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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.115.227025
J Pharmacol Exp Ther 355:386–396, December 2015
Minireviews
Cracking the Egg: Potential of the Developing Chicken as a
Model System for Nonclinical Safety Studies of Pharmaceuticals
Sigrid Bjørnstad,1 Lars Peter Engeset Austdal,1 Borghild Roald, Joel Clinton Glover,
and Ragnhild Elisabeth Paulsen
Received July 1, 2015; accepted October 1, 2015
ABSTRACT
The advance of perinatal medicine has improved the survival of
extremely premature babies, thereby creating a new and heterogeneous patient group with limited information on appropriate
treatment regimens. The developing fetus and neonate have
traditionally been ignored populations with regard to safety
studies of drugs, making medication during pregnancy and in
newborns a significant safety concern. Recent initiatives of the
Food and Drug Administration and European Medicines Agency
have been passed with the objective of expanding the safe
pharmacological treatment options in these patients. There is a
consensus that neonates should be included in clinical trials. Prior
to these trials, drug leads are tested in toxicity and pharmacology
studies, as governed by several guidelines summarized in the
multidisciplinary International Conference on Harmonization of
Technical Requirements for Registration of Pharmaceuticals for
Human Use M3 (R2). Pharmacology studies must be performed in
Introduction
Prior to the onset of clinical trials, new drug candidates are
vigorously assessed in nonclinical studies comprising mandatory toxicological, pharmacokinetic, and pharmacological testing. The multidisciplinary International Conference on
Harmonization of Technical Requirements for Registration of
Pharmaceuticals for Human Use (ICH) guideline M3 (R2),
“Guidance on Nonclinical Safety Studies for the Conduct of
Human Clinical Trials and Marketing Authorization for
Pharmaceuticals,” (ICH, 2009) gives an overview over the
This work was supported by the Norwegian Research Council [Grant 195484
to J.C.G. and R.E.P.] and Southern and Eastern Norway Regional Health
Authority (to B.R.).
1
S.B. and L.P.E.A. contributed equally to this work.
dx.doi.org/10.1124/jpet.115.227025.
the major organ systems: cardiovascular, respiratory, and central
nervous system. The chicken embryo and fetus have features
that make the chicken a convenient animal model for nonclinical
safety studies in which effects on all of these organ systems can
be tested. The developing chicken is inexpensive, accessible,
and nutritionally self-sufficient with a short incubation time and is
ideal for drug-screening purposes. Other high-throughput models have been implemented. However, many of these have
limitations, including difficulty in mimicking natural tissue architecture and function (human stem cells) and obvious differences
from mammals regarding the respiratory organ system and
certain aspects of central nervous system development (Caenorhabditis elegans, zebrafish).This minireview outlines the potential and limitations of the developing chicken as an additional
model for the early exploratory phase of development of new
pharmaceuticals.
toxicological and pharmacological studies that are required for
a drug lead to proceed into clinical trials and refers to specific
safety guidelines for further details. Estimates show that only
about 10% of agents entering clinical trials ultimately receive
marketing approval. Lack of safety contributes to 30% of
failures, whereas lack of efficacy is the other major cause of
attrition (Kola and Landis, 2004). The fact that several drug
candidates progressed well into clinical trials before rare and
potentially lethal adverse effects were detected led to an
extension of nonclinical trials and the foundation of safety
pharmacology as a discipline in the late 1990s (Pugsley et al.,
2008). One of the ICH M3 (R2) safety guidelines referenced is
S7A, “Safety Pharmacology Studies for Human Pharmaceuticals,” (ICH, 2009) which defines a core battery of vital organs
for which the impact of new drugs needs to be assessed. The
core battery comprises the central nervous system (CNS),
ABBREVIATIONS: CNS, central nervous system; EU, European Union; ICH, International Conference on Harmonization of Technical Requirements
for Registration of Pharmaceuticals for Human Use; NMDA, N-methyl-D-aspartate.
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Department of Pathology, Oslo University Hospital HF, Ullevål, Oslo, Norway (S.B., B.R.); Institute of Clinical Medicine
(B.R.), Department of Pharmaceutical Biosciences, School of Pharmacy (L.P.E.A., R.E.P.), and NDEVOR, Section of Physiology,
Department of Molecular Medicine, Institute of Basic Medical Sciences (J.C.G.), University of Oslo, Oslo, Norway; and
Norwegian Center for Stem Cell Research, Department of Immunology and Transfusion Medicine, Oslo University Hospital HF,
Rikshospitalet, Oslo, Norway (J.C.G.)
