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Analgesic models

The document discusses various analgesic models and their significance in pain research, including definitions of pain and classifications of analgesics. It details experimental models utilizing thermal, mechanical, electrical, and chemical stimuli, and the methodologies employed for studying analgesic responses in both acute and chronic pain settings. Additionally, it highlights the importance of animal models for developing novel analgesics and understanding pain mechanisms.

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ANALGESIC MODELS Presented By Dr. Puneshwar Keshari, Ist Year PG Scholar, Dept. of Dravya Guna, Guided By Dr. Prakash L. Hegde Prof. Dept. of Dravya Guna
CONTENTS • Definition • Functions of Pain • Pain classification • Analgesics • Classification of analgesics • Analgesic models • Experimental models • Discussion • Conclusion • References
Definition of Pain An unpleasant sensory or emotional experience associated with actual or potential tissue damage, or described in terms of such damage. (IASP)
Nociception Sherington, 1910 introduced the term nociception nocere – ‘to harm’
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Types of Human Pain Various types of pain are seen in humans: eg. – Somatic pain (arising from the skin, Muscles, joints, ligaments and bones) – Visceral pain – Referred pain – Neuropathic pain – Cancer pain etc.
Classification of Pain Acute Pain: • Source is soft tissue damage, infection or inflammation • Short duration • “Symptom of pain”
Chronic pain: • Lasts for six months or longer, • “The disease of pain” • Common causes are cancer, neuropathic and arthritic.
ANALGESICS • The drugs which possess significant pain relieving properties by acting in the central nervous system , or on the peripheral pain receptors without significantly affecting consciousness.
Classification of analgesics: NARCOTIC ANALGESICS NON NARCOTIC ANALGESICS: Act centrally Act peripherally Cause addiction Do not cause addiction Produce CNS depression Do not produce CNS depression Do not produce gastric irritation Produce gastric irritation Show no anti inflammatory effect Show anti inflammatory effect eg. Morphine, Tramadol, Pethidine etc. Diclofenac, Ibuprofen, Aspirin etc.
Analgesics in Ayurveda • ljbf/LuGwfk[lZgk0fL{a[xtLs06sfl/s}/08sfsf]nL rGbgf]zL/}nfdw'sfgLlt bz]dfGoËdb{k| zdgflg ejlGt . -r=;"=$÷$$_ • lkKknLlkKknLd"nrJolrqs>[Ëj]/dl/rfhdf]bfhuG wfhfhLu08L/f0fLlt bz]dflg z"nk|zdgflg ejlGt . -r=;"=$÷$%_ • zfns6kmnsbDakBst'Dadf]r/;lz/Lifj~h'n}njfn' sfzf]sf Olt bz]dflg j]bgf:yfkgflg ejlGt . -r=;"=$÷$&_
ANALGESIC MODELS • The animal models employed for screening of analgesic agents are analgesic models. • For animal models of pain, the word “model” refers to three entirely separate entities- – subject, – assay and – measure.
EXPERIMENTAL MODELS: 1. Models using thermal stimuli: – The tail flick model using radiant heat/immersion of the tail in hot water – Pain withdrawal test – Hot plate test – Pain state models using cold stimuli • Cold tail flick test • Cold ethanol tail flick test (CET) 2. Models using mechanical stimuli: – Haffner’s tail clip method – Randall Selitto Test – Strain gauges – Von Frey filaments – Inclined plane test
3. 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. 4. Models using chemical stimuli: – Formalin test – Acetic acid induced writhing test – Stimulation of hollow organs • Rat sigmoid colon model • Inflammatory uterine pain model
MODELS FOR CHRONIC PAIN • Neuropathic pain models • Vincristine induced neuropathy model • Diabetic neuropathy model • Persistent post thoracotomy pain model • Rat model of incisional pain • Rat model of bone cancer pain
IN VITRO METHODS • H – Naloxone binding Assay • Micro opiate receptor binding assay • Assay to study cannabinoids activity • Receptor binding of nociception • Bioassays for nociception • Vanilloid receptor binding
USE OF SHORT DURATION STIMULI (PHASIC PAIN) OR MODELS FOR ACUTE PAIN
A. PAIN STATE MODELS USING THERMAL STIMULI 1. The Tail Flick models: • Commonly accepted model for quantifying analgesic response in animals. • In this model, radiant heat is applied to a small surface of the tail or the tail is immersed in hot water.
a. Tail flick model using radiant heat: • Hardy et al. 1940 used on human • Then used in Rat (1953) • The tail flick test with radiant heat is a simplified method the application of thermal radiation to the tail of an animal provokes the withdrawal of the tail • The morphine like drugs are capable of prolonging the reaction time.
