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5
Advancing the 3Rs in
Neuroscience Research
The 3Rs (replacement, refinement, and reduction) play an increasingly
important role in animal research regulations. As previously described (see
Chapter 2), the revised European Union (EU) Directive includes a formal
introduction of the 3Rs as guiding principles. In addition, both U.S. and
Chinese regulations call for incorporation of the principles of the 3Rs in
experimental design. This session explored examples of how the 3Rs are
implemented in two fields of neuroscience research, spinal cord injury and
epilepsy. Speakers also discussed how systematic reviews could be applied
to preclinical research to help advance the 3Rs.
Sue Barnett, professor of cellular neuroscience at the University of
Glasgow, opened this session with a brief introduction to the 3Rs, the
framework for the humane use of animals in research first articulated by
Russell and Burch in 1959 (Box 5-1). (Session points are summarized at the
end of the chapter in Box 5-2.)
REPLACEMENT CASE EXAMPLE:
SPINAL CORD INJURY MODELS
Barnett described an example of a replacement strategy she is developing
for spinal cord injury research. Clinical strategies have primarily been pal-
liative care, including drugs (e.g., steroids) to dampen the immune response
during the acute phase, advanced rehabilitation strategies (e.g., physio-
therapy), and neural prostheses (e.g., functional electric stimulation [FES]).
The most common causes of spinal cord injury are motor accidents
(50 percent), falls (24 percent), and sports (9 percent). Spinal cord injury
43
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44 INTERNATIONAL ANIMAL RESEARCH REGULATIONS
BOX 5-1
Replacement, Refinement, and Reduction
Replacement
Methods to avoid or replace the use of animals in areas where they otherwise
would have been used, including
• sing non-animal alternatives such as human volunteers, computer models,
u
and in vitro techniques.
• sing animals of lower neurophysiologic sensitivities such as invertebrates.
u
Refinement
Improvement to scientific procedures and husbandry that minimize pain, suffering,
distress, or lasting harm and/or improve animal welfare, including
• mproved procedures (e.g., surgery).
i
• mproved anesthesia.
i
• mproved housing and husbandry.
i
• etter welfare assessment.
b
Reduction
Methods that minimize the number of animals used (or maximizing information
gained from a given number of animals), including
• ood environmental design and statistical analysis.
g
• issue sharing.
t
• maging.
i
SOURCES: Barnett presentation; Russell and Burch (1959).
is a complex event that begins within minutes of the mechanical injury and
progressively worsens over the subsequent weeks to months.
Repair Strategies
After an injury, formation of glial scars inhibit central nervous system
repair by creating both physical (e.g., cyst) and biochemical (e.g., inhibi-
tory signals) barriers to axonal growth. The goal of any repair strategy
is to fill any cysts, maintain glial/neuronal survival, limit scar formation,
promote axonal regeneration, and make functional reconnections. Using
animal models, researchers are studying injecting growth factors, blocking
inhibitory signals (e.g., anti-Nogo [described by Lemon]), transplanting
cells, bridging the gap using biodegradable scaffolds to align the axons, and
promoting plasticity/sprouting of any remaining intact fibers. No one treat-
ment alone is capable of repairing the spinal cord, Barnett noted. Current
thought is that a combination of strategies will be required.
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ADVANCING THE 3Rs IN NEUROSCIENCE RESEARCH
Three main laboratory strategies are currently used to treat a damaged
spinal cord. The first, neuroprotection, is to protect what is left and mini-
mize further damage. Second, especially for incomplete injuries, the strategy
includes remyelination or making the most of what remains. Repair is the
third strategy, which includes restoring communication, axonal regenera-
tion, and reconnection, often by cell transplantation or pharmacological
intervention.
Spinal Cord Research in Animals
Spinal cord research in humans is difficult and in some cases impos-
sible. There is no ability to biopsy tissue, imaging is limited, and studies
cannot be done on large groups of people with similar pathology. The only
way to investigate spinal cord injury, Barnett said, is to use animal models
or primary cells from animal tissue.
An example of an animal model of a spinal cord lesion is a wire knife
lesion, generated by inserting the knife into the dorsal column and pulling
up a piece of tissue. Barnett noted that this method is clean, accurate, and
consistent, resulting in a cavity and glial scarring that mimics human spinal
cord injury. By tracing regenerating axons using fluorescent labeling tech-
niques, Barnett has observed that while many axons enter and fill the lesion
site, they have limited ability to grow through the lesion, and few exit and
find their target. This, Barnett explained, is the major problem with many
of the spinal cord injury repair therapies.