The Chicken Model for Nonclinical Safety Studies of Drugs
Exposure of Pharmaceuticals to the Developing
Embryo, Fetus, and Neonate and the Use of
Juvenile Animal Models
The developing embryo, fetus, and neonate have previously
represented ignored populations with respect to drug safety
testing. However, the U.S. Food and Drug Administration and
European Medicines Agency have recognized the need for
increased knowledge of medicines in the pediatric population.
The Best Pharmaceuticals for Children Act, effective in the
United States since 2007, and the Pediatric Regulation in the
European Union (EU) of 2006 (EC 1901/2006 and 1902/2006;
EU 2006a, 2006b) were both passed with the objective to
facilitate development of new drugs with pediatric indications
and to improve the availability of information on the use of
medicines for children. A principal goal is to include children
in clinical trials. These trials should follow ICH guideline E11,
“Clinical Investigation of Medicinal Products in the Pediatric
Populations.” (ICH, 2000b). According to ICH M3 (R2) “Nonclinical Safety Studies for the Conduct of Human Clinical
Trials and Marketing Authorization for Pharmaceuticals,”
(ICH, 2009) section 12, the core battery of safety pharmacology
tests used in adult animals and safety data from adult human
exposure are usually available at the onset of pediatric clinical
trials. The guidelines recognize the potential differences in
safety profiles of drugs in adult versus those in young patients,
and the use of juvenile animal models is discussed. ICH E11
states, “The need for juvenile animal studies should be
considered on a case-by-case basis and be based on developmental toxicology concerns.” Apart from animal welfare and
ethics concerns, the reason behind the lack of a strict requirement for studies in juvenile animals might be that
results are difficult to extrapolate and, in some cases, have
been found to be of little relevance to humans (Anderson et al.,
2009). However, the interest in juvenile animal models in drug
development is increasing (Baldrick, 2010). ICH is currently
working (as of June 2015) on a new guideline S11, “Nonclinical
Safety Testing in Support of Development of Pediatric Medicines,” to provide guidance, for example, on the design of
studies in juvenile animals. This topic was endorsed by the
ICH Steering Committee in November 2014, and a completed
guideline is anticipated in 2018 (ICH, 2014). This highlights
the importance of studies related to pediatric pharmacology.
The progression of neonatal medicine has made it possible
to save infants born prior to gestational week 23 despite
extreme prematurity (Blencowe et al., 2012). This has created
an entirely new patient population for which increased
knowledge of pharmacology is strongly needed. Neonatal
therapeutic issues are often related to the cardiovascular
system, the respiratory system, and the CNS, and there is
an urgent demand for pharmacotherapy for conditions such
as epilepsy and bronchopulmonary dysplasia in the pediatric
population. This highlights the need for established prenatal and neonatal animal models in nonclinical studies
of drugs that may potentially be administered to humans
(Giacoia et al., 2012).
The Established Chicken Embryo Model
The Revival of a Well Established Model. The need for
juvenile and perinatal animal models prompts us to propose
the in ovo chicken embryo model as a valuable addition to
early safety studies. The chicken embryo and fetus constitute
a model system with a long and fascinating history. Since
Aristotle first started dissecting chicken embryos around the
year 350 BCE, this model system has contributed substantially to a foundation for our understanding of human development and has also contributed important insight into
immunology, cancer, and other areas of human disease (Stern,
2005; Datar and Bhonde, 2011; Kain et al., 2014).
Several features of the chicken embryo and fetus make it a
desirable animal model: it is phylogenetically closer to
mammals than, for example, zebrafish and the nematode
worm, it has a convenient size, a short incubation time of 21
days, and is readily accessible throughout in ovo or ex ovo
incubation. Development can be followed easily (Dohle et al.,
2009), which is convenient for imaging and microsurgical
procedures (Yalcin et al., 2010). Furthermore, the egg is
nutritionally self-sufficient, and the embryo and fetus develop
normally at 37–39°C and 45–55% air humidity without the
need for specialized facilities and equipment.
