Concepts of Preclinical Screening- In vitro/ In vivo screening of models for Pain

1. Pharmacological screening of
preclinical models of
pain/algesia
Part-2 of Pre-clinical Models for screening of drugs
Priyansha Singh
B. Pharm, M.S. (Pharm.)- Pharmacology & Toxicology

2. Introduction
1) According to IASP pain is an unpleasant sensory and emotional experience which is
associated with actual or potential tissue damage
IS PAIN GOOD OR BAD
1) Pain constitutes an alarm that ultimately has the protective role. it induces to learn
avoidance behaviors as a result, may limit the (potentially) damaging consequences
2) An analgesic or painkiller is defined as agent which selectively relieve pain by acting
in the CNS or by peripheral pain mechanisms without significantly altering
consciousness

3. How do we sense pain

4. How do we sense pain
Pain receptors- Nociceptors
1) Type of receptor at the end of a sensory neuron's axon that
responds to damaging or potentially damaging stimuli by sending
“possible threat” signals to the spinal cord and the brain
2) If the brain thinks the threat is credible, it creates the sensation of pain
to direct attention to the body part, so the threat can hopefully be
mediated. This process is called nociception

5. How do we sense Pain
Peripheral to Central Transmission
• The peripheral terminal of the
mature nociceptor is where the
noxious stimuli are detected and
transduced into electrical energy.
• When the electrical energy reaches a
threshold value, an action potential
is induced and driven towards the
central nervous system (CNS).
• This leads to the train of events that
allows for the conscious awareness
of pain.

6. Types of Nociceptors
1) Thermal: Thermal nociceptors are activated by noxious heat or cold at various
temperatures. The first to be discovered was TRPV1. The cool stimuli are
sensed by TRPM8 channels.
2) Mechanical: Mechanical nociceptors respond to excess pressure or mechanical
deformation. They also respond to incisions that break the skin surface. These
mechanical nociceptors frequently have polymodal characteristics. TRPA1
receptors detects mechanical nociception. both)

7. 3) Chemical: Chemical nociceptors have TRP channels that respond to a wide
variety of spices such as capsaicin. Like in thermal nociceptors, TRPV1 can
detect chemicals like capsaicin and spider toxins.
4) Sleeping/silent: Although each nociceptor can have a variety of possible
threshold levels, some do not respond at all to chemical, thermal or mechanical
stimuli unless injury actually has occurred. These are typically referred to as silent
or sleeping nociceptors since their response comes only on the onset of
inflammation to the surrounding tissue.
5) Polymodal: Many neurons perform only a single function, therefore neurons
that perform these functions in combination are given the classification
"polymodal. For ex: TRPV1 (Thermal and Chemical

8. Physiological vs Pathological Pain

9. SCREENING
MODELS

10. IN- VITRO
MODELS

11. In- Vitro Models
3H- Naloxone
binding assay
Inhibition of
Enkephalins
Receptor binding
bioassay of
Nociceptin
Cannabinoids
binding assay
3H- bremazocine
binding assay
Vanilloid/
Capsaicin
receptor binding
assay
m- opiate receptor
binding assay

23. Pain - state models using Thermal stimuli
* Hot plate test
* Tail-flick model using radiant heat
* Paw-withdrawal latency (Hargreaves) test
Pain –state models using mechanical stimuli
* von Frey hairs test
* Randall sellito test
Pain - state models using Electrical stimuli
* Electrical stimulation of the tail
* Grid - shock test
* Stimulation of the limbs
Pain - state models using Chemical stimuli
* Formalin test
Animal models for the assessment of analgesic activity

24. HOT – PLATE TEST
temperatures which are not damaging to skin.
2) The responses are jumping, withdrawal of the paw and licking of the
paws.
3) The responses is prolonged after administration of centrally acting
analgesics, whereas peripheral analgesics of the acetylsalicylic acid
or phenyl-acetic acid type do not generally affect these responses.
to heat at
Purpose and rationale
1) The paws of mice and rats are sensitive

25. Procedure
1) Groups of 10 mice (18-22g) are selected and divided into
standard, test & control group respectively
2) The temperature of the hot plate is maintained at 55° to
56°C.
3) The animals are placed on the hot plate & time (reaction
time) until either licking or jumping occurs is recorded.
4) The latency is recorded before & after 20, 60 and 90 min
after the administration of standard or test compound.

26. Evaluation
1) The prolongation of latency time between the test, standard
and control animals are compared.
2) Using various doses ED50 values can be calculated.

27. TAIL FLIC K MODEL
Purpose & evaluation
1) The tail flick test with radiant heat is an simplified
method.
2) The application of thermal radiation to the tail of an
animal provokes the withdrawal of tail.
3) The morphine like drugs are capable of prolonging the
reaction time.

28. Procedure
1) Wistar rats (170-210g) are selected and divided into
standard, test & control group
2) Appropriate temperature is maintained on the radiant source
3) The tail of the rat is placed on the radiant source & time
taken for the rat to withdraw its tail is recorded.
4) Usually withdrawal time is within 2-10s
5) The tail-flick latency (TFL) is recorded before & after the
administration of standard or test compound.

