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Screening of Analgesic Drugs pHARMACOLOGY.pptx

This document discusses various models of pain, including acute and chronic pain, and their underlying mechanisms involving nociception and neurotransmitters. It outlines different experimental methods to study pain in animals, such as hot plate tests, tail-flick tests, and models for neuropathic and cancer pain. The conclusion emphasizes that while several pain models exist, none perfectly emulate human pain experiences, particularly in assessing the affective components of neuropathic pain.

Health & Medicine◦
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Dr. Devesh pandey MBBS, MD
 Introduction  Pathway of pain  Animal models of acute pain  Animal models of chronic pain  Conclusion
 Pain is an unpleasant bodily sensation evoked due to external/ internal noxious stimuli. Various types of pains are seen in humans like somatic pain, visceral pain etc  Pain is produced by excitation of receptors, the nocireceptor, which respond to a broad spectrum of physical and chemical noxious stimuli. Hence called as nociception  The afferent fibres that are excited by noxious stimuli are non-myelinated C-fibres with low conduction velocity & Aδ myelinated fibres with rapid conduction
 Pain are of two types : acute or chronic Acute pain is of short duration, and pain which lasts 6months or longer is chronic pain, it includes cancer pain, arthritic pain
 From the site of pain generation, the pain senses are carried by Aδ & C fibres whose cell bodies are situated in the dorsal root ganglion  The central processes of the neuron, lying in the sensory root of the spinal nerve enters the dorsal horn to terminate in the substantia gelatinosa rolandi situated in the tip of dorsal horn  From the SGR, 2nd order neuron arises crosses to the opposite side then moves up through the white matter of spinal cord to reach the brain  Most of these 2nd order neurons travel up as spinothalamic tract then ultimately terminates in the proper area of the thalamus
 From these specific nuclei of thalamus, next order neuron arises to terminate on cerebral cortex as well as cingulate gyrus  The neurotransmitter at synapse between Aδ and 2nd order neuron is glutamate while between C fibres and 2nd order neuron is substance P  Other 2nd order neuron also cross to the opposite side to reach different area of brain  The tip of the dorsal horn plays a key role in modification of pain perception called gate, so also controls the SGR involved in the incoming traffic of pain
Hot Plate Model Animal models of acute pain
Hot plate method : widely used to evaluate opioid analgesics Procedure mice is placed on hot plate ↓ Temperature of the hot plate is maintained 55 -56◦ C ↓ Responses like jumping , withdrawal of paws and licking of paws are seen ↓ Time period between the animal placed and response occur is recorded by stop watch ↓ Test compound is administered orally or subcutaneously, values are compared with the values before administration of the test drug
The tail-flick test : In this heat is used as a noxious stimuli. The stimulus causes the rat to flicks its tail away from the heat source The two variant of the tail-flick test : -Applying radiant heat to a small surface of the tail -Involving immersion of tail in water at a predetermined temperature
Procedure Rodents are placed into small cages , leaving tail exposed ↓ mouse tail is held gently by the observer, a light beam is focused to the proximal third of the tail ↓ The mouse try to pull the tail away and rotates the head, the reaction time of this movement is recorded ↓ Test and standard are given and reaction time is noted ↓ Lengthening of reaction time interprets as an analgesic action of test drug, (codeine, pethidine morphine are the drugs used as standard)
Procedure Wistar rats are placed into cage such that their tail hanging freely, the distal part is marked and immersed into a cup of warm water. Withdrawal reflex is noted along with reaction time Test substances are given and reaction time noted Conclusion : Centrally acting analgesic drugs are capable of prolongation of tail withdrawal reflex and reflex > 6 sec in test animal denotes positive analgesic response ↓
In this test, tail of rat is immersed in a cold mixture of water & ethylene glycol at -10◦C ↓ The reaction time is measured as the time taken by the rat to deflect its tail Cold ethanol tail –flick test In this test -20◦C is selected as a noxious stimulus ↓ Male sprague- dawley rats are used and their tail are submerged halfway into the bath containing a solution of ethanol ↓ Reaction time is measured until the rat removes its tail from the bath ↓ Time from immersion to tail removal is measured
Tooth Pulp test : In this test electrical stimulus is given in rabbits tooth pulp Procedure Rabbits are used and anaesthetized, using drill tooth pulp chamber are exposed close to the two upper incisors ↓ Clamping electrodes are placed into drilled holes ↓ Current is started with 0.2mA and increased until animal start licking ↓ Test compound is administered, after 2hrs threshold current is measured and compared with the threshold current prior to drug administration
Formalin test : 10% formalin is used as a noxious stimuli. By injecting the formalin solution into the paw of rat, a model of persistent pain caused by peripheral tissue injuries and inflammation is created Procedure In the dorsum of front paw of wistar rat 10% formalin is injected and observe for the pain responses which will be in form of elevation of paw, excessive licking and biting Pain response scoring is done and after administration of test drug again scoring is done for comparison
Writhing test
 A model of visceral or peritoneal pain in animals, involves administration of algogenic agent, used to detect peripheral analgesic activity of test compound  Pain is induced by intraperitoneal administration of chemicals that irritate serous membrane and provoke a stereotyped behaviour in mouse Procedure : Rodents are injected i.p. with 0.25 ml of suspension composed of (Phenylquinone 0.02% suspended in 1% suspension of carboxymethyl cellulose)