Animal Welfare and Ethics with Regards to the
Chicken Embryo Model. Nonclinical testing should be
carried out without unnecessary use of animals and other
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cardiovascular system, and respiratory system. Traditionally,
these studies have been performed on selected drug candidates late in the nonclinical phase and continued during firstin-human trials. In an attempt to reduce costs and accelerate
development, there has been a shift toward performing safety
pharmacology studies earlier in the discovery phase (Hamdam
et al., 2013). Safety pharmacology endpoints may also be
incorporated into other nonclinical toxicological and kinetic
studies. For early screening purposes in the exploratory phase
of drug development, it is thus beneficial to have efficient and
inexpensive model systems suitable for high throughput in
which functional tests, biochemical markers, and morphology
can be assessed. This will increase the likeliness of a drug
succeeding in later phases of nonclinical studies. A number of
early high-throughput models have been proposed to improve
productivity and efficiency in the early nonclinical phase,
including Caenorhabditis elegans, zebrafish (Danio rerio), and
human stem cells. However, animal models phylogenetically
closer to mammals with well characterized CNS, cardiovascular, and respiratory systems are needed to better predict
alteration in organ function relevant for humans. Established
animal models in safety pharmacology may include pigs,
monkeys, primates, and dogs, models that require substantial
care and tend to stir public opinion (Pugsley et al., 2008). It
would therefore be advantageous to develop additional models
that, in the early exploratory phase, could help reduce the
number of drug leads that later need to be tested in the
established animal models. Ideally, model systems should be
adjustable with respect to parameters such as organ function
and age (Jacobs, 2006). The developing chicken has several
advantageous features for drug discovery. Here, we summarize areas within pharmaceutical research where the use of
chicken embryos is already established, and we suggest an
extended use of the developing chicken as a model system for
the CNS and respiratory system, two of the major organ
systems for which safety needs to be assessed. The chicken
embryo as a cardiovascular model system was recently
discussed by Kain et al. (2014).
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chicken embryos instead of mammalian embryos obviously
reduces the size of the experimental group, in line with
international ethical guidelines. A further reduction is possible by monitoring effects on several major organ systems in
each individual simultaneously. An important feature of the
chicken embryo in this regard is that the development and
maturation of the CNS and respiratory system overlap to a
large extent temporally, as will be discussed in the following
sections. It is thus possible to monitor several organ systems
at the same time in one embryo—in line with the goal of
reduction. In this respect, the chicken embryo model could
serve as a replacement for other perinatal and juvenile animal
models. However, the experimental use of embryonic stages is
no longer considered an acceptable replacement for adult
animals. Partial replacement by using, for example, tissue and
cell culture, perfused organs, and tissue slices should be
preferred instead of replacement with other live laboratory
animals. All of these techniques are applicable to and described in the chicken.
Administration of Pharmaceuticals to the Developing Chicken. The physical isolation of the chicken embryo
and fetus from the mother is an advantage in studies of
mechanisms of pharmaceuticals. Drug administration is
easily controlled, and both primary and secondary pharmacology, off-target effects, and toxicity can be tested. However,
the chicken model does have limitations in this respect. As a
rule, the route of drug administration in an animal model
should appropriately mimic the intended therapeutic route of
administration in humans. Historically, however, studies of
acute toxicity have been performed by parenteral administration in addition to the intended clinical administration, as
discussed in ICH M3(R2; ICH, 2009). In the developing
chicken, various drug delivery systems and formulations have
been evaluated, and topical administration onto the chorioallantoic membrane as well as systemic administration intravenously or via injections into the yolk sac, amnion, or
peritoneum have been tested (Vargas et al., 2007). Drug
inhalation in the developing chicken can only be done in the
postpipping stage after E19, whereas oral drug administration can be performed after hatching. As in all nonmammalian
animal models, challenges arise when attempting to translate
results to a similar setting in mammals. For example, drug
metabolism in the mother and the placental barrier in
mammals may, depending on properties of the pharmaceutical, protect the fetus against exposure, a feature which cannot
be mimicked correctly in avian models. Further, there is no
excretion from the egg until hatching, a feature that may
cause a prolonged effect of a single exposure. In studies of
pharmacokinetics, the chorioallantoic membrane has raised
interest in testing drug delivery systems and has been
suggested as a valuable tool in nonclinical studies, thoroughly
reviewed by Vargas et al. (2007). One has to be aware of the
possible differences in pharmacokinetics in chickens compared with humans (Nowak-Sliwinska et al., 2014).
Established Use of the Chicken Model in Nonclinical
Safety Studies. Avian embryos are established models in
teratology and developmental toxicology. Such issues are
addressed by the ICH guideline S5 (R2) “Detection of Toxicity
to Reproduction for Medicinal Products and Toxicity to Male
Fertility.” (ICH, 2000c) Several apoptosis assays (activated
caspase-3, terminal deoxynucleotidyl transferase–mediated
digoxigenin-deoxyuridine nick-end labeling assay) in addition
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resources. Unanesthetized animals are preferred, and the
number of animals in each experimental group should not
exceed what is scientifically justified (ICH S7A; ICH, 2000a).
