Replacing animal experiments:choices, chances and challengesGill Langley,1* Tom Evans,2 Stephen T. Holgate,3 and Anthony Jones4

SummaryReplacing animal procedures with methods such as cellsand tissues in vitro, volunteer studies, physicochemicaltechniques and computer modelling, is driven by legisla-tive, scientific and moral imperatives. Non-animal ap-proaches are now considered as advanced methods thatcan overcome many of the limitations of animal experi-ments. In testingmedicines andchemicals, in vitro assayshave spared hundreds of thousands of animals. Incontrast, academic animal use continues to rise and theconcept of replacement seems less well accepted inuniversity research. Even so, some animal procedureshave been replaced in neurological, reproductive anddentistry research and progress is being made in fieldssuch as respiratory illnesses, pain and sepsis. Systematicreviews of the transferability of animal data to the clinicalsetting may encourage a fresh look for novel non-animalmethods and, as mainstream funding becomes available,more advances in replacement are expected. BioEssays29:918–926, 2007.� 2007 Wiley Periodicals, Inc.

Introduction

Public disquiet about animal experiments has a long history

and has intensified in the last two decades. Concern focuses

primarily on the suffering caused to sentient animals, both

during experiments and by the nature of confinement in a

laboratory setting. Critics also point out that the validity of

animal experiments is often assumed rather than proven(1)

and that they are very demanding in costs and time. Animal

research policies that command both public and scientific

support remain elusive and a 30-year downward trend in

numbers of experiments is reversing.

A key emerging solution is the development of novel or

adapted research and testing methods that not only avoid

animal use, but can replace animal experiments. As part of the

internationally accepted Three Rs concept (replace, reduce

and refine animal experiments), the development of replace-

ment techniques is neither new nor untried: since 1986,

national(2) and European(3) legislation has required that

equivalent non-animal techniques must be used in place of

experiments on animals. In the USA, a similar imperative

operates at the institutional level. In the European Union,

the Commission and Member States have a further legislative

duty to encourage research into methods that could achieve

equivalent research objectives, but using fewer animals or

none at all. For this reason, the European Centre for the

Validation of Alternative Methods (ECVAM) was created in

1991, and has successfully validated more than 18 full or

partial replacement methods, eight of which have already

gained regulatory acceptance.

To promote the Three Rs, in 2004 the British government

established the National Centre for the Replacement, Reduc-

tion and Refinement of Animals in Research, whose ultimate

aim is the replacement of all animal experiments. There are

similar centres in Germany, Austria, the Netherlands, Japan

and elsewhere. There is a growing acceptance that non-

animal research methods are advanced techniques, having

potential to improve scientific andmedical progresswith fewer

demands on time and finance. As the British government has

said, ‘‘. . .alternative methods are often, in reality, ‘advanced

methods’ broadening the scope and overcoming some of the

limitations of existing animal models.’’(4)

This review discusses the origins and applications of some

successful replacementmethods already used in fundamental

medical research and in regulatory testing, and looks to three

fields of research (sepsis, respiratory disease and pain) for

chances to further replace animal experiments and to the

future for upcoming developments.

Replacing animal procedures—significant

successes

Replacing animal procedures is a well-established concept in

industry, especially in the pharmaceutical, chemical and

1Dr Hadwen Trust for Humane Research, Hitchin, Hertfordshire, UK.2Division of Immunology, Infection and Inflammation, University of

Glasgow, UK.3Infection, Inflammation and Repair Division, School of Medicine,

University of Southampton, Hampshire, UK.4Human Pain Research Group, Manchester University, Rheumatic

Diseases Centre, Clinical Sciences Building, Hope Hospital, Salford,

UK.

Tom Evans is in receipt of a Grant from the Dr Hadwen Trust.

*Correspondence to: Gill Langley, Dr Hadwen Trust, 84A Tilehouse

Street, Hitchin, Herts. SG5 2DY, UK.

E-mail: gill@drhadwentrust.org

DOI 10.1002/bies.20628

Published online in Wiley InterScience (www.interscience.wiley.com).

