Pain Models-Screening Assay in vitro & in vivo

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 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 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 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 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 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. 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. 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. 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. 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. S CR EE NING MODELS

10. IN- VITRO MODELS

11. In- Vitro Models

12. 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

13. HO T – P L A TE TE S T temperatures which are not damaging to skin. T he r e spons e s a re j u m p i ng, w it hdr a w a l of t he p a w a nd licking of the paws. The responses is prolonged after administration of centrally acti ng a n al g e s ic s, wh e r ea s p e r i ph e r a l a n al g e s ic s of t he acet y l s alic y li c ac i d or ph e ny l - ace ti c ac i d t ype do not generally affect these responses. t o h ea t at Purpose and rationale 1) The paws of mice and rats are sensitive

14. P r o ce du r e Groups of 10 mice (18-22g) are selected and divided into standard, test & control group respectively The temperature of the hot plate is maintained at 55° t o 56°C. The animals are placed on the hot plate & time (reaction time) until either licking or jumping occurs is recorded. The latency is recorded before & after 20, 60 and 90 min after the administration of standard or test compound.

15. Evaluation The prolongation of latency time between the test, standard and control animals are compared. Using various doses ED 50 values can be calculated.

16. TA I L F L I C K M O D EL Purpose & evaluation The tail flick test with radiant heat is an simplified method. The application of thermal radiation to the tail of an animal provokes the withdrawal of tail. The morphine like drugs are capable of prolonging the reaction time.

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

18. Evaluation T he t a i l fli ck l a t ency i n t he t es t , s t anda r d and con tr ol animals are compared Using various doses ED 50 values can be calculated

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

20. GRID-SHOCK TEST 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

21. 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.

22. 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.

23. 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.

24. Formalin test PURP OS E AND R A TIO NA L E 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. The test drug is administered simultaneously either sc. or orally. 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.

25. EVALUATION f or p r o tecti on c an be Using various doses, ED50 values calculated. T he for mali n te st i d e n ti f ie s mai n l y ce n t r all y acti ve 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

26. 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

27. 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.

28. 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 % i nh i b iti on = Mc – Mt x 100 (or % protection) Mc Mc = mean of wriths produced in control group Mt = mean of wriths produced in test group

29. GATE CONTROL THEORY OF PAIN 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 Therefore, stimulation by non-noxious input is able to suppress pain Proposed by Melzack and Wall in 1965 Science article "Pain Mechanisms: A New Theory” 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

30. 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

31. Gate control theory Activation of nerves which do not transmit pain signals, aka non-nociceptive fibers, can interfere with signals from pain fibers, thereby inhibiting pain. Afferent pain- receptive nerves, those that bring signals to the brain, comprise at least 2 kinds of fibers: 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

32. Proposed Neural Circuit diagram 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 The nonnociceptive fibers indirectly inhibit the effects of the pain fibers, 'closing a gate' to the transmission of their stimuli In other parts of the laminae, pain fibers also inhibit the effects of nonnociceptive fibers, 'opening the gate'. This presynaptic inhibition of the dorsal nerve endings can occur through specific types of GABAA receptors

33. Clinical Scoring of Pain Pain is subjective in nature