All use of laboratory animals is subject to strict regulations
with certain national variations. Guidelines regarding animal
ethics and best practice aim to ensure minimal pain, suffering,
and distress for the animal.
There is a consensus among scientists that the avian
embryo gains the ability to experience pain at a certain point
of development in ovo. The first sensory afferent nerves in
chicken develop from embryonic day 4 (E4); however, synaptic
connection with the dorsal horn is not established until E7,
making perception of pain during the first trimester impossible (Eide and Glover, 1995, 1997; Rosenbruch, 1997). The
development of tractus spinothalamicus is not fully characterized in the chicken embryo, so the exact time point at which
the chicken embryo could begin experiencing pain is not
known. At E13, the chicken fetus has developed a functional
brain, and a few days prior to hatching, it is considered fully
conscious (Animal Care and Use Committee California State
Polytechnic University, 2012; Institutional Animal Care and
Use Committee University of Louisville, 2012). Despite the
uncertain onset of nociception and pain perception in the
developing chicken, the American Veterinary Medical Association guidelines for euthanasia state that avian embryos that
have attained .1/2 of the total incubation period have a
sufficiently developed CNS for pain perception and should
be euthanized by similar methods as used in neonates—for
example, by anesthetic overdose, decapitation, or prolonged
exposure to CO2. Eggs at ,1/2 of the total incubation period
may be destroyed by cooling (,4°C for 4 hours), freezing, or
prolonged exposure to CO2. The EU legislation on the protection of animals used for scientific purposes (2010/63/EU;
EU, 2010) defines embryos or fetuses developed to .2/3 of
their total gestational period as neonates. Also included and
protected by the requirements of this legislation are embryonic or fetal forms at a developmental stage less than 2/3 of
their gestational length if they are subjected to experimentation during these two trimesters and allowed to live into the
third trimester. This includes nonmammalian vertebrates,
such as birds.
In accordance with the 3R principles of animal ethics
(reduce, refine, replace), appropriate anesthesia of animals
subjected to potentially painful procedures should be provided. The knowledge on nociception and distress during
chicken embryonic development is relatively new and has
led to new protocols for humane anesthesia of chicken
embryos. These protocols have helped to refine the model to
a higher ethical standard (Aleksandrowicz and Herr, 2015).
In mice and rats, litter size varies across breedings and
cannot be accurately predicted in advance, whereas in general,
a hen lays only one egg per day. To obtain a sample size of
animals large enough for sufficient statistical power, additional matings are often required in mice and rats, causing
distress for the mothers and potentially resulting in an
unnecessarily large number of animals being handled, manipulated, and destroyed. In chicken hatcheries, however, the
daily production of fertilized eggs is more predictable, making
it easier to assign the appropriate number to experimental
groups. Moreover, a broader genetic variation can be obtained,
mimicking the clinical setting more closely. Additionally, the
mother is excluded from experimentation. Thus, the use of
The Chicken Model for Nonclinical Safety Studies of Drugs
Expanding the Use of the Chicken Embryo Model
The Developing Chicken as a CNS Model. Conditions
affecting the nervous system are common in neonates; one
typical sequela of dysregulated brain development is the
occurrence of seizures, which requires treatment with antiepileptic drugs (Giacoia et al., 2012). During CNS development,
cell division, cell death, and cell differentiation are all tightly
regulated (Marzban et al., 2014). To avoid administering
neuroactive drugs that may alter CNS development, safety
studies are recommended to investigate key functional domains of the developing CNS (Himmel, 2008). These studies
should be performed in the early nonclinical phase of drug
development.