918 BioEssays 29.9 BioEssays 29:918–926, � 2007 Wiley Periodicals, Inc.

Abbreviations: ECVAM, European Centre for the Validation of

Alternative Methods; Three Rs, the replacement, reduction and

refinement of animal procedures; FE, finite element; TMS, transcranial

magnetic stimulation; PET, positron emission tomography; COPD,

chronic obstructive pulmonary disease.

Challenges

cosmetics sectors, but seems less well known in academia.

Animal experiments conducted by industry in Britain have

fallen significantly over several years, but those carried out by

universities and medical schools have risen by 52% in the last

decade.(5) The following successful examples of replacement

havebeenselected frommany, to illustrate different routes and

reasons for the development and implementation of replace-

ment methods.

Regulatory testing of medicines and chemicalsSafety or quality testing of medicines or chemicals are areas

where the replacement of animal-based methods has wide

currency, both in industry and the regulatory authorities.

In the regulatory arena, new tests are validated by an

internationally agreed, formal process.(6) Several replacement

techniques have been successfully validated and approved.

Since 2000, three in vitro or synthetic methods have gained

regulatory acceptance for testing chemical corrosivity: the rat

skin assay and human skin model assay and the Corrositex

model(7) have replaced severe in vivo tests on rabbits. The cell-

based neutral red uptake assay for photo-irritancy is accepted

internationally, avoiding tests on mice.(8) Data from the in vitro

method of assessing skin penetration of drugs, pesticides and

chemicals are accepted worldwide, replacingmany studies on

rodents. In 2007, ECVAM endorsed two in vitro tests using

human skin models as replacements for in vivo skin irritation

tests on rabbits.

Other techniques have beenvalidated scientifically and are

in the regulatory pipeline, such as new in vitro pyrogenicity

assays. Parenteral medicines must be tested for pyrogens to

exclude the possibility of bacterial contamination. The original

in vivo test, introduced in the 1940s, measures fever response

by a rectal probe in rabbits restrained in stocks. Some animals

may suffer fever, respiratory problems, organ failure or fatal

shock. The test’s detection limit is above the human fever

threshold; it is time-consuming and costly, and unsuitable for

important new therapeutic areas such as cellular products.(9)

A secondmethod, conducted ex vivo on blood samples from

horseshoe crabs (Limulus polyphemus), was first developed in

the 1970s. Although its range is limited, between 1988 and

1998, the Limulus assay led to a decrease from 78,000 to 9,500

in the annual number of rabbit tests carried out in Britain.

An understanding of the mechanisms of the human fever

reaction, together with advances in cell biology techniques,

prompted the development of new in vitro pyrogenicity tests

usinghumanbloodcells.Basedon theactivation ofmonocytes

in response to pyrogens, the new assays, now fully validated

by ECVAM, avoid problems of species-specificity and are

more sensitive, more accurate, quicker and more cost-

effective.(10) They detect a wider range of pyrogens than the

Limulus test, and are being adapted for use with medical

devices and for environmental air pollutants; yet few people

believed earlier that a complex function such as fever could be

transferred to the ‘test tube’. The European Commission

estimates that more than 200 laboratories worldwide are

already implementing the in vitro methods, which will replace

some 200,000 rabbit tests a year in Europe alone.

In the manufacture and quality control of vaccines and

biological medicines, hundreds of thousands of animals

worldwide have been spared by the introduction of cell-based

methods. Historical cases enable a clearer analysis of causes

and outcomes, from a longer-term perspective. Examples

include testing for residual toxicity of diphtheria vaccine(11) and

potency testing of yellow fever vaccine. The original potency

test for yellow fever vaccine, comprising intracerebral inocu-

lation of immune serum and virus in mice, was introduced in

the 1950s. This lethal dose method was never fully stand-

ardised and had poor reproducibility, attributed to varying

sensitivities to the virus inmice of different ages, to differences

in the route of virus introduction, and to the type of animal

serumused.(12) In the late 1970s, a cell culture assaybasedon

plaque formation to detect neutralising antibodies was found

to be more practical, sensitive and reproducible,(13) finally

replacing an estimated 1,500 mice each year.