One aspect of spinal cord injury that researchers want to mimic is
the glial scar. A useful model would have a lesion surrounded by reactive
astrocytes that express molecules of interest; axons would be inhibited from
entering or exiting the scar and would become demyelinated; and there
would be activated microglia.
Several disadvantages to rat models of spinal cord injury include the
need for large numbers of animals, the severity of the procedure, and
the distress and discomfort to the animals, Barnett said. Additionally, there
is a long time frame for results and the experiments are expensive and time
consuming. To address this, Barnett is working to replace animals in her
experiments.
Replacing Animals with Cell Culture
Barnett described her in vitro model of spinal cord injury in which
disassociated embryonic spinal cord cells from rats are layered on top of an
astrocyte monolayer derived from embryonic tissue (Sorensen et al., 2008).
Growth in culture over time leads to complex axonal/glial interactions re-
sulting in myelinated neurons. This system allows for the study of contact
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46 INTERNATIONAL ANIMAL RESEARCH REGULATIONS
between astrocytes and how they communicate with the axons, which is
necessary for understanding these problems in spinal cord injury. Barnett
and colleagues next induced lesions in the cell culture by cutting with a
scalpel to studying axon density and myelination adjacent to the lesion
and cell growth into the damaged area. To validate the model, Barnett
has studied several molecules previously tested in vivo to see if they could
promote outgrowth or repair in the vitro system.
Overall, the findings from the in vitro model of spinal cord injury cor-
relate with in vivo findings, including the formation of features typical of a
glial scar, neurites that do not cross the boundary of the scar, and myelina-
tion and neurite density that is decreased adjacent to the lesion. The cells
in culture respond to reagents that have been reported to promote axonal
growth in rat models of spinal cord injury. This model also could be used
to prescreen combinations of biological and pharmacological agents for
potential therapy for repair of spinal cord injury. Barnett noted that getting
the model published so others can become aware of it has been successful,
but also challenging (Boomkamp et al., 2012).
REFINEMENT AND REDUCTION CASE EXAMPLE:
EPILEPSY MODELS
Gavin Woodhall, reader in neuropharmacology at Aston University,
discussed refinement and reduction strategies, using his work in epilepsy
research as an example. Refinement can improve research findings, he
noted, and often results in reduction as a “byproduct.” Simple refinements
can have significant effects on the study results. Enriching the cage environ-
ment, for example, by adding a few tunnels or a bit of nesting material to a
rat cage, improves the neurological development of rats. Rats reared in an
environment that contains no enrichment show different somatic mecha-
nisms of memory than rats that have been reared in an enriched environ-
ment. Studies have also shown that cross-fostering to equalize litter sizes
impairs cortical neuronal network function. Other examples of refinement
include substitution of non-invasive approaches for more invasive ones; use
of analgesia preoperatively, not just postoperatively; habituating animals to
procedures, such tail-vein blood sampling, so that they are less stressed; and
reducing the severity of protocols.
Animal Models of Epilepsy
In the United Kingdom, 450,000 people, or 0.5 to 1 percent of the
population, suffer from epilepsy, with approximately 30,000 new cases
diagnosed each year. One-third of patients do not respond to any of the
currently available drugs and 20 to 30 percent do not improve with sur-
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ADVANCING THE 3Rs IN NEUROSCIENCE RESEARCH
gery. In the developing world, 60 to 90 percent of epilepsy is undiagnosed
or untreated.
A variety of in vivo and in vitro models of epilepsy exist, including
spontaneously induced epileptic mouse strains, chemically or physically
induced models, and cultured neurons. Woodhall’s research relies on a
long-established technique called lithium-pilocarpine epileptogenesis, which
uses a chemical insult to provoke development of epilepsy over an extended
period of time and results in a chronic epileptic syndrome in an animal.
Brain slices are then obtained from the animals for testing. After injection
of the drugs, the rodent goes into acute status epilepticus defined as con-
tinuous seizures with very short gaps in between. In many laboratories, this
phase is allowed to continue anywhere from 90 minutes to 6 or 7 hours,
Woodhall said. Seizures are then arrested with a sedative. The animal enters
a quiescent period that lasts 1 or 2 weeks before they begin to exhibit spon-
taneous recurrent seizures. A conservative estimate of mortality from this
approach is 5 to 50 percent; however, in some laboratories mortality rates
are more than 80 percent, Woodhall noted. This extremely high mortality
rate prompted Woodhall to focus on how this model could be refined and
survival improved.