TailFlick-Low.wmv.mp4
Procedure –The tail of Wistar rat (170- 210gms) is placed on the radiant source and time taken for the rat to withdraw its tail is recorded. Withdrawal of the tail from heat source is referred to as ‘ tail flick latency’. Exposure of the tail with radiant heat is done for 10-20 seconds.
Evaluation: • The tail flick latency in the test, standard, and control animals are compared • A lengthening of the reaction time is interpreted as an analgesic action. • Using various doses, ED-50 values can be calculated.
Merits • Effective for screening Morphine like analgesics • Simple technique, which does not require any special skill • Results of experiments are quite accurate and less time consuming.
Demerits: • Tail flick response is prone to habituation • Efficient only for revealing the activity of opioid analgesics • Prolonging exposure to radiant heat beyond 20 seconds may cause burning of tail.
Modification • USG induced tail flick procedure • Ear withdrawal by applying radiant heat • Tail flick formalin test in rodents: changes in skin temperature
b. Tail flick model using immersion of the tail: • Distal 5 cm of the tail of wistar rat is marked and immersed in a cup of warm water ( 55°Cto56°C) for 15 seconds. • The reaction time is determined periodically after administration of test drug (0.5, 1, 2, 3, 4, and 6 hours)
Tail flick model using immersion of the tail Mouse Tail-Flick, Allegheny College.mp4
• Response and Evaluation: - Withdrawal of tail or attempt to escape or abrupt movement of tail and sometimes the recoiling of whole body is seen. • Centrally acting analgesics are capable of causing prolongation of tail withdrawal reflex( more than 6 sec.). • Modification: –Cold tail Flick test –Cold ethanol Tail flick test
2. Hot Plate Model • Introduced by Woolfe and Mc Donald in 1944 • Mice are put on the hotplate(temp. 55° to56 °C)and then latency is noted following administration of test drug. • Response – jumping or paw licking.
Hot plate Analgesic.mp4
EVALUATION • The prolongation of latency time between the test, standard and control animals are compared (by using t-test) • Using various doses ED-50 values can be calculated.
B: PAIN STATE MODELS USING MECHANICAL STIMULI: 1. Haffner’s Tail Clip Method: • Preferred sites for applying nociceptive mechanical stimuli are the hind paw and the tail • Highly sensitive for centrally acting drugs • Tests using constant pressure have been abandoned progressively for those applying gradually increasing pressures
PROCEDURE • An artery clip is placed at the root of tail of mice. • A quick response is seen as biting the clip or tail, where clip has been placed. The reaction time is noted by a stopwatch. • The test compounds are administered subcutaneously or orally. • Then after 15, 30, and 60 minutes, the same procedure is repeated and the reaction time is measured.
EVALUATION • Reaction time of the test animals, greater than the cut off time denotes a positive response, indicating analgesic activity.
PAIN STATE MODELS USING ELECTRICAL STIMULI: Grid shock method -Blake et al (1963) -Used for central and peripheral analgesics -Stimulus is given in the form of square wave pulses to male mice . The output of the stimulator is connected to the wires of grid with fixed resistance which is kept parallel to oscilloscope -Pain thresholds are determined in each individual mouse twice before and after administration of the test drug. -Response- on oscilloscope , a starling reaction , increase locomotion or attempt to jump.
EVALUATION • Current in milli-ampere is recorded in each mouse before and after administration of drug is noted. • The average pain threshold values for each group at each time interval are calculated and statistically compared with the control values.
D. PAIN STATE MODELS USING CHEMICAL STIMULI • Slow, progressive, and irreversible form of stimulation • Closest in nature to clinical pain
1. Formalin Test • 10% formalin solution is used as noxious stimulus which is injected into the paw of rat or mouse and inflammation is created as a model of persistent or chronic pain. • Two phases reactions are shown – Early phase starts immediately after injection, and lasts for 3-5 minutes • The second phase starts after 10—15 mins. of injection and lasts for 20-40 mins. • Opioid analgesics are effective in both phases, while NSAIDs are effective in the second phase.