Physicochemical techniques, such as colorimetric assays

and high-performance liquid chromatography, have been

introduced as quality control tests for biological medicines.

Digitalis used to be routinely tested for potency on pigeons and

guinea pigs, by a lethal method involving intravenous injection.

In the late 1980s, this was replaced by a chemical colorimetric

assay which directly measured the content of digitoxin.

Similarly, the mouse convulsion method, by which every batch

of insulin was originally tested on 600 mice per sample, was

first refined and eventually replaced by high-performance

liquid chromatography. This was introduced as amore precise

technique(14) and accepted as a replacement by the British

Pharmacopoeia in 1990.(15) Chromatographic assays also

replaced animal bioassays for growth hormone, oxytocin and

lypressin.

In most examples pre-dating the late 1980s, the develop-

ment of non-animal assays was driven by dissatisfaction with

existing animal tests and the need for improved precision,

range or reproducibility.(12–14) The replacement techniques

werealso less costlyandproduced faster results. In the caseof

cosmetics and chemicals, high-profile consumer campaigns

have driven the pace and European directives have now

mandated or facilitated the replacement of animal-based

methods.(16,17) In the last decade or so, as the Three Rs

concept has gained widespread currency, a concern to spare

animals from suffering has become an increasingly powerful

driver for additional efforts to replace them.(18)

Computer simulations in medical research:fetoplacental physiology and orthodonticsAnimal models of human fetoplacental physiology are of

limited value, due to the species specificity of features such as

Challenges

BioEssays 29.9 919

(1) placental structure, permeability and blood flows, (2)

responses to hypoxia, (3) amniotic vascularisation, and (4)

amniotic fluid composition and dynamics.(19,20) This is

particularly so in the case of twin–twin transfusion syn-

drome,(21) a serious condition in which identical twins receive

unequal vascular supplies in the uterus.

The limitations of animal studies prompted the develop-

ment of computer models, based originally on clinical

measurements of fetal growth rates, phenotypic features

and weight discordance, obtained using ultrasound. Colour or

power Doppler imaging of blood flows and vascular anasto-

moses in pregnant women provided further data, as did in vitro

and ex vivo human organ and tissue studies, such as placental

dye injection and microscopy.

The first models addressed the causes of pregnancy-

induced hypertension and pre-eclampsia. By 1995 they had

successfully explained the mechanism underlying the diagnos-

tic ‘notch’ in uterine artery blood flow in women at risk of pre-

eclampsia.(22) In pinpointing the cause as an abnormality in

artery wall elasticity, the computer simulations overturned the

previous hypothesis. Mathematical modelling also demon-

strated howan imbalance in someanastomoses in the placenta

leads to twin–twin transfusion syndrome,(23) revealing the links

between fetofetal transfusion and amniotic fluid imbalance, the

hallmark of the syndrome. This finding led to a test to predict

women’s susceptibility and provided a rationale for classifying

severity so that optimal treatments could be selected.(24)

The models continue to improve and provide important

insights into the physiology of human pregnancy. They now

incorporate features such as imbalances in chorionic vascu-

lature,(25) fetal fluid dynamics, fetoplacental growth and

circulatory alterations, fetal heart failure and aspects of the

rennin–angiotensin system.(26) These developments are

expected to clarify the progression of conditions such as

twin–twin transfusion syndrome, and help determine the

efficacy of current and potential therapies.

Finite element (FE) analysis is a technique borrowed from

engineering. It is a theoretical approach in which a structure is

mathematically modelled by subdividing it into a mesh of

elements, bounded by sets of nodes. Computational proce-

dures are used to determine effects such as strains and

stresses caused byapplied loads. These are thenvisualised to

identify their magnitude and precise locations in the structure.

Since 1973, when it was introduced into dental biomechanical

research, FE analysis has been very widely applied in studies

of dental materials, oral and maxillofacial surgery, orthodon-

tics, dental restorations and more.(27) It is used to predict,

quantitatively and in three dimensions, the stresses and

strains imposed on tissues of the teeth and jaws, to help

assess the safety and efficacy of dental treatments and

appliances (see Fig. 1).