As a major model in developmental biology, the chicken
embryo has also played a pivotal role in the study of neurodevelopment. Famously, the neural crest was first discovered
in the chicken embryo by Wilhelm His in 1868. Rita LeviMontalcini shared the Nobel Prize in Medicine or Physiology
in 1986 for her discovery of nerve growth factor, and her work
was primarily performed in the chicken embryo. Her mentor,
Viktor Hamburger, together with Howard Hamilton described
the 46 stages of chick development, a staging system that is
still used today (Fig. 1), and performed seminal studies of the
development of motility and behavior as well as trophic
interactions (Hamburger and Hamilton, 1951). Several aspects of the stages of neuronal development in chickens are
characterized. As an example, the efferent motor neurons
develop early in chickens. This gives the embryo the ability to
move spontaneously from E3.5, when early movements start
in the neck before spreading to the trunk. At E6.5, motility in
the legs appears (Hamburger and Balaban, 1963; Bekoff,
1976). Nicole LeDouarin invented and developed the technique of quail-chick chimeras to launch a wide-reaching series
of fate-mapping studies that have helped elucidate the fates
and developmental roles of neural crest cells (Dupin et al.,
1998; Le Douarin, 2004; Le Douarin et al., 2008; Le Douarin
and Dieterlen-Lievre, 2013). Viral approaches for genetic fate
mapping were introduced in the late 1980s (Price et al., 1987;
Gray et al., 1988; Sanes, 1989). More recently, the chicken
embryo has been used to study neural induction and early
patterning (Le Douarin, 2004; Teillet et al., 2008). Fluorescent
and other tracers have been developed to map the structural
and functional development of axon projections and neural
circuits (Glover et al., 1986; O’Donovan et al., 1994). The
tradition of utilizing the chicken embryo in neurodevelopmental studies has continued to progress to embrace molecular
genetic approaches, including in ovo electroporation for gainand loss-of-function studies (Nakamura et al., 2004) and a
genetic toolbox of DNA constructs and viral vectors for
targeted tracing and functional manipulation of specific
neuronal subtypes (Hadas et al., 2014). The chicken embryo
and fetus are accessible for in ovo kinematic, electrophysiological, and noninvasive imaging studies of neurophysiological
and behavioral development (Bekoff, 1992; Ryu and Bradley,
2009; Balaban et al., 2012). Last, the chicken embryo has been
used as a recipient for xenotypic grafting studies of human
neural stem and progenitor cells (Glover et al., 2009; Boulland
et al., 2010).
Given its proud history in the field of neurodevelopment, it
is surprising that, when it comes to nonclinical neuropharmacological studies, the chicken model is not yet routinely used.
An area where the advantages of the chicken embryo model
have been highlighted is developmental endocrinology (De
Groef et al., 2008). It is of particular benefit that the only
endocrine influence the embryo receives from its mother is
through the egg yolk. This facilitates external manipulation of
the endocrine system and the study of whether pharmaceuticals and environmental chemicals cause endocrine disruption
(Mathisen et al., 2013). Tight regulation of the endocrine
system is crucial for correct development of the CNS. The
chicken embryo can be a valuable tool in early screening of
drug candidates to determine whether they alter endocrine
profiles.
Epilepsy is one of the most serious conditions affecting
human neonates. A homozygous strain of epileptic chickens,
the Fayoumi strain, has been used in epilepsy research for
over 30 years (Nunoya et al., 1983). Epileptic chickens show
differences in electroencephalography compared with normal
chickens from E17 (Guy et al., 1995). They have been used for
magnetic resonance imaging (Fabene and Sbarbati, 2004) and
evaluation of anticonvulsant drugs (Loscher, 1984; Johnson
et al., 1985) and N-methyl-D-aspartate receptor (NMDA)
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to echocardiography and morphologic changes in organs have
been used to test effects of toxicants (Smith et al., 2012). The
chicken embryo has also been used to study the effects of
retinoids that alter patterns of gene expression, neuronal
differentiation, and axonal projections. In situ hybridization,
RNA sequencing, immunostaining, and retrograde labeling
techniques have been used to assess such effects (Forehand
et al., 1998).
A challenge in nonclinical studies is poor translation of
results between species. Retinoids are known teratogens that
also affect the nervous system in the human embryo (Adams,
2010). Here, there is good concordance between the chicken
and other animal models. In general, proper cross-species
translation of results from animal models requires knowledge
of comparative biology and careful selection of relevant and
translatable endpoints before concluding. Observation of
similar results in animal models of different species increases
the likeliness of the results being translatable to humans.
As another example from the area of developmental toxicology, the effect of mammalian cardiac inward rectifying
potassium channel blockers has been shown to produce digit
malformations in developing rats. Knowledge of the comparative biology of expression of the human Ether-à-go-goRelated Gene (hERG) channel was essential for translation
of these results into a human drug-development context.
This channel is important in the chicken embryo as well
(Krogh-Madsen et al., 2005; Danielsson et al., 2007), which
makes it possible to study primary or off-target effects of
pharmaceuticals on this channel in the developing chicken.
One area of drug development where the chicken model has
been more firmly implemented is in ocular toxicology testing.
