Progress in pain research has been aided by thedevelopment of increasingly sophisticated animalmodels. Although models of acute nociception innaive animals, such as the tail-flick, hot-plate andwrithing tests, were used to develop all of theclassic analgesic drugs, more recently developedmodels represent clinically relevant pathologicalconditions. More importantly, these models of in-flammatory or neuropathic pain can identify po-tent and effective drugs that have the potential tobe successful in the clinic while leaving animalsin general good health and without excessive distress, weight loss or general behaviouralchanges.

Measuring pain in an animal is not an easytask. Acute pain is a normal defensive functionthat is not associated with a pathological condi-tion but signals threat to the integrity of the or-ganism. It is evoked when ‘nociceptor’ primary afferents are activated, which generally havehigher thresholds than the sensory afferents thatsignal innocuous touch or temperature infor-mation. However, pain, hyperalgesia and allody-nia1 (otherwise indifferent or pleasant stimuli thatbecome painful) can also develop as constantsymptoms of disease, particularly in associationwith inflammation (inflammatory pain) and afterperipheral nerve damage (neuropathic pain).Recently, it has been recognized that inflamma-tory and neuropathic pain might have differentcomponents in their pathomechanisms (Fig. 1).

The species of choice in behavioural modelsof pain is usually the rat or mouse, however, withthe increasing availability of genetically engi-neered animals, issues of species or strain dif-ferences are of major importance2.

Measurement of pain in animalsThe measurement of ‘pain sensation’ in animalsis largely indirect. There is no way to assay the‘quality’ of pain (shooting, stabbing, lancing, etc.)in current animal models. With the exception ofaversive behaviour (vocalization or biting/lick-ing/shaking the affected limb) to potentially nox-ious stimuli, all measurements are based on thenocifensive reflex. This reflex is measured as thelatency of hindpaw withdrawal on exposure to in-creasing heat or as the intensity of pressure thatproduces withdrawal. Both acute and chronicmodels use these measurements. The tail-flickand hot-plate models use direct thermal stimulusto elicit a flick of the tail or paw licking at noxious

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Animal models for pain researchKatharine Walker, Alyson J. Fox and Laszlo A. Urban

Figure 1. Schematic diagram of the major components of chronic inflammatory and neuropathic pain. (a)Persistent inflammatory pain models. The most important component of chronic pain syndromes, which havedominant inflammatory pathomechanism, is the constant activation and sensitization of polymodal (chemo-thermo-mechano-sensitive) C-type primary afferents. These fibres release neuropeptides, such as sub-stance P and calcitonin-gene-related peptide (CGRP), which contribute to neurogenic inflammation in the periphery and which might produce increased spinal excitability (windup). Inflammatory pain models arecharacterized by increased sensitivity to mechanical and thermal stimuli (hyperalgesia). The strong periph-eral component is supported by the effectiveness of non-steroidal anti-inflammatory drugs (NSAIDs) as anal-gesics in these models. (b) Persistent neuropathic pain models. After peripheral nerve injury, some C fibreslose their synaptic contact in the spinal cord and develop axonal degeneration. Perhaps, as a compensatorymechanism, large, myelinated fibres sprout to the site of the synaptic loss, namely into the superficial dorsalhorn of the spinal cord. In parallel with the collateral sprouting, large fibres also develop abnormal ectopic dis-charges, which might be the major generator of neuropathic pain. This theory is supported by experimentalevidence that low concentrations of local anaesthetics that block ectopic discharges produce significant anal-gesia in animals and in some clinical neuropathic pain conditions. The presence of mechanical allodynia inneuropathic models further supports the role of large, myelinated fibres. NSAIDs have little effect in thesemodels. Another component of neuropathic pain might be the loss of inhibitory function in the spinal cord [de-generation of g-aminobutyric acid (GABA)-ergic cells in the dorsal horn] owing to excitotoxicity by the ex-cessive release of glutamate and aspartate. Some of these models also produce cold allodynia. The pres-ence of a sympathetic component in these models is likely, but the importance of the sympathetic nervoussystem in the development of chronic neuropathic conditions is not considered to be general. DRG, dorsalroot ganglion; SP-LI, substance P-like immunohistochemistry; WDR, wide dynamic range neurons.

Spinal cord• Increase in SP levels• Central sensitization

Spinalcord

Periphery (C fibres)• Excitation• Sensitization• Neurogenic inflammation

a

C fibre

A fibreDRG

WDRcell

Spinal cord• Increase in SP binding• Increase in sensitivity to SP• Decrease in total SP levels• Collateral sprouting

A fibres:• Start to express SP• Develop spontaneous

activity• Peripheral SP-LI basketsC fibres:• Sprouting in the DRG• Loss of synaptic contact

in spinal cord

b

C fibre

A fibreDRG

WDRcell

Spinalcord

Collateralsprouting

Mol

ecul

ar M

edic

ine

Tod

ay

(unpleasant) intensities of thermal stimulation(45–528C). These methods can detect mechani-cal and thermal hyperalgesia and characterizethe effects of analgesic and anti-hyperalgesicdrugs.