Based on the known structural properties of human tooth,

bone and ligament, and of dental materials such as ceramic

and titanium, FE analysis allows simulations of the outcomes

of corrective treatments, in terms of fracture properties,

stresses and strains at junctions, bond strengths, influences

of thermal or mechanical loads and failure problems.

A recent example is a FE-based computer model of a

titanium screw developed for implantation into the jawbone as

an anchor for applying orthodontic pressure.(28) A simulation

of the screwand surrounding bone predicted the displacement

causedwhen corrective pressurewas applied to the implanted

screw. Themodel showed the highest stresseswould be at the

neck of the screw, and in the bone at the level of the first screw

thread. The results suggested that the screw would provide

adequate anchorage, and helped to inform clinical decisions

about the best use of the implant.

Some animal experiments conducted in dentistry research

are invasive, long term and cause significant suffering.(29)

However, morphological and biomechanical dissimilarities

between species mean that animal studies may provide only

a ‘‘crude indication of the likely biomechanical consequences

in patients’’.(30) Ideally, FE models should be informed by and

validated using human data.(31)

Simulations can generate important insights: for example,

FE modelling of a human tooth and ligament under load,

validated using data from a human volunteer study, revealed

that the periodontal ligament was the location of the largest

strains. This suggested that initial tooth displacement is

Figure 1. Finite element-based three-dimensional simula-

tion of bone adaptation around a screwthread implant. The

colour scale represents predicted variations in bone density in

response to the implant. Density is directly related to bone

strength and, after implant insertion, evolves by a damage/

repair process that continues until a mechanobiological

equilibrium is reached. Computer simulations can give deep

insight into the complex feedback mechanisms and various

parameters involved, predicting the rate of osseointegration

and hence durability of the implant, over more than 80 days.

With permission from Georges Limbert and John Middleton.

Challenges

920 BioEssays 29.9

mediatedvia the ligament rather than bycellular remodelling in

the bone.(32)

The widespread use of FE simulations in dentistry has

replacedsomeanimal experiments, particularly in researching

novel orthodontic appliances and materials, and in cases

where competing hypotheses would otherwise have been

explored using animal models.

Transcranial magnetic stimulation in functionalbrain researchTranscranial magnetic stimulation (TMS) is a technique used

in functional brain research which, applied to human volun-

teers, is widely accepted as a replacement for some invasive

experiments on non-human primates.(33) Using a coil, TMS

applies to the head amagnetic field which, by inducing random

neural activity, transientlyandsafelydisrupts a targetedareaof

the brain. This creates a ‘virtual lesion’ and during the tens of

milliseconds (or more) of disruption, volunteers perform

cognitive, visual or other tasks to determine the effect of the

‘lesion’ on their normal performance. The usual function of that

part of the brain can thus be inferred.

Magnetic stimulation of the human nervous system was

originally conceived in the mid-1980s(34) as a technique for

clinical assessment of central nervous function (especially

motor function), in patients with conditions such as multiple

sclerosis and demyelinating neuropathy. The conventional

method used painful electrical stimulation through the skin.

Magnetic stimulation, by contrast, was painless and suitable

both for clinical assessment and research.(35) The technique

was developed in human volunteers, by comparison of

magnetic versus electrical stimulation in the same individu-

als.(36) Experiments using dogs and primates were also

conducted(37) although not from a need to demonstrate safety

or to validate the technique, since these studies had already

been conducted with humans.

The research applications of TMS have since expanded

beyond motor function to include central visual and cognitive

processing aswell as cortical development and plasticity. As a

virtual lesioningmethod, it can demonstrate whether or not an

area of the human brain is actually necessary for a certain

function, rather than merely correlating neural activity with an

outcome, as is the case with functional magnetic resonance

imaging or positron emission tomography (PET) studies. TMS

combines good spatial and temporal resolutions, and has a

number of advantages in functional brain research.(38)

