know about pain!!!!!

1. screening of analgesics & drugs used in
arthritis & neuropathic pain
Made by: Meenakshi
Gupta

2. ANALGESICS

3. INTRODUCTION
 Pain is a complex unpleasant phenomenon
composed of sensory experiences originating from
damaged tissue or abnormal physiological
condition.
 Analgesics are those agents which selectively
relieve pain by acting in the CNS or peripheral pain
mechanisms without significantly altering
consciousness. Analgesics may be narcotic or non-
narcotics.
Narcotic analgesic Non-narcotic analgesic
Act centrally Act peripherally
Cause addiction Do not cause addiction
Produce CNS depression Do not produce CNS
depression
Show no anti-inflammatory
effect
Show anti-inflammatory
effect
e.g. Morphine, tramadol,
pethidine
e.g. Diclofenac, ibuprofen,
aspirin

5. EVALUATION TECHNIQUES
IN-VIVO MODELS:
 Pain state models using thermal stimuli
 The tail-flick model
 Paw-withdrawal test
 Hot-plate test
 Pain- state models using cold stimuli
 Pain state models using mechanical
stimuli
 Strain gauges
 Von- frey filaments

6.  Pain state 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.
 Pain state models using chemical stimuli
 Formalin test
 Acetic acid induced writhing test
 Stimulation of hollow organs

7. IN-VITRO MODELS:
•
3
H-Naloxone binding assay
•
3
H-Dihydromorphine binding to µ opiate receptors
in rat brain
•
3
H-Bremazocine binding to κ opiate receptors in
guinea pig cerebellum
• Isolated tissue preparation

8. IN-VIVO MODELS:
The tail- flick model
Principle: The application of thermal stimulation to
the tail of an animal provokes the withdrawal of the
tail by a brief vigorous movement. The withdrawal of
the tail from the heat source is referred to as ‘tail-flick
latency’.
Procedure:In this model, timer is started at the same time as the application
of the
heat source
Time taken by the rat to withdraw the tail is recorded.
Usually withdrawal time is within 2 to 10s

9.  The lengthening of this reaction time by the animal
seen after the administration of drug is interpreted
as an analgesic action
Merits:
 Effective for screening morphine like analgesics
 Simple technique & does not require any special
skill
 Results are quiet accurate & less time consuming
Demerits:
 Tail- flick response is prone to habituation
 Truly efficient only for revealing the activity of
opioid analgesics
 It is advisable not to prolong the exposure to
radiant heat beyond 20s.

10.  Modification:
1. Ultra- sound induced tail-flick procedure
2. Ear-withdrawal by applying radiant heat
3. The tail flick formalin test in rodents

11. The hot plate method
Principle: A plate heated to a constant temperature
produces two behavioral components that can be
measured in terms of their reaction time, namely paw
licking & jumping. Both are considered to be supra-
spinally integrated responses.
Procedure:
Introduction of rat or mouse into an open-ended cylindrical
open space with a floor consisting of metallic plate that is
heated by a thermode or a boiling liquid.
The paw licking behaviour is affected only by opioids
whereas the jumping reaction is increased equally by
less powerful analgesics especially when the
temperature is 50 degree or less.

12.  The specificity & sensitivity of the test can be
increased by measuring the reaction time of the first
evoked behaviour. Chaotic defensive mechanisms in
rats includes sniffing, licking fore paws & hind paws,
straightening, stamping of feet.

13. Mechanical method:
Procedure:
The preferred sites for applying nociceptive
mechanical stimuli are the hind paws and the tail.
a pressure of increasing intensity is applied to a
punctiform area on the hind paw or on the tail
The paw or tail is jammed between a plane
surface & a blunt point mounted on top of a
system of cogwheels with a cursor that can be
displaced along the length of graduated beam

14. Disadvantages:
 Sometimes difficult to measure the intensity of
stimulus with precision.
These devices permits the application of increasing
measurable pressures and the interruption of test,
when threshold is reached
The measured parameter is the threshold for the
appearance of a given behaviour
When the pressure increases, the reflex
withdrawal of the paw, or a complex movement of
the animal to release its trapped limb & finally a
vocal reaction is observed.

15.  A non negligible level of the variability of responses.
 Repetition of a mechanical stimulus can produce a
diminution or conversely an increase in the sensitivity
of the stimulated part of the body.

16. Electrical stimulation of tail
Principle: Electrical stimuli of gradually increasing intensity can
be applied in sequence through subcutaneous electrodes placed in
tail of the rat or the mouse. When such gradually increasing intensity
of electrical stimuli are applied from constant voltages, one can
observe a reflex movement of tail & vocalisation.
 Morphine or morphine like drugs are effective in this model.
 There may be chances of death of the animal due to the
electrical stimulation.
Modifications: ultrasonic stimulation of the tail may be used
in placed of electrical stimulation. This method is fast, simple
& precise.