29. Evaluation
1) The tail flick latency in the test, standard and control
animals are compared
2) Using various doses ED50 values can be calculated

30. Testing for mechanical Allodynia & Mechanical
hyperalgesia
von Frey hair test/Electronic von Frey
Anesthesiometer for mechanical allodynia
Randall selitto test for mechanical
hyperalgesia

31. GRID-SHOCK TEST
1) Purpose and rationale
The electric grid shock test in mice has been described by Blake et al.
The analgesic properties of drugs like morphine, acetylsalicylic acid can be
measured by the flinch–jump in response to electric shock in rodents

32. Procedure
Male mice (18-20g) are selected and placed individually
in plastic chamber
The floor of the box is wired with stainless steel wire
The stimulus is given in the form of pulses (30 cycles per
second)
With increase in shock intensities the mice flinch, exhibit
startling reaction & increase locomotion or attempt to
jump.
The fixed resistance is placed with the grid & parallel to
an oscilloscope to allow calibration in milliamperes.

33. 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.

34. Evaluation
• The current measured in milliamperes is recorded for
each animal before and after administration of the drug.
• The average pain threshold values for each group at
each time interval are calculated and statistically
compared with the control values.

35. Formalin test
PURPOSEAND RATIONALE
◦ The formalin test in rats has been proposed as a chronic pain model which is sensitive
to centrally active analgesic agents.
PROCEDURE:
Male Wistar rats weighing 180–300 g are administered 0.05 ml of 10% formalin
into the dorsal portion of the front paw.
Thetestdrug isadministeredsimultaneouslyeithersc.ororally.
Readings are taken at 30 and 60 min and scored according to a pain scale.
Pain responses are indicated by elevation or favoring of the paw or excessive
licking and biting of the paw.

36. EVALUATION
for protection can be
◦ Using various doses, ED50 values
calculated.
◦ The formalin test identifies mainly centrally active drugs,
whereas peripherally acting analgesics are almost ineffective.
◦ Therefore, the formalin test may allow a dissociation between
inflammatory and non-inflammatory pain, a rough classification
of analgesics according to their site and their mechanism of
action

37. WRITHING TESTS
Purpose and rationale
Pain is induced by injecting irritants like acetic acid into
peritoneal cavity of mice.
The animals react with characteristic stretching behavior which is writhing.
The test is suitable to detect analgesic activity of peripherally acting drugs.
Models for peripheral analgesic activity

38. Procedure
Mice (20-25g) are selected and divided into
standard, test & control group respectively
Appropriate volume of acetic acid solution is
administered to the mice (control group) and
placed individually in the glass jar.
The onset of writhing, abdominal contractions &
trunk twist response are recorded for 10 min.
The test and standard drug is administered 15 min
prior to the acetic acid administration.

39. Evaluation
 The writhing period is recorded and compared with the control
group.
 Writhing response in the drug treated must be less when
compared to the acetic acid treated control.
Analgesic activity was evaluated
% inhibition = Mc – Mt x 100
(or % protection) Mc
Mc = mean of wriths produced in control group
Mt = mean of wriths produced in test group

40. GATE CONTROL THEORY OF PAIN
1) The gate control theory of pain asserts that non-painful input closes the
"gates" to painful input, which prevents pain sensation from traveling to
the central nervous system
2) Therefore, stimulation by non-noxious input is able to suppress pain
3) Proposed by Melzack and Wall in 1965
4) Science article "Pain Mechanisms: A New Theory”
5) Gate control theory is considered to be one of the most influential
theories of pain because it provided a neural basis for pain perception
and modulation and ultimately revolutionized the whole pain research

41. 1. Authors proposed that both thin (pain) and large diameter (touch, pressure,
vibration) nerve fibers carry information from the site of injury to two
destinations in the dorsal horn of the spinal cord:
1) transmission cells that carry the pain signal up to the brain, and
2) inhibitory interneurons that impede transmission cell activity.
2. Thin fiber activity impedes the inhibitory cells (tending to allow the
transmission cell to fire) and large diameter fiber activity excites the inhibitory
cells (tending to inhibit transmission cell activity).
3. So, the more large fiber (touch, pressure, vibration) activity relative to thin
fiber activity at the inhibitory cell, the less pain is felt
Gate control theory
Proposed mechanisms

42. Gate control theory
1. Activation of nerves which do not transmit pain signals, aka non-nociceptive
fibers, can interfere with signals from pain fibers, thereby inhibiting pain.
2. Afferent pain- receptive nerves, those that bring signals to the brain, comprise at
least 2 kinds of fibers:
1) a fast, relatively thick, myelinated "Aδ" fiber that carries messages quickly with
intense pain, and
2) a small, unmyelinated, slow "C" fiber that carries the longer-term
throbbing and chronic pain.
3) Large-diameter Aβ fibers are non-nociceptive (do not transmit pain stimuli) and
inhibit the effects of firing by Aδ and C fibers
Nociceptive vs Non-nociceptive Fibers

43. Proposed Neural Circuit diagram
1) Some areas in the dorsal horn of the
spinal cord that are involved in receiving
pain stimuli from Aδ and C fibers, called
laminae, also receive input from Aβ
fibers
2) The nonnociceptive fibers indirectly
inhibit the effects of the pain fibers,
'closing a gate' to the transmission of
their stimuli
3) In other parts of the laminae, pain fibers
also inhibit the effects of nonnociceptive
fibers, 'opening the gate'.
4) This presynaptic inhibition of the dorsal
nerve endings can occur through specific
types of GABAA receptors

44. Clinical Scoring of Pain
 Pain is subjective in nature