Compared to animal lesioning experiments, TMS is

superior in providing a chronology of activity in several brain

areas and the ability to use within-subject controls. It permits

analysis of a lesion effectwithout the complicationof functional

re-organisation, which occurs after brain lesioning in conven-

tional animal experiments. The spatial resolution of TMS does

not compare with cell-level experiments using electrodes in

animals. But in cognitive psychology research, for example,

with a systems-level focus, some experts believe that human

functional studies could provide virtually all the information

needed. This would replacemany experiments on non-human

primates.(38) Being safe and non-invasive, TMS has opened

the door to a wide range of human brain studies(39) without the

complications of species variations.(40)

Replacing animal procedures—further

opportunities

Three areas of medical research—sepsis, respiratory illness

and pain—have been selected to illustrate where significant

progress is being made in replacing animal experiments.

In each area, experiments may involve substantial animal

suffering, therapeutic breakthroughs are urgently needed;

and the validity of the animal models has been criticised.

Research into sepsisSepsis is a complex host response to severe infection.(41)

Despite considerable advances in intensive care treatment

and thedevelopment ofmoreeffectiveantibiotics, themortality

of this condition remains about 20–50%. An estimated 31,000

patients were admitted with sepsis to Intensive Care Units in

2004 in England,Wales andNorthern Ireland, of whom14,000

died before discharge, a mortality rate of 45%.(42) There is a

major need to developnew therapies targetedat this condition.

Animals have been used extensively to study the patho-

physiologyof sepsis, and asmodels for the disease to test new

therapies as they are developed.(43) Two major classes of

model have been developed. Compounds derived from

microbes, such as the bacterial component lipopolysacchar-

ide, canbe used tomimicmanyof the features of sepsis, and in

a high enough dose these are fatal. Alternatively, experimental

infections with live microbes are used to establish systemic

infection and the development of sepsis.

Both thesemodels producemajor physiological changes in

the animals used and can be rated substantially severe in

Britain by the Home Office, which regulates animal experi-

ments. Animal models have provided a large body of evidence

to establish the major pathophysiological mechanisms that

operate during sepsis, and as a springboard in the develop-

ment of new therapies. However, they are imperfect models.

There are important differences in the responses of different

animals, including humans, to sepsis. For example, rodents,

the most-utilised species in such work, are about 1,000-fold

more resistant to the toxic effects of lipopolysaccharide than

humans.(44) Additionally, there have been a number of high-

profile experimental treatments that have worked well in

animal models but failed in clinical trials.(45)

Given these ethical and scientific concerns, there is a need

to replace and refine animalmodels in the study of sepsis, both

to reduce animal suffering and to improve the predictive power

of models in developing new therapies for human sepsis. The

complex nature of sepsis represents a real challenge to

Challenges

BioEssays 29.9 921

develop alternatives to animal experimentation that will

provide meaningful biological information. However, given

the lack of success of animal models of sepsis in predicting

outcome in humans(45) there is a real need to develop better

models to predict this. There are a number of approaches that

hold promise for the future.

The in vitro tools of molecular and cellular biology will still

providemuch important information in the future. The ability to

manipulate such systems in a controlled fashion allows firm

inferences to be drawn and new hypotheses tested. Newer

methods of cell culture using three-dimensional supports hold

promiseas better models of tissue function (seeFig. 2),(46) and

advances in stem cell biology may well allow quite complex

tissues to be constructed entirely in vitro. Similarly, progress

being made in computer modelling of sepsis may also allow

modelling of septic processes without the use of animals.(47)

The last 50 years have seen a considerable decline in the

field of clinical experimentationwith human subjects. Although

the growth of much greater control over human studies is to

be welcomed, it seems this has led to a decline in such

approaches, both in living patients and in material removed

for subsequent in vitro analysis. Both these approaches can

shed considerable light on the pathophysiology of sepsis. For

example, studies investigating blood flow in human volunteers

have investigated the mechanisms underlying venodilatation

following cytokine administration, shedding light on the role of

nitric oxide in living humans.(48)

An important study used biopsies from children with

meningococcal sepsis to study the role of the anticoagulant

proteinC in sepsis.(49) The finding that endothelial activation of

protein C was impaired in this setting helped underpin the

development of activated protein C as a therapy of sepsis with

some demonstrable benefit. This is a concrete example where

data derived directly from human studies have aided in the

development of a useful therapy. Similarly, muscle biopsies

taken from septic patients demonstrated severemitochondrial

dysfunction, suggesting novel targets for therapeutic inter-

vention.(50) A greater use of human subjects and material in

sepsis research could contribute to the reduction of animal

experimentation, while providing mechanistic insights into this

serious medical problem.