17. Grid-shock model
Procedure:
Male mice weighing around 18-20g are individually
placed in clear plastic chambers
The floor of the box is wired with tightly strung
stainless steel wire, space about 1mm apart
The stimulus is given in the form of square wave
pulses, 30 cycles per second.
The output of the stimulator has to be connected to
alternate wires of the grid. A fixed resistance is
placed in series with grid and in parallel to

18. The behavior is accurately reflected on the oscilloscope by marked
fluctuations of the displaced pulse and defined as pain threshold
response. Pain threshold are determined in individual mouse twice
before administration of test drug and 15, 30, 60, 90 & 120 minutes
after dosing.
Modification: fractional escape procedure
With increasing shock intensities, the mice flinch,
exhibit a starling reaction, increased locomotion &
attempt to jump

19. Electrical stimulation of the tooth pulp
Principle: This method is based on the stimulation of the
tooth pulp of the animal by applying the electrical current.
Stimulation of the tooth pulp produces characteristic reactions
such as licking, biting, chewing & head flick due to induction of
pain.
Procedure:
Rabbits of either sex are anesthetized with 15 mg/kg thiopental
Pump chambers are exposed close to the gingival line in the lateral
margins of the two front upper incisors with a high speed dental-drills.
On the day of experiment, clamping electrodes are placed into the
drilled holes
After an accommodation period of 30 min, stimulation is
started to determine the threshold value. The stimulus of
frequency of 50 Hz is applied for the duration of 1s. The
electrical current is started with 0.2 mA & increased till licking
occurs.

20. Monkey shock titration test
Procedure:
Doses of 3.0 mg/kg i.m. morphine, 1.7 mg/kg i.m. methadone & 10
mg/kg i.m. pentazocine were found to be effective. It may be used for
the final evaluation of a new compound before administration to man.
Monkeys are seated in restraining chairs. Electrical current is delivered by
shockers through electrodes coupled to two test tube clamps, which are
attached to the shaved portion of the tail.
The current ranges from 0 to 4 mA through 29 progressive steps. The
monkey presses the bar to interrupt the shock. A stable baseline
shock level is established for each monkey on the day prior to drug
administration
After drug administration, shock titration activity is rated according to
the change in maximum level of median shock intensity attained for
the drugs as compared to control levels.

22. Formalin test
Principle: chemical stimulation involving the
administration of algogenic agents represents a slow,
progressive and irreversible form of stimulation.
Procedure:
Painful behavior can be assessed on the four-level scale related to
posture: 0 denotes normal posture; 1denotes the injected paw
remains on the ground but not supporting the animal; 2 denotes the
injected paw clearly raised and 3 denotes the injected paw being
licked, nibbled & shaken.
Formalin is injected into the front paw and reaction is recorded as
excessive licking & biting of the paw.
Analgesic response or protection is indicated, if both paws rest on
the floor

23. Acetic-acid induced writhing
Pain is often induced in rats or mice by injecting certain irritants such
as phenyl quinone or acetic acid into the peritoneal cavity. The
animal reacts with a characteristic stretching behaviour which is
called writhing. The i.p. administration of agents that irritate serous
membrane elicit abdominal contractions.
Modifications:
 Use of phenyl benzoquinone & acetyl choline
 Use of substance P & capsaicin in rats in knee joints
 Intraplantar injection of
LPS, bradykinin, carragen-
an, TNF-α etc.

24. IN-VITRO MODELS:
3H-Dihydromorphine binding to µ receptors
Purpose and rationale
µ Receptors are considered to mediate the supra-spinal activity of
opioids. 3H-Dihydromorphine (3H-DHM)exhibits some selectivity for the µ
receptor, a high affinity opiate binding site.
Tissue preparation: Male Wistar rats are sacrificed by decapitation &
whole brain homogenated.
Assay
• 850 µl tissue suspension
• 80 µl distilled water
• 20 µl vehicle, or levallorphan, or appropriate concentration of drug
• 50 µl [3H]DHM.
Evaluation
• Specific binding is defined as the difference between total binding
and binding in the presence of 0.1 mM levallorphan.
• IC50 values are calculated from the percent specific binding at each
drug concentration.

25. NEUROPATHIC PAIN

26. ‘The most terrible of all tortures, which a nerve wound may inflict’.
Neuropathic pain is characterized by the sensory abnormalities
such as unpleasant abnormal sensation (dysesthesia), an
increased response to painful stimuli (hyperalgesia), and pain in
response to a stimulus that does not normally provoke pain
(allodynia).
Classification of models:
a. Central pain models: Weight drop model
Excitotoxic spinal cord injury
b. Peripheral nerve injury models: Chronic constriction injury
model
Partial sciatic nerve ligation
L5/L6 spinal nerve ligation
model
c. Cancer pain models: Cancer invasion pain model
Vincristine induced peripheral neuropathic
pain

27. Weight drop model
In this model, the spinal cord at lower thoracic-lumbar level is
exposed and a constant weight is dropped over the nerve to
produce an injury, which is characterized by severe paraplegia
and complete segmental necrosis.
Procedure:
After anesthesia, laminectomy is performed at the vertebral lower
thoracic-lumbar region (T-10) level to expose dorsal spinal cord
A brass guide tube (15 cm in length) is positioned perpendicularly
above the exposed cord, and a cylindrical 10 g steel weight (2 mm in
diameter) with a rounded tip is suspended within the tube
The weight is allowed to drop on the exposed cord, at the T12–13
segmental level, to produce cord injury.

