Models of Nociception: Hot‐Plate, Tail‐Flick, and Formalin Tests in Rodents

Anthony W. Bannon1, Annika B. Malmberg2

1 Icagen Inc., Durham, North Carolina, 2 Amgen, Inc., Cambridge, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 8.9
DOI:  10.1002/0471142301.ns0809s41
Online Posting Date:  October, 2007
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Abstract

Experimental models of pain include tests of response thesholds to high intensity stimuli (acute pain tests) and changes in spontaneous or evoked behavioral responses in animals with peripheral injury or inflammation (persistent pain models). Acute thermal pain is modeled by the hot‐plate and tail‐flick test, while persistent pain can be modeled by the formalin test. This unit presents protocols for all three of these tests, including preparation of animals (rats or mice), administration of a compound being tested for its analgesic properties and data collection. Curr. Protoc. Neurosci. 41:8.9.1‐8.9.16. © 2007 by John Wiley & Sons, Inc.

Keywords: pain; nociception; tail flick; formalin; hot plate; rat; mouse; animal model; analgesia

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Acute Pain Using the Hot‐Plate Test
  • Basic Protocol 2: Measurement of Acute Pain Using the Tail‐Flick Test
  • Basic Protocol 3: Measurement of Persistent Pain Using the Formalin Test
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Acute Pain Using the Hot‐Plate Test

  Materials
  • Animal: 30‐ to 35‐g male CD1 mice, group‐housed (e.g., Charles River Labs) or 200‐ to 250‐g male Sprague‐Dawley rats
  • Test compounds in saline or other appropriate vehicle
  • Reference analgesic compound (e.g., morphine, to be given 10 mg/kg, i.p.)
  • 1‐ml syringes with 27‐G needles
  • Hot‐plate apparatus or metal plate in a controlled temperature water bath
  • Stopwatch or timer

Basic Protocol 2: Measurement of Acute Pain Using the Tail‐Flick Test

  Materials
  • Animals: 25‐ to 30‐g male C57BL/6 or CD1 mice, or 200‐ to 250‐g male Sprague‐Dawley rats
  • Test compound and control solution of appropriate vehicle
  • Reference analgesic compound (e.g., morphine, to be administered subcutaneously at 10 mg/kg)
  • Tail‐flick apparatus with automated timer or stopwatch for manual use (Fig. )
  • 1‐ml syringes with 30‐G needles for injecting mice or 27‐G needles for injecting rats
  • Towel for gentle restraint of the animals or Plexiglas tube for same purpose

Basic Protocol 3: Measurement of Persistent Pain Using the Formalin Test

  Materials
  • Animal: 25‐ to 30‐g male C57BL/6 or CD1 mice, or 250‐g male Sprague‐Dawley rats, group‐housed (e.g., Charles River Labs)
  • Test compounds in saline or appropriate vehicle
  • 5% formalin (see recipe)
  • Individual clear containers to hold animals (e.g., shoebox cages with tops; see Fig. )
  • Mirrors
  • 0.5‐ml syringes with 28.5‐G needles (for rats) or 50‐µl Hamilton syringe with 30‐G needles (for mice)
  • 1‐ml syringes with 27‐G needles (for rats) or 26‐G needles (for mice)
  • Hand‐held counters (one per animal)
  • Clock or timer
  • Electronically controlled timing lights (optional)
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Figures

  •   FigureFigure 8.9.1 Apparatus used for the tail‐flick test.
  •   FigureFigure 8.9.2 Apparatus used for formalin test.
  •   FigureFigure 8.9.3 Change in pain score through phases 1 and 2 in an animal injected with formalin.
  •   FigureFigure 8.9.4 Effects of morphine on paw lick and jump latency in the hot‐plate assay in mice. Separate experiments were conducted for each endpoint.
  •   FigureFigure 8.9.5 Data collected in the automated hot‐plate test, which measures response to acute thermal pain. Because experiments were performed at different times, data are expressed as change from control for each experiment calculated as [change = (mean value − mean control value)/(mean control value) × 100]. For the experiments presented, the mean jump latency for vehicle alone (control) was 67.7 sec with a standard deviation of ±18.4.
  •   FigureFigure 8.9.6 Effect of morphine in the tail flick test in mice. The mice were injected subcutaneously (s.c.) 30 min before the tail‐flick test. n=5 to 8 animals per dose.