Questions persist as to whether these models are good models of tem-
poral lobe epilepsy, or of epilepsy in general, and whether the pathology is
similar to that seen in humans. There are also concerns about reproducibil-
ity, as measurement of key indicators can be highly variable. For example,
γ-amino butyric acid (GABA)–mediated levels, an indicator of inhibitory
action, in this model have been shown to decrease, increase, or remain
unchanged. In addition, Sloviter (2005) showed that when animals are al-
lowed to remain in acute status epilepticus for 6 or 7 hours, large areas of
hemorrhage and damage were visible in brain slices. This raises questions
about the seizures the model elicits, specifically whether these seizures as
a result of gross global damage are a true model of human epilepsy, said
Woodhall.
Refinement
Woodhall raised several questions regarding refinement of the current
epilepsy model: whether the severity of this approach can be reduced;
whether acute status epilepticus can be avoided altogether; whether more
“ethical value” can be gained from the model; and whether other ap-
proaches could be used.
Seizure activity feeds from the cortex, through the basal ganglia, and
back into the cortex, to create a positive feedback loop during epilepto-
genesis. Seizures then become uncontrolled and spread to the brainstem,
killing the animal, Woodhall explained. Use of the central muscle relaxant,
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48 INTERNATIONAL ANIMAL RESEARCH REGULATIONS
xylazine, reduces the intensity of the seizure activity and instantly reduces
mortality rates. The other critical point in the process is arrest of the sei-
zures. A massive dose of diazepam is currently used, which can stop the
heart. Instead, a cocktail of very low doses of synergistic drugs, acting at
different receptor systems, can more controllably terminate the seizures,
Woodhall explained. It turns out, he said, that acute status epilepticus can
be avoided. To make the most of the model, Woodhall identified several
ways to increase the use of the fragile brain slices obtained from animals.
Enlisting multiple researchers on one day to extract as much data as pos-
sible from each individual rat reduces the number of animals needed during
the experiment. Methods for production and storage of slices were also
improved. Together, these refinements led to development of a new model,
low-dose lithium-pilocarpine-xylazine epileptogenesis, with a very brief
period of acute status epilepticus, much longer quiescent period, and less
than 2 percent mortality. The new model, which mimics the unique features
of pediatric epilepsy, was validated using brain slices from children who had
surgery for intractable epilepsy, Woodhall noted. In addition to refining the
models themselves, Woodhall said that data sharing among researchers is
another aspect of refinement and overall reduction as well.
Refinement presents some challenges, Woodhall noted. The new epi-
lepsy model, for example, takes longer to achieve recurrent seizures and is
therefore more expensive, and there is more variability. Woodhall concurred
with Barnett that it can be challenging to publish refinements to methods
that have been broadly used for decades.
SUPPORTING THE 3Rs WITH PRECLINICAL SYSTEMATIC REVIEWS
Clinical systematic reviews combine the results of many different
studies, increasing the power of analysis and confidence in the conclusions.
Meta-analysis of clinical trials has long been used in drug development
to gain a fuller picture of the potential efficacy of an investigational com-
pound. Meta-analysis has, for example, identified shortcomings of indi-
vidual trials, identified toxicities that were not significant in a single study,
influenced how future trials should be designed, and clarified responses of
different subpopulations of patients.
Anne Murphy, associate professor at the University of California, San
Diego, suggested that systematic reviews of preclinical data and transla-
tional animal studies could assist with replacement, refinement, and re-
duction of animal use in neuroscience research. A systematic review is a
formulaic, statistically based approach to analyzing preclinical data. The
formulaic approach to systematic reviews minimizes bias and maximizes
transparency; the results are objective and quantitative. In general, the steps
of a systematic review are
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ADVANCING THE 3Rs IN NEUROSCIENCE RESEARCH
• Conduct exhaustive search for published and unpublished relevant
data.
• Select studies for inclusion that meet predetermined criteria.
• Critically appraise studies, evaluate quality, and extract data.
• Combine data and apply appropriate statistical analysis.
• Draw conclusions and write manuscript.
• Update review as additional relevant studies emerge.