28. The hypersensitivity to light mechanical stimulation of the skin has been
noted to develop within 1 day of injury and parallels the allodynia
experienced by patients rapidly following spinal injury
Alternatively, a longitudinal incision is made on the midline of the back
to expose T8 vertebrae by dissecting the paravertebral muscles. It is
followed by a four-level T6–T7 laminectomy to expose the spinal cord,
and injury is
produced by extradural compression of the spinal cord using an
aneurysm clip with a closing force of 24 g

29. Excitotoxic spinal cord injury
Intraspinal injections of a-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid (AMPA) metabotropic receptor agonist
quisqualic acid (QUIS) have been made to simulate injury-induced
elevations of excitatory amino acids.
Procedure:
Intraspinal or intrathecal injections of glutamate,N-methyl-D-
aspartic acid, kainic acid, dynorphin A, serotonin, and tryptamine
have also been reported to produce SCI-related pain behaviors.
The unilateral injections are made between the dorsal vein and dorsal
root entry zone at depths ranging from 300 to 1200 mm below the
surface of the spinal cord, at the levels ranging from T10 to L4
The intraspinal injection of QUIS has been made to produce
excitotoxic injury resulting in neuronal loss in specific regions of the
spinal gray matter and produces ‘spontaneous’ and/or ‘evoked’ pain
behaviors

30. Chronic constriction injury model
The behavioral signs of spontaneous pain guarding, excessive
licking, and avoidance of placing weight on the injury side,
mechanical and thermal hyperalgesia, chemical hyper-reactivity
and cold allodynia have been noted to develop within a week.
Under anesthesia, about 3-cm long blunt dissection is made into the
skin exposing the common sciatic nerve at the level of the mid-thigh
level and loose ligation of nerve with three ligatures (chromic silk,
Ethicon), at about 0.5 mm spacing, proximal to trifurcation of the nerve
This constriction of the sciatic nerve is associated with intraneural
edema, focal ischemia, and Wallerian degeneration

32. Partial sciatic nerve ligation
The left hind leg of rat is shaved and dissection is made to expose the
sciatic nerve at the upper-thigh level.
The dorsal one-third to half of the sciatic nerve is tightly ligated with a silk
suture just distal to the point at which posterior biceps semitendinosus
nerve branches off
The behavioral signs of spontaneous pain in the form of paw guarding
and licking on the injury side have been reported. The behavioral
alterations like cold allodynia, chemical hyper-reactivity, and mechanical
hyperalgesia have been noted to occur within 1 week after the surgery
and most of the changes persist for 6 weeks

34. Vincristine induced peripheral
neuropathic pain
Procedure: Administration of multiple doses of
vincristine (20, 75, 100 or 200 µg/kg i.v.) by tail vein
injection, followed by 500µl of saline to prevent
vessel deterioration, was reported to result in rapid
onset painful neuropathy. The dosage regimen of 75
lg/kg i.v. has been preferred because of maximal
hyperalgesia and relative absence of motor
impairment in rat.

35. ARTHRITIS

36. Canine anterior cruciate ligament
transection model
Principle:
Procedure:
ACL transection in the dog knee & joint instability
Cartilage erosion, fibrillation & formation of osteophytes
Dog anaesthetized with 30 mg/kg sodium pentobarbital i.v. followed by
continuous inhalation of halothane+ nitrous oxide+ oxygen
After shaving & sterilizing the knee joint externally, it is bent in a bent
position at 90º
Scalpel blade is inserted deep into the joint space diagonally posterior to
the ACL and parallel to the lateral border of the patellar ligament

37. Evaluation:
Microscopic inspection of cartilage & osteophytes are
recorded.
By the rotation of blade, ACL is dissected & blade withdrawn & the
wound is closed
After 8-12 weeks, fibrillation & erosion of cartillage

38. Spontaneous OA model in STR/IN mice
Procedure:
Mice of age 10 weeks are trained to walk on a slowly rotating cylinder
Record the mean walking time of each mouse
Mice showing moderate activity like neither dropping off too soon nor
staying on for longer time are selected
In 8-10 animals, the drug is applied systemically applied for 8 weeks
Mobility of each animal is recorded once or twice a week on rotating
cylinder experiment. Body weight recorded regularly.

39. Evaluation: mean walking time decreases with age &
disease progression.
At the end of experiment, animals are sacrificed, both knees are dissected,
fixed, decalcified & embedded in defined orientation for histology.

40. Thank you