 Rodents react with a characteristic stretching behaviour accompanying with turning movement and extension of hind limb  In each test group prior to administration of suspension, test drug is administered orally. The mice are placed individually into glass chambers and no. of writhes are recorded for 10min in each  Acetic acid, acetylcholine, bradykinin, prostaglandin E1 are several other chemicals give rise to writhing behaviour
Haffner’s Tail Clip Method: Nociceptive mechanical stimulus is applied on the hind paw and tail Procedure : An artery clip is placed at the root of the tail of the mice to apply noxious stimulus ↓ A quick response is seen as biting of the clip or tail ↓ Reaction time between application of the clip and response is noted by a stop watch ↓ Test compound administered, after 1 hr procedure is repeated and reaction time is measured
 Inflammation is induced by subcutaneous administration of brewer’s yeast and then pressure is applied which increases the intensity of pain Procedure: Wistar rats are divided into two groups – test and control. In test group, test agents are administered and vehicle is administered in control group  After ½ hr 0.1 ml of Brewer’s yeast (20%) in distilled water is injected subcutaneously into plantar surface of left hind paw of the rat  After 3hr using a special apparatus (Randall Selitto apparatus) pressure is applied to the plantar surface of rat’s foot at a constant rate until animal struggle
 In both groups each animal is tested for its control pain threshold and then comparison is made between the two groups
Colburn et.al. evaluated a mechanical visceral pain model, where chronic intermittent intestinal distension is produced in the rat using a chronic indwelling intraduodenal balloon catheter
 Neuropathic pain models: Represents disorder of neuropathic pain sensation, partial somatosensory nerve injury is the cause of causalgiform pain disorders in man Procedure :  Sprague- Dawley rats are anaesthetized, incision is given and sciatic nerves of both legs are exposed at the level of mid thigh  Chromic gut sutures are tied loosely with a square knot around the right sciatic nerve. In next few days animal shows a mild aversion of the affected paw and foot drop  Halogen projector lamp is focused at the plantar area of one hind paw, as soon as heat is applied a withdrawal response of hind paw is seen
The time interval between exposure of heat and withdrawal response is measured  A rat model of partial sciatic nerve injury has also been produced. Unilateral tight ligation of about half of sciatic nerve rapidly produces sympathetically dependent neuropathic pain that last many months and resembles causalgia in humans  An animal model of persistent neuropathic pain has also been produced involving spared nerve injury. This involves a lesion of two of the three terminal branches of the sciatic nerve
 For experimental study of painful peripheral neuropathies in patients, hyperalgesia induced by Vincristine in the rat provides a good model Procedure : Vincristine 100μg/kg is administered daily for two weeks in rats. A decrease in mechanical nociception threshold and hyperalgesia occur after the second day of administration Increased response to stimuli during the second week of Vincristine administration Responses gradually return to baseline following discontinuation of treatment
 Painful diabetic neuropathy is one of the most common complications of insulin-dependent diabetes in man  Streptozotocin –induced diabetic rat has been a model of chronic pain as signs of hyperalgesia observed in diabetic humans Procedure: Streptozotocin 75mg/kg is administered, animal subjected to various pain stimuli, with follow up of four weeks with clinical symptoms and hyperglycemias. A decrease in threshold to noxious heat stimuli and non-painful thermal and mechanical stimulation is observed 4 wks after the induction of diabetes, score obtained in rats by injection of formalin, if score greater than those in normal rats indicates hyperalgesia
Rat model of incisional pain: Model helps to understand mechanism of sensitization caused by surgery and investigate new therapies for post operative pain. Model revealed that both the sural and tibial nerve are responsible for transmitting the inputs from the incision that produces hyperalgesia Procedure Longitudinal incision of 1cm through skin of plantar surface of hind paw. Withdrawal responses are measured using von frey filament at different areas of wound before its surgery. Results of tests for withdrawal responses suggest that a surgical incision of the rat foot causes mechanical hyperalgesia lasting for several days after surgery
Rat model of bone cancer pain :  Sprague- Dawley rats are given intra-tibial injections of syngeneic MRMT-1 rat mammary gland carcinoma cells, control rats receive heat killed cells  Sprague –dawley develop behavioural signs indicative of pain including mechanical allodynia and mechanical hyperalgesia  Development of bone tumor and structural damage of bone was monitored  Intra-tibial injection produced a rapidly expanding tumor within the boundaries of the tibia
 No tumor growth, no changes in body weight was observed after the injection of heat killed MRMT-1 cells  The general activity of animals after injection with live or heat killed MRMT-1 was higher than that of the control group  However the activity of MRMT-1 treated group declined during the progress of the disease
 Although different pain models based on nociceptive stimuli (electrical, thermal, mechanical or chemical) have been used but none of them is ideal. However test using chemical stimuli closely mimic acute clinical pain  As far as neuropathic pain is concerned it is difficult to design a model in animal for both technical and ethical reason that could measure affective component of pain
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