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Literature Cited

Literature Cited
   Bannon, A.W., Gunther, K.L., and Decker, M.W. 1995. Is epibatidine really analgesic? Dissociation of the locomotor activity, temperature, and analgesic effects of (±)‐epibatidine. Pharmacol.Biochem. Behav. 51:693‐698.
   Berge, O.‐G., Garcia‐Cabrera, I., and Hole, K. 1988. Response latencies in the tail‐flick test depend on tail skin temperature. Neurosci. Lett. 86:284‐288.
   D'Amour, F.E. and Smith, D.L. 1941. A method for determining loss of pain sensation. J. Pharmacol. Exp. Ther. 41:419‐424.
   Dickenson, A.H. and Sullivan, A.F. 1987. Peripheral origins and central modulation of subcutaneous formalin‐induced activity of rat dorsal horn neurons. Neurosci. Lett. 83:207‐211.
   Dubuisson, D. and Dennis, S.G. 1977. The formalin test: A quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 4:161‐174.
   Hargreaves, K., Dubner, R., Brown, F., Flores, C., Jorrs, J. 1988. A new sensitive method for measuring thermal nociception in cutaneous hyperalgesia Pain 32:77‐88.
   Hunskaar, S., Fasmer, O.B., and Hole, K. 1985. Formalin test in mice, a useful technique for evaluating mild analgesics. J.Neurosci. Methods 14:69‐76.
   Janssen, P.A.J., Niemegeers, C.J.E., and Dony, J.G.H. 1963. The inhibitory effect of fentanyl and other morphine‐like analgesics on the warm water induced tail withdrawal reflex. Arzneim. Forsch. 13:502‐507.
   Jett, M.F. and Michelson, S. 1996. The formalin test in rat: Validation of an automated system. Pain 64:19‐25.
   Jourdan, D., Ardid, D., Bardin, L., Bardin, M., Neuzeret, D., Lanphouthacoul, L., and Eschalier, A. 1997. A new automated method of pain scoring in the formalin test in rats. Pain 71:265‐270.
   Jourdan, D., Alloui, A., and Eschalier, A. 1999. Pharmacological validation of an automated method of pain scoring in the formalin test in rats. J. Pharmacol. Toxicol. Methods 42:163‐170.
   Malkmus, S., Lu, X., Bartfai, T., Yaksh, T.L., and Hua, X.Y. 2005. Increased hyperalgesia after tissue injury and faster recovery of allodynia after nerve injury in the GalR1 knockout mice. Neuropeptides 39:217‐221.
   Mogil, J.S., Kest, B., Sadowski, B., and Belknap, J.K. 1996. Differential genetic mediation of sensitivity to morphine in genetic models of opiate antinociception: Influence of nociceptive assay. J. Pharmacol. Exp. Ther. 276:532‐544.
  Murray, C.W., Porreca, F., and Cowan, A. 1988. Methodological refinements to the mouse paw formalin test. An animal model of tonic pain. J. Pharmacol. Methods 20:175‐186.
   Sufka, K.J., Watson, G.S., Nothdurft, R.E., and Mogil, J.S. 1998. Scoring the mouse formalin test: Validation study. Eur. J. Pain 2:351‐358.
   Tjøsen, A., Lund, A., Berge, O.‐G., and Hole, K. 1989. An improved method for tail‐flick testing with adjustment for tail‐skin‐temperature. J. Neurosci. Methods 26:259‐265.
   Wheeler‐Aceto, H. and Cowan, A. 1991. Standardization of the rat paw formalin test for the evaluation of analgesics. Psychopharmacol. 104:35‐44.
   Yaksh, T.L., Ozaki, G., McCumber, D., Rathbun, M., Svensson, C., Malkmus, S., and Yaksh, M.C. 2001. An automated flinch detecting system for use in the formalin nociceptive bioassay. J. Appl. Physiol. 90:2386‐23402.
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