Research into respiratory diseasesThemajority of diseases affecting the lung are environmental in

origin, whether produced by infectious, immunological or

toxicological mechanisms. The conducting airway and alveolar

epithelium, with the surface area of a tennis court, provides the

interface between the external environment and the internal

tissue milieu which, if disturbed, leads to tissue damage and

disease. The epithelium serves a barrier function both physi-

cally, by excluding environmental insults, and functionally

through the release of several bioactive molecules and a range

ofmetabolic activities to protect against or inactivate chemicals.

New therapeutic agents are needed for asthmaand chronic

obstructive pulmonary disease (COPD), but animal models

are limited because of structural and physiological differences

in the airways ofmice and humans.(51) These two diseases are

good examples of environmentally associated disorders that

reveal themselves through increased genetic susceptibility

and a range of environmental insults e.g. tobacco smoke,

allergens, chemicals and infectious agents.(52) In both

diseases, there are major changes in structure and function

of the epithelium. The epithelium also expresses many

candidate susceptibility genes for these diseases, identified

by positional cloning. For example, in asthma, a strong

association has been found with polymorphisms of several

genes that are preferentially expressed in the epithelium.(53)

Based on gene–environmental interactions, it has been

possible to reproduce some of the characteristics of asthma

and COPD using tissue engineering. Epithelial cells brushed

from the airways of volunteers at fibreoptic bronchoscopy can

be cultured to confluence in tissue culture over 2–3 weeks.

These provide a resource for investigating basal character-

istics as well as responses on exposure to pertinent stimuli,

e.g. air pollutants,(54) tobacco smoke,(55) viruses.(56,57) Epi-

thelium reconstituted in vitro from asthmatic patients exhibits

increased permeability due to reduced formation of tight

junctions, and increased susceptibility to oxidant pollutants. It

is also more susceptible to damage by common cold viruses

(due to reduced b- and l-interferon production).

In the case of COPD, epithelial cells at baseline and

following exposure to tobacco smoke extract also exhibit

Figure 2. Renal tubule grown in vitro. Human primary renal

tubule epithelial cells were grown in a three-dimensional

collagen matrix in the presence of hepatocyte growth factor

to encourage tubulogenesis. The cells were fixed and stained

for the actin-associated protein ezrin (green), which is

concentrated in the brush border (arrowheads) facing a central

tubular lumen (L). Such three-dimensional cell cultures can be

used to study tissue damage in sepsis. Bar is 50 mm.

Challenges

922 BioEssays 29.9

markers of injuryand repair that occur invitro in theairwaycells

of patients, but not in those fromnormal volunteers. The profile

of protective oxidant genes expressed by epithelial cells from

COPD patients at baseline and when exposed to tobacco

smoke extract also map onto disease severity in vivo.(55) As in

asthma, epithelial cells cultured from COPD patients also

exhibit increased sensitivity to the damaging effects of

common cold viruses that might explain why such patients

are more vulnerable to virus-induced exacerbations. In both

conditions, the disease-related phenotypes persist in tissue

culture over several passages, suggesting that they are

primary rather than secondary abnormalities.(57)

Building upon the in vitro monolayer system, it has been

possible to produce a fully differentiated airway epithelium

by growing cells on inserts and, after they have formed

monolayers, bringing the cells up to the air/liquid interface.(58)

On removal of growth factors and addition of retinoic acid,

these cells differentiate after 3–4 weeks into a fully stratified

epithelium with functional ciliated and mucus-secreting goblet

cells. If grown in the presence of the pro-asthmatic cytokine IL-

13, a high proportion of the columnar cells transform into

goblet cells with evidence of active mucus secretion.(59)

Differentiated epithelial cells grown from asthmatic airways

behave differently from those of normal airways in their defi-

cient formation of tight junctions, accompanied by a parallel

reduction in transepithelial electrical resistance indicating a

more ‘leaky’ epithelium (STH, unpublished observations).