Can Systematic Reviews Assist with the 3Rs?
Systematic reviews could also assist with replacement, refinement, and re-
duction, Murphy suggested. Preclinical systematic reviews could potentially:
• Replace animal use by
o roviding evidence of the validity of studies by comparing in
p
vitro, invertebrate, or in silico data with data from traditional
animal studies.
• Refine experimental procedures by
o ighlighting how differing methodologies affect measures of
h
efficacy.
o roviding a platform for setting a standard for the methodology
p
of a particular model and unifying the reporting requirements.
o roviding evidence of the effectiveness of refinements.
p
• Reduce the ineffective use of animals by
o voiding duplication, preventing further studies of ineffective
a
interventions.
o roviding a more precise estimate of treatment effect, thereby
p
informing future power analysis.
Systematic reviews do have weaknesses, however, noted Murphy. The
value of the review for the development of therapeutics for humans depends
on the quality of included preclinical studies. Systematic reviews can be-
come outdated rather quickly and must be regularly updated as new data
become available. This requires some sort of repository or electronic ware-
house for the data so that modifications can readily be made. Systematic
reviews can still be susceptible to bias in the selection of studies, especially
if the predefined rules are not followed. Finally, there is the challenge of ob-
taining unpublished data; in particular, negative data are difficult to collect.
Preclinical Studies
A fundamental problem with the use of animals in research is that
efficacy in animal models of disease does not necessarily equal efficacy in
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humans, Murphy noted. Many compounds come through animal studies
only to fail in the clinic. Clinical trials fail for a variety of reasons. For
example, they may be underpowered or they may underestimate the vari-
ability of the endpoint measures, leading to inconclusive results. However,
sometimes the treatment regimen for humans differs from that of the animal
model. In stroke, for example, the majority of preclinical data suggested
a short therapeutic window; however, it generally is not possible to see a
patient within 15 minutes after the start of a stroke. This, Murphy sug-
gested, is one of the reasons that many stroke compounds have failed in
the clinic.
Some animal studies also have methodological bias. As an example of
empirical bias in the design of experimental stroke studies, Murphy noted
that studies are generally done in young, healthy, male animals, while
humans who have strokes are generally older with comorbidities (Crossley
et al., 2008).
The quality of preclinical studies is highly variable. A recent survey
found that 40 percent of 271 randomly chosen articles did not state a hy-
pothesis or objective, or the number and characteristics of animals (e.g.,
species, strain, sex, age, weight). The survey also found that more than
85 percent of studies did not report randomization or blinding and 30 per-
cent did not report statistical methods (Kilkenny et al., 2009). Study quality
influences measures of efficacy. The assessment of study quality is an inher-
ent part of a systematic review, Murphy noted.
Murphy suggested that preclinical systematic reviews could help ad-
dress some of these issues. A systematic review by Perel and colleagues
(2007) comparing treatment effects in animal experiments and clinical
trials found systematic reviews of preclinical data could identify low-quality
animal studies and better predict success or failure of compounds in clini-
cal trials.
Integrating Systematic Review into Preclinical Translational Research
In summary, Murphy said, systematic review could be applied to pre-
clinical data in order to improve the overall quality and value of animal
studies, support the 3Rs, and inform clinical trials. The path to implemen-
tation of systematic reviews as a matter of routine potentially includes
the Food and Drug Administration, pharmaceutical companies, research
institutions, and publishers.
Murphy suggested two strategies for supporting systematic reviews of
preclinical research. The first is to raise awareness of the power of applying
systematic reviews to animal studies. Conducting reviews can inform and
improve the timing, design, and quality of studies and better inform sub-
sequent clinical trials. The second is to secure support from publishers and
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ADVANCING THE 3Rs IN NEUROSCIENCE RESEARCH
journal editors. Access to useful data might increase with more rigorous
application of requirements for publication and rejection of low-quality or
incomplete studies. In addition, the support for the publication of negative
data would enable increased sharing of primary data, regardless of the
outcome.
IMPACT OF THE 3Rs ON DRUG DISCOVERY AND DEVELOPMENT
Jackie Hunter of OI Pharma Partners discussed how the evolving phar-
maceutical industry may change animal research, specifically, how human
studies could lead to opportunities for increased application of the 3Rs
and how changes in business models could lead to greater data sharing
and hence, opportunities for reduction in the numbers of animals used
as well. The pharmaceutical industry faces many challenges in bringing a
new product to market. Hunter noted that over the past 10 to 15 years,
the number of approvals of new drugs for nervous system disorders has
dropped and the pharmaceutical industry in general is moving away from
neuroscience research.