The chicken embryo is an established model for the study of
retinal development (Vergara and Canto-Soler, 2012). The
chicken has large eyes during development and at birth, and
retinal cells can be cultured in vitro (Daston et al., 1991). The
isolated chicken eye is an accepted method for toxicology
testing in both the U.S. and the EU (Dholakiya and Barile,
2013). Toxicity is assessed by a qualitative examination of
opacity of the cornea, swelling, and damage measured by
fluorescein staining (OECD, 2009).
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Fig. 1. Developmental events during the embryonic and fetal period in humans and chickens. Whereas a human pregnancy takes 40 weeks, chickens
hatch after only 21 days of incubation. The Hamburger-Hamilton (HH) classification is a useful research tool to stage chicken embryos and fetuses based
on morphologic landmarks rather than chronological age. The early development of efferent motor neurons of the chicken makes the embryo able to move
spontaneously from E3.5. Sensory afferents, however, do not establish synaptic connections with the dorsal horn until E7, making perception of pain
during the first trimester impossible (the exact time point at which the chicken embryo starts experiencing pain is not known). The human cerebellum
grows exponentially toward the end of gestation (Guihard-Costa and Larroche, 1990), whereas in chickens, a cerebellar growth spurt occurs earlier
(around E13–E17) and slows before hatching, thus creating a near sigmoid-shaped growth curve (unpublished observations). In humans, staging of fetal
lung development based on morphology is well established. The human lung develops from an embryonal stage to a pseudoglandular, canalicular stage,
and then in the last trimester into a saccular stage. The chicken lung, on the other hand, develops atrio-infundibular and air-capillar lungs during the
last trimester. This appears coincidentally with chicken fetal respiratory movements starting around E18, much later than the movement of the legs,
trunk, and neck.
antagonists (Pedder et al., 1990). Studies have been carried
out to examine how generalized seizures alter the development of the chicken brain (Gong et al., 2003).
For studies of neural development in chicken, the cerebellar
cortex is an important model system (Nieuwenhuys, 1967;
Solecki et al., 2006). The structure of the cerebellar cortex was
first described in 1911 by Ramon y Cajal, who found it to be so
conserved among species that he proposed it to be a “law of
biology” (Sultan and Glickstein, 2007). The cerebellum is
considered an appropriate model that reflects well the general
aspects of CNS development. Cerebellar granule neurons
are numerous, constitute the most homogenous neuron
The Chicken Model for Nonclinical Safety Studies of Drugs
although few are firmly established. The transcription factor
PAX6 is important for cerebellar development and migration
(Engelkamp et al., 1999), and perturbations of PAX6 expression have been linked to autism (Umeda et al., 2010). Also, the
role of PAX6 has been studied and found to be important for
brain development in the chicken embryo (Mathisen et al.,
2013). Other markers that have been suggested and partly
implemented in studies of neurotoxicity are Calbindin D28k
and microtubule-associated protein-2 (Haworth et al., 2006).
Calbindin D28k is a calcium-binding protein highly expressed
in the cerebellum across various species, has been studied in
chickens (Hunziker and Schrickel, 1988; Koszka et al., 1991;
Marzban et al., 2010; Flace et al., 2014), and is associated with
autism (Whitney et al., 2008) and hereditary ataxia (Koeppen
et al., 2013) in humans. Microtubule-associated protein-2 has
roles in neuronal growth and plasticity (Johnson and Jope,
1992), has been studied in chickens (Pinkas et al., 2015), and
has been shown to be important in human traumatic brain
injury (Mondello et al., 2012) and Rett syndrome (Johnston
et al., 2001). Therefore, these are examples of markers that
could serve as endpoints in safety studies with results
relevant for conditions seen in humans. Other relevant
neuronal markers are listed in Table 1. We thus argue that
the chicken cerebellum is a valuable tool for the study of
neurodevelopment that could successfully be implemented
in early nonclinical studies of new pharmaceuticals.
The Developing Chicken as a Lung Model. In the 17th
century, the lung was suggested to be a link between vital air
particles in the atmosphere (later determined to be oxygen)
and the blood (Partington, 1956). Since then, it has been
acknowledged as a prerequisite for life, and the respiratory
system is a natural component of the core battery of safety
pharmacology studies (Murphy, 2002; Dhokarh et al., 2012).