In addition to hyperalgesia, mechanical andthermal allodynia can develop when innocuous(not painful) stimuli evoke limb withdrawal. Thiscan be measured by the use of von Frey fila-ments, which are used to apply slight pressure to

the skin. In the presence of allodynia, this slightpressure becomes unpleasant and the animalmoves the affected limb. Mechanical allodynia isone of the most common symptoms in clinicalconditions such as neuropathic pain.

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Table 1. Commonly used animal models of pain a

Model Similarities to human disease Differences from human disease Further comments

Acute pain

Tail-flick/hot-plate No equivalent Does not model any human pathology Used to study nociceptive modalities and to develop analgesic

drugs

Persistent/central pain

Capsaicin (s.c.) Skin inflammation by irritants; A fine model for certain mechanistic Used as a valuable tool to study selective activation of polymodal

represents spinal component components of pain but is of no nociceptors and ‘central sensitization’ in the spinal cord

of pain real relevance to any clinical syndrome

Formalin (i.p.) No human equivalent; No human equivalent An extensively used model as it features both peripheral and

mechanism is largely unknown central components; easy to use with a high throughput capacity;

no relevance to chronic pain conditions

Chronic/inflammatory pain

FCA Rheumatoid arthritis, severe Symptomatology and Only monoarthritic or localized form is used for pain research;

inflammation of joints and pathomechanism is different from long-lasting, reliable model for inflammatory pain; time course

soft tissue; it is most relevant human rheumatoid arthritis might differ between species

to monoarthritis

Carrageenan and Subacute inflammation of Short duration compared to chronic Produce inflammation with a 3–7 day duration and substantial

turpentine models joints and soft tissue inflammatory pain in humans oedema formation

UV-irradiation Sunburn, moderate burn injury Not known Various irradiation periods with UV-B produce skin inflammation

with different time courses

Chronic/neuropathic pain

Bennett model (loose Spontaneous pain, allodynia and Difficult to relate to any particular Frequently used model, particularly for behavioural studies;

chromic ligature of protective posture all present in neuropathic pain condition; nerve high frequency of autotomy is a disadvantage

the sciatic nerve) this model; pharmacological compression and direct mechanical

profile is similar to that of neuronal damage might be relevant

clinical neuropathic pain clinical comparisons

Seltzer model (partial Same symptoms as above but As above High reproducibility, easy to perform, excellent for behavioural

tight ligation of the less severe; minimal studies; however, difficult to study changes in the DRG as

sciatic nerve) inflammatory process damaged and undamaged primary afferents are mixed in the

nerve

Chung’s model General model of neuropathic As above; root compression might be Fairly extensive surgery with muscle damage might complicate the

(tight ligation of one pain a relevant clinical comparison pathomechanism; excellent model for in vitro use as the damaged

of the two spinal and undamaged fibres of the peripheral nerve originate from

nerves of the sciatic distinct DRGs

nerve)

Diabetic neuropathy Diabetic neuropathy Many features and underlying Produces severe distress to the animal with deterioration of

(STZ) mechanisms differ from diabetes general condition; difficult to interpret data or obtain clear pain

mellitus scores; insulin treatment abolishes pain and hyperalgesia

aAbbreviations: DRG, dorsal root ganglion; FCA, Freund’s complete adjuvant; i.p., intraplantar; s.c., subcutaneous; STZ, streptozotocin.

Animal models of painAnimal models of pain can be generally classifiedas either somatic pain models or models of vis-ceral pain (such as gastrointestinal pain, urinarybladder dysfunction). Somatic pain models arethe most widely used and include acute nocicep-tive models (hot-plate, tail-flick) and pathologicalpain models. In the latter category, pain can be in-duced in several ways: (1) persistent central paincan be induced by formalin or capsaicin; (2)chronic inflammatory pain by carrageenan, tur-pentine, UV-irradiation or Freund’s complete ad-juvant (FCA); and (3) chronic neuropathic pain bydamage or disturbance to a peripheral nerve. Aseparate group of pain models representsparticular diseases that feature pain as a promi-nent symptom (e.g. diabetic neuropathy).

Models of somatic painAcute nociceptive modelsModels of acute pain measure the behavioural re-sponses of naive animals to noxious stimuli.Noxious heat is the most common stimulus usedin these tests, and analgesic drugs, such as opi-ates, can modify the behavioural responses pro-duced. Anaesthetic drugs are also effective inthese models. However, many common ‘pain-killers’, such as non-steroidal anti-inflammatorydrugs (NSAIDs), are not effective in models ofacute pain. These drugs interact with mechanismsthat develop during pathological conditions.Therefore, relying on models of acute nociceptionalone might prevent the discovery of potentiallyimportant new classes of pain-relieving drugs.

Pathological pain modelsIdeally, a drug that does not affect the nocifensiveproperties of the nervous system but that canreset the nociceptive thresholds to normal levelsin pathological conditions provides the greatestclinical benefit. Persistent stimulation of nocicep-tive primary afferents produces sensitization inthe central nervous system owing to the processof ‘windup’. This fundamental spinal process in-volves a dominant N-methyl-D-aspartate (NMDA)receptor component and underlies the develop-ment of hyperalgesia. Mechanistic studies of thephysiological and pharmacological mechanismsof chronic pain have depended largely on the useof the capsaicin and the formalin models of per-sistent pain. In particular, the formalin model hasthe advantage of the easy assessment of spon-taneous nocifensive behaviours3.