Since these cell culture systems maintain aspects of

disease phenotypes, they can be used to look for novel

molecular targets using genomic and proteomic platforms.

They can also be used as test systems for novel therapeutics

such as human recombinant b-interferon in restoring resist-

ance to the common cold virus.(56) Taken together, these

studies provide a solid basis of utilising airway epithelial

cultures from well-phenotyped patients, to discover novel

therapeutic targets and test new therapeutics for these

diseaseswhere there is an urgent need for greater innovation.

Computer simulations have also been used to explore

mechanisms of asthma pathology and predict the efficacy of

potential treatments, helping further to replace some animal

experiments.(60)

Research into painPain canonly be described andconfirmed inhumans in termsof

an integratedsensory, affectiveandmotivational experience.(61)

Although most pain is generated by activation of specialised

nerve endings (nociceptors), pain can also result from damage

to the nervous system or from psychological stress.

Functional brain imaging experiments in volunteers have

demonstrated a matrix of higher cortical centres implicated in

the complex cognitive integration that results in pain.(62,63)

Some of these higher centres, such as the cingulate cortex,

are poorly developed in many non-primates.

The assumption that discoveries can be easily transferred

from one species to another has to be challenged by a long list

of drugs that are effective at reducing nociceptive responses in

animals, but have failed as analgesics in clinical trials. The

challenge now is to determine how we can develop new and

safe analgesics more efficiently.

Animalmodels have provided uswith key information about

the detailed anatomical connections of nociceptive path-

ways(64,65) and potential physiological mechanisms of pain

perception.(66–68) However, the focus of studies in animals has

been on nociception within structures downstream of the

brain stem. Animal models of pain have been well reviewed

recently.(69) Generally, animal behaviour is observed, or

responses in the nervous system are recorded, whilst a

noxious stimulus is applied before and after some kind of

physiological intervention. The main problem with the inter-

pretation of animal models is that behavioural observations

are limited to motor responses and these cannot easily be

extrapolated to a change in pain experience. Animal pain

models can therefore only yield some possible clues to

potential mechanisms of pain in humans.

Pain is perceived as a result of integrated activity of well-

defined brain structures and there are two main systems: the

medial and lateral pain systems. Functional brain imaging

studies in humans have provided substantial information

about how these structures contribute to normal and abnormal

human pain perception in volunteers and patients (see

Fig. 3).(62,63) For instance, PETstudies that indirectly measure

synaptic activity in the brain have been used to establish that

the medial and lateral pain systems are mainly concerned

with emotional pain processing (e.g. unpleasantness), and

sensory-discriminative processing (e.g. pain localisation),

respectively.(70) Using a different PET technique that images

receptor binding on neurons, it has been possible to measure

changes in activity of natural pain-killers called endorphins,

in different types of chronic pain.(71,72) Complementary

approaches include post-mortem tissue analysis, which

provides a single ‘snapshot’ of chemical differences in

peripheral and central neurons (e.g. spinal cord), in relation

to sensory abnormalities in patients with pain due to nerve

damage.(73)

The challenge is to use these different types of information

to develop new therapies for painmore effectively.We suggest

some approaches to achieve this. First, there needs to be a

shift from anatomical-, disease- and time-based classification

of pain to a more physiological definition of clinical pain

syndromes, using physiological methods such as functional

brain imaging techniques. This can be achieved by careful

physiological and psychological measurement in patients with

different types of pain syndrome. For instance, we (AJ) have

recently identified an inability to alter the way that they attend

to pain in patientswith a type of widespread chronic pain called

fibromyalgia.(74)

Challenges

BioEssays 29.9 923

To achieve a more physiological approach to pain classi-

fication, it will be necessary to identify the normal physio-

logical(70,71) and pathophysiological mechanisms of pain

perception.(75,76) For example, PET studies have shown a

selective reduction in receptors for natural opiates in thebrains

of patients with severe pain due to stroke.(76) Such studies

require substantial investment and greater collaboration

between the pharmaceutical industry and academia.