• Male wistar rats ( 180-300gms) are administered subcutaneously with 0.05 ml of 10% formalin in dorsum of the front paw. • Each animal is placed into separate cages for observation of pain responses in early and late phases. • Responses are as elevation or favoring of paw, or excessive licking and biting of the paw PROCEDURE
• Scoring is done according to the pain scale • Then test drug is administered and again scoring is done after 30, 60, minutes for comparison • If both paws of animal are allowed to rest on the floor with no obvious favouring of the injected paw, this is taken as positive analgesic response. Contd..
b. Writhing Test • Model of visceral or peritoneal pain in animals • Test is issued to detect peripheral analgesic activity • Pain is induced by intra peritoneal administration of chemicals, which irritate serous membrane and provoke a stereotype behaviour in the mouse or rat known as writhing. • Chemicals used are acetic acid, acetylcholine, bradykinin, aconitine, 4% NaCL, etc.
PROCEDURE • Acetic acid solution is administered to the mice(20- 25gm.) and placed individually in the glass jar. • The test and standard drug is administered 15 minutes prior to the acetic acid administered. • Responses are noted.
Writhing Test writhing test.mp4
EVALUATION • The writhing period is recorded for 10 minutes and compared with the control group. • Writhing response in the drug treated must be less when compared to the acetic acid treated control.
IN VITRO MODELS BIOASSAYS FOR NOCICEPTION Purpose and rationale: Nociception receptors in the periphery can be characterized by studies in isolated organs(Guerrini et al.1998,Bigoni et al.1999) . The guinea pig ileum , according to Paten 1957, the mouse vas deferens according Hughes etal. 1975, the rabbit vas deferens according to Oka et al. 1980, the guinea pig renal pelvis according to Giuliani and magi 1996.
PROCEDURE • Tissues are taken from male Swiss mice, guinea pigs, Sprague Dawley rats, and New Zealand albino rabbits. • Suspended in 10 ml organ baths containing Krebs solution oxygenated with 95% oxygen and 5% carbon-dioxide. • Temperature is set around 33-35 degrees and a resting tension of 0.3-1 gm is applied • The tissues are stimulated through two platinum ring electrodes
• The electrically evoked contractions are measured isotonically with a strain gauge transducer and recorded on a multichannel chart recorder. • After equilibration period about 60 mins, the contractions induced by electrical field stimulation are stable. At this time, cumulative concentration response curves to nociception or opioid peptides are performed. Contd..
• Four electrical field stimulation are performed with each tissue at 30 min intervals. • Agonists and antagonists are added to the bath • Contractile response to electrical field stimulation are expressed as % increment to the spontaneous activity of the tissue. • The biological effects of the application of agonists or antagonists are expressed as % inhibition of electrical field stimulation induced contraction. Contd..
EVALUATION • The agonists and antagonists potencies are recorded and expressed as means of +- SEM.
MODELS OF CHRONIC PAIN: • Rat Model of Incisional Pain: • This model helps to understand mechanisms of sensitization caused by surgery and investigate new therapies for post operative pain • A longitudinal incision of 1 cm is given through the skin, fascia and muscle of the plantar surface of the hind paw in halothane anesthetized rats. • Withdrawal responses are measured using Von Frey filaments at different areas around the wound before surgery and for the next 6 days. • The results of tests for withdrawal responses suggest that surgical incision of the rat foot causes mechanical hyper-algesia lasting for several days after surgery.
RESEARCH ARTICLES • The necessity of animal models in pain research • Jeffrey S. Mogil a,*, Karen D. Davis b,c, Stuart W. Derbyshire • IASP PAIN 151 (2010) 12–17 • Abstract-There exists currently a fair degree of introspection in the pain research community about the value of animal research. This review represents a defense of animal research in pain. We discuss the inherent advantage of animal models over human research as well as the crucial complementary roles animal studies play vis-à-vis human imaging and genetic studies. Finally, we discuss recent developments in animal models of pain that should improve the relevance and translatability of findings using laboratory animals. We believe that pain research using animal models is a continuing necessity–to understand fundamental mechanisms, identify new analgesic targets, and inform, guide and follow up human studies–if novel analgesics are to be developed for the treatment of chronic pain.
Animal models of pain : progress and challenges Jeffrey S. Mogil Nature Review, Neuroscience; Vol. 10, April 2009; pg 283-94 Abstract: Many are frustrated with the lack of translational progress in the pain field, in which huge gains in basic science knowledge obtained using animal models have not led to the development of many new clinically effective compounds. A careful re-examination of animal models of pain is therefore warranted. Pain researchers now have at their disposal a much wider range of mutant animals to study, assays that more closely resemble clinical pain states, and dependent measures beyond simple reflexive withdrawal. However, the complexity of the phenomenon of pain has made it difficult to assess the true value of these advances. In addition, pain studies are importantly affected by a wide range of modulatory factors, including sex, genotype and social communication, all of which must be taken into account when using an animal model.