Refinement Stemming from Target Validation in Humans
In drug development, animal research plays a role in target valida-
tion, screening of compounds to optimize pharmacokinetics and efficacy,
and safety and toxicology testing. Advances in technologies, however,
are enabling increased target validation in man, potentially reducing the
need for animals. Studies of the genetics of rare diseases, imaging studies,
genome-wide association studies (GWAS), pharmacogenomics, and stem cell
research are informing industry decisions to pursue particular drug targets.
For example, researchers are modeling schizophrenia using human-
induced pluripotent stem cells, identifying new pathways and potential
drug targets that have not been previously associated with schizophrenia
(Brennand et al., 2011). Studies of mutations in individuals with rare
diseases or isolated syndromes who exhibit a gain or loss of function also
can help focus drug discovery efforts. For complex disorders involving
multiple genes, GWAS are beginning to cluster pathways, identifying con-
vergent nodes on these pathways that may be important in terms of disease
progression.
While increased target validation in humans is unlikely to replace ani-
mal models, it will allow refinement of the questions asked of the models,
Hunter said. For example, knowledge from human validation studies could
lead to an increased focus on models of mechanism, rather than models of
disease. Refined models could also help identify unwanted target-related
effects, allowing a target to be invalidated early in the process.
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Animal Models of Mechanism Versus Models of Disease
Few animal models faithfully represent the full complexity of the dis-
ease being modeled. This is especially true for nervous system disorders and
diseases, for which animal models are limited in predicting drug efficacy,
Hunter noted. Animal models may provide conflicting data in terms of ex-
posures of drugs required, and result in false negatives. As a result, products
are frequently tested in multiple models.
Hunter suggested a need to move toward more mechanistic models.
Increasing disease knowledge allows for better identification of key mecha-
nisms. The focus then should be on developing mechanistic in vivo assays
that can be translated to humans. Such assays could demonstrate com-
pound effects on the mechanism, define the exposures required for effi-
cacy on the mechanism, and allow comparison of pharmacodynamics with
pharmacokinetics. This could lead to a reduction in the number of models
and experiments needed, Hunter opined.
This approach requires a different mindset, Hunter said. For progres-
sion into human trials, if a molecule works in an animal model of disease,
it is often necessary to show that it works in several models. On the other
hand, if a molecule works on a particular mechanism, only one experiment
may be needed to take it forward.
Precompetitive Collaborations
The current economics of drug development are not sustainable, Hunter
commented. One approach to help move discovery forward is the concept of
precompetitive collaborations. A number of efforts are underway globally to
share more data and information. One example is the Innovative Medicines
Initiative (IMI),1 a public–private partnership between the European Federa-
tion of Pharmaceutical Industries and Associations and the European Union.
Large consortiums facilitated by IMI share information on existing animal
data, developing new models, and standardizing models across different
companies and institutions. The NEWMEDS Consortium, for example, is
working to develop both new preclinical models and translational experi-
mental medicinal models for schizophrenia and depression.
In summary, Hunter stressed that advances in technology and creative
approaches to precompetitive collaboration and data sharing are providing
real opportunities to refine animal models.
1 See http://www.imi.europa.eu/.
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ADVANCING THE 3Rs IN NEUROSCIENCE RESEARCH
BOX 5-2
Summary of Session Points
3Rs
• dvances in technology have and will continue to provide opportunities for
A
replacement, refinement, and reduction (3Rs).
• ncreased understanding of disease mechanism may help in development of
I
replacement and refinement strategies.
• Replacement: In vitro cell culture models can be used to test reagents and
potential therapeutic candidates, including prescreening combinations of bio-
logical and pharmacological agents.
• Refinement: Simple refinements can improve study results while positively
impacting concerns about animal care and use.
• Reduction is often a “by-product” of refinement.
Systematic Reviews
• ystematic reviews of preclinical data could potentially:
S
o mprove the quality and value of animal studies and support the 3Rs.
I
o etter inform the timing, design, and benefit of clinical trials.
B
• he path to implementation of systematic reviews of preclinical data might in-
T
clude the Food and Drug Administration, pharmaceutical companies, research
institutions, and publishers.
SOURCE: Individual panelists and participants.
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