain after pressure.
Mechanical visceral pain:
Ethical constraints prevent repeated assessments in a single
animal by phenylquinone induced writhing tests based on acute
inflammation and it is a noxius stimulus.
To overcome these constraints, a model for mechanical visceral
pain was developed based on repeatable and reversible
distension of duodenum in the rat by intraduodenal balloon
catheter.
43. Peripheral Analgesic Activity
Antagonism Against Local Effects of Bradykinin
• Deffenu et al. (1966) and Blane (1968) used the
bradykinin-induced effects after intra-arterial injection
in rats as an assay for analgesic drugs.
• Paravascular sensory nerves which accompany blood
vessels to end in unmyelinated free-branching
terminals close to the capillaries and venules most
likely carry the chemoreceptors of pain.
• The criterion for protection is the disappearance of the
bradykinin effect after at least 2 consecutive doses of
bradykinin.
• Using groups of 10 rats for various dose levels, ED50
values are calculated.
44. Peripheral Analgesic Activity
Effect of Analgesics on Spinal Neurons-
• . Medial articular nerve (MAN) exposed at knee joint. Bipolar
electrodes are inserted at the MAN near the knee for stimulation
of articular afferents.
• The saphenous nerve is cut in the inguinal fossa for recording.
• For recordings from spinal cord neurons the
spinal segments T12–L7 are exposed by laminectomy.
• Acute arthritis in the right knee joint is induced
several hours before recordings are started by injecting
0.3–0.5ml of 4%kaolin suspension and 15–20 min
later 0.3ml of 2%carrageenan solution. Acute arthritis
develops within 1–3 h.
• Effects of the test substance on ongoing and mechanically evoked
activity (by movements, pressure stimuli) are determined.
45. Peripheral Analgesic Activity
Antagonism to Nerve Growth Factor
• The expression of NGF is high in injured and
inflamed tissues, and activation of the receptor
tyrosine kinase trkA in nociceptive neurons triggers
and potentiates pain signaling by multiple
mechanisms.
• NGF is the major mediator of both inflammatory
and neuropathic pain.
• An effective pain therapeutic needs to prevent the
activation of TrkA by NGF, may be done by
molecules that remove free NGF, prevent binding,
prevent activation of TrkA.
46. Antagonism to Nerve Growth Factor
In Vivo Methods
• Ma and Woolf (1997) reported that the progressive tactile
hyperalgesia induced by peripheral inflammation is nerve growth
factor dependent.
Nerve Ligation Injury
Intrathecal Catheter Placement
Thermal Sensitization
Evaluation of Tactile Allodynia
In vitro methods
Radiolabeled Neurotrophin and Receptor Preparation
Chemical Cross-Linking of 125I-NGF to TrkA
and/or p75NTR in the Presence of Antagonists and
Immunoprecipitation.
Neurotrophin Receptor Binding
TrkA Phosphorylation Assay
NGF Protomer Cross-Linking
47. Chronic pain models
• Neuropathic pain – Partial injury to
somatosensory nerves sometimes
causes causalgia in humans.( spontaneous
burning pain combined with
hyperalgesia and allodynia and usually follows
an incomplete peripheral nerve injury.)
Chronic nerve constriction injury
Peripheral nerve injury
Spared nerve injury
Spinal cord injury
Neuroma formation
48.
49. Chronic Pain Models
Chemotherapy-Induced Pain-
• Vincristine, an antineoplastic agent widely used in
cancer therapy, was found to be neurotoxic for all
treated patients and can induce peripheral neuropathy.
• Vincristine (100mcg/kg) administered daily for 2 weeks,
decrease in mechanical nociceptive threshold and
Hyperalgesia occurs after the 2nd day of administration.
• Responses gradually return to baseline following
discontinuation of treatment.
Trigeminal Neuropathic Pain Model-
neuropathy is produced by a chronic constriction injury of
the Infraorbital Branch Of The Trigeminal Nerve.
50. Chronic Pain Models
Craniovascular Pain in cats by stimulating
• The superior sagittal sinus and monitoring trigeminal
neuronal activity using electrophysiological
techniques.
• The suppression or activation of cell firing was
determined from both peri-stimulus and post-
stimulus histograms using the criteria of a shift of
>30% from baseline.
51. Post surgical pain model Brennan et al
•Anaesthetized rats with ether and the plantar
surface of the left hand paw prepared in a sterile
manner.
•A 1 cm longitudinal incision made with a number
10 scalpel, through skin and fascia of the plantar
aspect of the paw, starting 0.5 cm from the
proximal edge of the heel and extending toward
the toes.
•The plantaris muscle was elevated and incised
longitudinally. Following hemostasis with gentle
pressure, the skin was opposed with 2 single
interrupted sutures using 5-0 nylon. The wound
site was covered with povidone-iodine and
antibiotic powder.
•Radiant heat ,Mechanical stimulatiion, Cold
stimulation applied .
•Paw Withdrawal Latency measured.
52. Persistant Post Thoracotomy Pain Model
• Chronic post thoracotomy pain recurs /persists
after thoracotomy incision at least 2 months
following surgical procedure.
• Allodynic response (Radiant heat ,Mechanical
stimulatiion, Cold stimulation ) and
Histopathologic changes after Thoracotomy and
Rib Retraction are observed to evaluate anti
nociceptive potential of test drugs.