Following identification ofmechanisms in humans, targeted

drug development should be narrowly based on modulation

of those mechanisms. Having identified and measured the

pathophysiological mechanisms, proof-of-concept trials will

be much more cost-effective. Preferably no drug should be

developed without establishing that it reaches the target organ

in humans, prior to clinical trials. In many cases, this can be

achieved using molecular imaging e.g. PET.

In summary, techniques exist to begin to reclassify human

pain physiologically and to identify candidate pathophysiolog-

ical mechanisms in volunteers. By working back from these

mechanisms to drug development, some animal experiments

maybe replaced. This is not beyond currentmolecular imaging

capacities but will require a sea-change in the way we think

about pain.

Where next?

The drive to replace animal procedures has historically involved

humaneand scientific aspects, and this is still so. Acceptance of

the capacityof animals toexperience pain anddistressprovides

a growing impetus for progress, most apparent in regulatory

testing where non-animal method development has been

embraced by toxicologists, legislators and regulators. Individual

scientists concerned about animal use have made a significant

impact in particular fields, such as vaccine testing.

Dissatisfaction with the quality of animal data remains a

powerful motivator for change, and the search for superior

alternatives is increasingly attracting government funds.

The European Commission now supports a programme of

research to replace animal-based toxicology, and promotes

this as a contribution to the quality of science and the safety of

consumers as well as preventing animal suffering.

In academia, with an emphasis more on fundamental

medical research, change has been slower. There are several

reasons: (1) open-ended research questions are perceived as

being more difficult to pursue without animal experiments, (2)

there are few avenues for consumer pressure to be exerted,

and (3) academic research is less shaped by legislative and

regulatory initiatives. However, recent systematic reviews of

the translation of animal research into clinical benefit may well

signal a change.

Several such reviews have demonstrated that animal

studies were poorly predictive of human outcomes. In

particular, discordances were found in animal and human

data for (1) corticosteroids in head injury, (2) anti-fibrinolytics in

bleeding, (3) tirilazad for stroke,(77) and (4) neuroprotective

drugs in stroke.(78) A systematic review of 76 highly cited

animal studies published in seven leading journals, found that

only 37% translated into successful human trials.(79) In these

articles and others, one of the key questions is whether the

animal models sufficiently mimic the human diseases.

Change is on itsway. Legislative developments, such as the

new chemical regulatory framework in the European Union,

and the revision of European legislation on animal experi-

ments, may force the pace. The perception that research to

replace animals is a niche field is gradually being overcome,

and ideally the replacement concept will become embedded

in mainstream journals. In the USA, the Interagency

Figure 3. Sections of brain areas at 4 mm and 6 mm demonstrating increased activation within the areas of the medial pain system in

patientswith arthritic pain (AP) condition (AP - EP), compared to the experimental pain (EP) condition (EP - AP). pACC, perigenual anterior

cingulate cortex; aMCC, anterior mid-cingulate cortex; PCC, posterior cingulate cortex; SGC, subgenual cingulate cortex.

Challenges

924 BioEssays 29.9

Coordinating Committee on the Validation of Alternative

Methods has just drafted a five-year plan to develop and

validate alternatives to animal experiments; and Japan

established a centre for alternative methods in 2005. Major

funders such as the Wellcome Trust are taking their first

tentative steps to support this research. Influential organisa-

tions such as the Royal Society(80) and the Nuffield Council on

Bioethics(81) increasingly acknowledge, in their publications,

the limitations of animal data in medical research.

Replacing animal experiments is a cultural challenge

requiring flexibility and openness to new ideas, and a scientific

challenge needing a fresh, cross-disciplinary approach. The

research needed also has potential to boost science, improve

medical progress and better protect the safety of patients and

consumers.

Acknowledgments

We are very grateful for assistance from Nevil Chimon and

Alison Watson, and to Georges Limbert and John Middleton

for permission to reproduce Figure 1.

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