In nonclinical testing of drugs on laboratory animals, clinical
observation alone to assess respiratory function is not adequate. Thus, several parameters of respiratory function,
including monitoring both the pumping apparatus and the
gas-exchange unit, should also be quantified using appropriate methods, as discussed by Murphy (2002, 2014). Important
conditions in neonatology that affect the respiratory system
include neonatal respiratory distress syndrome, apnoe of
prematurity, and persistent bronchopulmonal dysplasia, and
call for immediate treatment. Fetal hypoxia is an important
concern during pregnancy and is often present in combination
with premature birth and other conditions, such as chorioamnionitis, preeclampsia, and maternal diabetes. Premature
birth occurs in .10% of all live births worldwide (Blencowe
et al., 2012), and drugs that have minimal adverse effects
in healthy and normally developed individuals can be lifethreatening in individuals with a diseased or immature
respiratory system. Both the pumping apparatus and the gasexchange unit are essential for respiration. Mild respiratory
depressant drugs may produce serious adverse effects if given
to a patient with a ventilatory defect such as apnea, often seen
in low-birth-weight neonates with an immature CNS. Drugs
that mildly hamper lung development and maturation may be
mortally dangerous to premature individuals with marginally
developed lungs. Adverse effects on the respiratory system in
newborn babies after drug administration to the mother during
pregnancy may also occur. Selective serotonin reuptake inhibitors are associated with an increased risk of pulmonary hypertension in the newborns of women receiving this therapy
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population in the brain, are easily accessible, and may be
purified and grown in vitro. They undergo all key stages of
neural development and thus provide a model of choice for
studying mechanisms of neuronal proliferation, apoptosis and
survival, differentiation, and migration (Contestabile, 2002).
A key feature of cerebellar development is the migration of
granule neurons through layers of the cerebellar cortex—from
the external granule layer to the internal granule layer. To a
large extent, this occurs postnatally in some mammals,
including humans. Alterations in migration may be indicative
of impaired neurodevelopment and could be monitored by
histologic methods in safety studies to assess whether a drug
candidate is likely to be safe for children or during pregnancy.
Over time, cerebellar granule neurons in vitro differentiate
and acquire the characteristics of mature neurons, express
NMDA receptors, and rapidly develop excitotoxicity in culture
(Contestabile, 2002). Exposure of these cultured neurons to a
drug candidate can determine at an early stage of preclinical
testing whether the drug is toxic to neurons and, for example,
initiates apoptosis (Jacobs et al., 2006). An important aim in
drug development is to discover detrimental effects as early as
possible (Pugsley et al., 2008). This is of particular importance
when testing new antiepileptic drugs that interfere with
glutamate (as NMDA antagonists) or GABA signaling, or to
discover off-target effects of other potential drugs.
As pointed out by Ramon y Cajal, the structure of the
cerebellar cortex is highly conserved among species, which
makes histologic examination of alterations in the cortex
relevant to a similar setting in humans. However, there are
some differences. The vermis (a medial expansion of the
cerebellum) is only seen in mammalian species (Butts et al.,
2014), and the migration of granule neurons exhibits different
patterns in different species. In the human embryo, the
external granule layer reaches its peak thickness by gestational week 25, and the process of migration continues well
into the first postnatal year. In the chicken cerebellum, the
migration of cerebellar granule neurons occurs prenatally
(Aden et al., 2008, 2011; Volpe, 2009; Mathisen et al., 2013), in
contrast to the situation in the rodent cerebellum where the
external granule layer is formed postnatally, and granule
neurons migrate between postnatal days 4 and 15 (Fujita,
1967). In fish, the migration of granule neurons follows yet a
different pattern from that of mammals and chicken or is
absent altogether (Butts et al., 2014). That the migration of
cerebellar granule neurons in chicken resembles the human
counterpart more closely than what is seen, for example, in
zebrafish makes the chicken embryo cerebellum a more
appropriate model than the fish cerebellum for highthroughput screening in safety studies of the CNS. That
granule neuron migration in the chicken occurs at a fetal
stage is another advantage of the chicken embryo model that
facilitates the study of insults to cerebellar development. This
has been used to assess the neurodevelopmental effects of
glucocorticoids (Aden et al., 2008, 2011), a clinically relevant
neonatal issue (Roberts and Dalziel, 2006; Brownfoot et al.,