Evaluation of the Analgesic and Anti inflammatory Activity of Kadamba (Anthocephalus cadamba miq.) – an experimental study Thesis dissertation by Dr. Prakash L Hegde, submitted to the RGUHS • Used model – Hot Plate Method • Conclusion - Alcoholic extract of Kadamba has potent analgesic and anti-inflammatory activity almost like standard drug. • Thus kadamba is a freely available safe drug showing efficacy in single use both in extract and kashaya form on rats. Same has to be reconfirmed on human beings.
EXPERIMENTAL MODELS IN AYURVEDA
; ;DoS;DkGgdGg+ ;'k/LlIft+ ljz'¢dUGoflbif' k|fu'kgLt+ lzlvgf b[i6dlek|f]lIft+ k|f]If0f}M k'/M l:ytf] /fhg+ x:ta¢f}iflw/Tg+ ef]ho]t c=; +=;"= *.$ How the Vaidya examines and certifies the King’s food: First, he should test it by fire, then, by feeding it to a peacock to determine whether it is poisonous or not. Then, he should chant holy hymns and chants to ward of evil, and should ensure that the king has donned and sacred stones and medicaments as
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DISCUSSION • Pain – – Common unpleasant sensory and emotional experiences. – Acute, chronic, somatic, visceral, Neuropathic etc are different ways of experiences of pain. – In Ayurveda pain is described as Vedana, Ruja, Shoola, Angamarda, Arti etc. – Nociception – Pain due to noxisious stimuli
• Analgesics – – Various types – New drugs are introducing continuously due to limitations and adverse effects of existing one. – Bright future for Ayurvedic drugs in field of Analgesics – Ayurveda system seeking potent analgesic drugs for different types of pain • Ayurveda and Analgesic – Acharyas mentioned different drugs in Vedana- Sthapaka, Shoolprashaman, Angamardaprashaman Gana. – These drugs should be tested by analgesic models and Contd..
Contd..
• Analgesic models – Analgesic models are equally applicable for phasic or acute pain as well as tonic or chronic pain. – Centrally acting and peripherally acting analgesics. both can be evaluated by analgesic models. – Different thermal, mechanical electrical and chemical stimuli are used. – In-vitro models are also used.
– Haffner’s tail clip method, hot plate method, tail immersion method, grid shock test, formalin test in rats etc. are used mainly for central analgesic activity. – Responses given by the subject of the analgesic models helps to determine analgesic effect. – Writhing test, Randall Selitto test, mechanical/visceral pain models in the rat etc. Are used mainly for peripheral analgesic activity. – Animal models are necessary for pain research, having various challenges. – In Ayurveda, birds and other animals were used as experimental model in few extent. Contd..
CONCLUSION • Analgesics have to be tested not only for their in vitro activity, but also for their in vivo analgesic potential • The most commonly used methods for measuring peripheral analgesics activity are the writhing test in mice. • Tail flick method, Hot plate method and Haffner’s tail clip method are common methods for measuring central acting analgesic effects.
• Herbs described as vedanastahpaka, vedanahara, shothahara, shoolprashaman,Angamardaprashaman could be tested for their respective analgesic effects by using analgesic models. • Limitations of different models should be tackled with modification in subject, assays and measures.
References • Charak Samhita • Astanga Sangraha • K. D. Tripathi. Essential of Medical Pharmacology • Drug Screening Methods : SK Gupta • Dr. Prakash L. Hegde; Evaluation of the Analgesic and Antiinflammatory Activity of Kadamba (Anthocephalus codamba Miq.): An Experimental Study • DANIEL LE BARS,MANUELAGOZRIU, AND SAMUELW.CADDEN-Animal Models of Nociception,The American society for pharmacology and experimental therapeutics : vol 53 no.4 ; 597- 612 • Parle Milind, Yadav Monu. Laboratory Models for Screening Analgesics. International research Journal of Pharmacy. 2013;4 (1): p-15-19 • Jeffery, S. Mogil, Karen D. Davis, Sturt W. Derbyshire The necessity of animal models in pain research – comprehensive review, PAIN 151 (2010); p.12-17 • Jeffery, S. Mogil – Animal models of pain: progress and challenges, Nature Reviews / Neuroscience; 2009 April: (10); p-283-294
Analgesic models