Chronic inflammatory andneuropathic pain models

More sophisticated pain models, which producecomplex time-dependent pathomechanisms, in-clude models of inflammatory pain. Most inflam-

matory pain models induce hyperalgesia by thesubcutaneous injection of inflammatory agentsinto the hind paw of rats or mice (Table 1).Alternatively, exposure of the rat hind paw to UVirradiation produces highly reliable and persistentallodynia that is particularly useful as a model ofinflammatory pain that is associated with first- andsecond-degree burns4.

The development of chronic pain following nerveinjury is a formidable clinical problem as it is stub-bornly resistant to existing pharmacotherapies, suchas the NSAIDs. Several peripheral nerve-injury models5 have been developed and are usedto study the underlying mechanisms of neuropathicpain (see Table 1). Although all of these models fea-ture some degree of direct neuronal damage, theydiffer in the time course and pathomechanisms thatare associated with the development of hyperalge-sia. The study of these models has contributed sig-nificantly to the understanding of pathological painmechanisms, such as collateral sprouting orchanges in primary afferent phenotype6,7. Moreover,these models have been used extensively to de-velop novel drugs for clinical neuropathies.

Models of visceral painAnother area of unmet clinical need is that of vis-ceral pain. The pathomechanisms of visceral painmight differ from that of somatic pain. Althoughnot nearly as well studied as somatic pain, at-tempts have been made to develop models of vis-ceral pain that have pathological mechanismssimilar to those of irritable bowel or irritable uri-nary bladder syndromes.

Disease models for the study ofsymptomatic pain

The rat streptozotocin (STZ) model is the mostcommonly used model of pain associated with diabetic neuropathy8. STZ treatment destroysLangerhans cells in the rat pancreas and pro-duces the rapid development of hyperglycaemia.However, recent evaluation of this model sug-gests that STZ treatment produces severe dis-tress and morbidity in rats, which brings to ques-tion the interpretation of the results of behaviouralexperiments9. Genetic strains that develop spon-taneous diabetes might offer a more valid modelof diabetic neuropathy.

Validation of pain modelsUltimately, animal models should provide a basisfor the better understanding of clinical conditions,the pathomechanisms of human disease and infor-mation on the therapeutic value of novel drugs.Therefore, the clinical predictability of pain modelsis a major issue. A valid model should produce re-producible behavioural indices of pain, such as hy-peralgesia or allodynia. Reliable models of inflam-

mation and neuropathic pain should show a simi-lar developmental time course and pharmacologi-cal profile to that observed in clinical conditions(see Table 1). Models of pain that can be repro-duced in different species are of the greatest valueto drug development, as protein or receptor het-erogeneity between different species is always apossibility10.

References1 Bennett, G.J. (1994) Neuropathic pain , in

Textbook of Pain (Wall, P.D. and Melzack, R., eds),pp. 201–224, Churchill Livingstone

2 Mogil, J.S. and Griesel, J.E. (1998) Transgenicstudies of pain , Pain 77, 107–128

3 Tjølsen, A. et al. (1992) The formalin test: anevaluation of the method , Pain 51, 5–17

4 Perkins, M.N., Campbell, E.A. and Dray, A. (1993)Antinociceptive activity of the bradykinin B1and B2 receptor antagonists, des-Arg 9, [Leu 8]-BK and HOE 140, in two models of persistent hy-peralgesia in the rat , Pain 53, 191–197

5 Babbedge, R., Dray, A. and Urban, L. (1995)Complex regional pain syndromes (CRPS):mechanism and therapy from experimentalmodels , Pain Rev. 2, 298–309

6 Hokfelt, T. et al. (1997) Phenotype regulation indorsal root ganglion neurons after nerve injury:focus on peptides and their receptors , inMolecular Neurobiology of Pain (Borsook, D., ed.),pp. 115–143, IASP Press

7 Woolf, C.J., Shortland, P. and Coggeshall, R.E. (1992)Peripheral nerve injury triggers central sproutingof myelinated afferents , Nature 355, 75–77

8 Hounsom, L. and Tomlinson, D.R. (1997) Doesneuropathy develop in animal models? Clin.Neurosci. 4, 380–389

9 Fox, A. et al. (1999) Critical evaluation of thestreptozotocin model of painful diabetic neuropathy in the rat , Pain 81, 307–316

10 Campbell, E.A. et al. (1998) Selective neurokinin-1 receptor antagonists are anti-hyperalgesic ina model of neuropathic pain in the guinea-pig ,Neuroscience 87, 527–532

Katharine Walker PhDLaboratory Head

Alyson J. Fox PhDLaboratory Head

Laszlo A. Urban* PhD, MDDeputy Unit Head

Novartis Institute for Medical Sciences, 5 Gower Place, London, UK WC1E 6BN.

Tel: 144 171 333 2126Fax: 144 171 387 4116

*e-mail: laszlo.urban@pharma.novartis.com

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