2013). The same model has been used to address developmental disruption caused by bisphenol A, with results in concordance with those reported in a mouse model (Mathisen et al.,
2013). Histopathology remains the gold standard (Sasseville
et al., 2014) for assessing detrimental effects on the developing
CNS in chickens (Table 1). In addition, there are several
biomarkers that could be useful for early screening purposes,
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TABLE 1
Examples of specific parameters available in chickens for quantitative evaluation of drug responses
Specific Tests/Endpoints
CNS function
Physiologic
EEG
EMG
Structural
Brain weight
PET scan
MRI
Morphology, measuring thickness
of the cerebellar EGL and IGL
Biochemical
Transcription factor PAX6
Calbindin D28k
NeuN
S-100
Respiratory function
Physiologic
Tidal volume
Breathing rate
Minute ventilation
Oxygen consumption
Structural
Lung weight
Morphology with staging of fetal
lung development (embryonal,
pseudoglandular, atrio-infundibular
or air-capillar)
Biochemical
Pulmonary surfactant components
(spB, PC, DSPL)
Aquaporin 5
Caveolin 1
VEGF
Chicken References
Monitoring brain function, epileptic activity, and
focal brain disease
Monitoring neuromuscular function and motility
Guy et al., 1995; Di Pascoli et al., 2013
CNS growth
Structural and functional neuroimaging relevant in
diffuse and focal brain disease
Cerebellar abnormalities are clinically important
causes of neurodevelopmental disabilities in
premature babies, often manifested by cognitive,
behavioral, attentional, and socialization deficits
(Volpe, 2009)
McCutcheon et al., 1982; Fabene and
Sbarbati, 2004; Aden et al., 2008, 2011;
Balaban et al., 2012; Mathisen et al., 2013
Important for cerebellar development and migration Cicero and Provine, 1972; Hunziker and
Schrickel, 1988; Koszka et al., 1991;
(Engelkamp et al., 1999); linked to autism
Marzban et al., 2010; Mezey et al., 2012;
(Umeda et al., 2010)
Highly expressed in the cerebellum; associated with
Mathisen et al., 2013; Flace et al., 2014;
autism (Whitney et al., 2008) and hereditary
Pinkas et al., 2015
ataxia in humans (Koeppen et al., 2013)
Neuronal growth and plasticity (Johnson and
Jope, 1992)
Marker for neuronal maturation (Sarnat et al., 1998)
Marker enabling monitoring of brain injury in blood
and CSF (Sun et al., 2012)
Monitoring respiratory function
Mortola and Labbe, 2005; Szdzuy and
Mortola, 2007
Pulmonary growth
Insufficient lung development is associated with
respiratory distress syndrome
Hylka and Doneen, 1983; Maina, 2004a;
Bjornstad et al., 2014
Surfactants increase compliance, lower surface
tension, and prevent atelectasis of the lungs; lack
of surfactants causes respiratory distress of the
newborn
Expressed in human type I pneumocytes, vital for
gas exchange (Kreda et al., 2001)
Expressed in mature gas exchange tissue of the
human lung (Kaarteenaho et al., 2010)
Important for sufficient vascularization of the gas
exchange membrane
Hylka and Doneen, 1983; Makanya et al.,
2007, 2013; Been et al., 2010;
Bjornstad et al., 2014
CSF, cerebral spinal fluid; DSPL, disaturated phospholipids; EEG, electroencephalography; EGL, external granule layer; EMG, electromyography; IGL, internal granule
layer; MRI, magnetic resonance imaging; PC, phosphatidylcholine; PET, positron emission tomography; spB, pulmonary surfactant protein B; VEGF, vascular endothelial
growth factor.
during the last trimester of pregnancy (Moses-Kolko et al.,
2005; Chambers et al., 2006). Administering drugs to newborn
children and pregnant women is thus a major safety concern.
There is a need for animal models to study whether pharmaceuticals affect the respiratory system in fetal and neonatal
life. As conditions affecting the respiratory system can be
severe, there is also a clinical need for new treatment options.
Models in which lung development is well characterized can
provide an important research tool to identify targets for
future therapy.
The respiratory anatomy and physiology of birds differs
from that of mammals, and the morphology of the developing
chicken lung is well described (Maina, 2003a,b, 2004b). The
parabronchial bird lungs are connected to air sacs that serve
as reservoirs for oxygen-rich air and are used for respiration
during exhaling. This results in a very efficient respiration
that is highly useful, for example, during long flights. In
chickens, the respiratory movements start around E18 of
development. The human fetus, by contrast, already makes
breathing-like movements from week 9, and the circulation of
amniotic fluid into the airways is essential for sufficient lung
development. Unlike mammals at birth, birds adapt to air
breathing gradually, with an overlap of chorioallantoic membrane and lung-dependent gas exchange lasting from hours to
even days during hatching. Partly because of these major
anatomic and physiologic differences, the chicken has traditionally not been implemented as a model system in safety
studies of respiratory function. However, the commercial
broiler industry, which uses in ovo vaccination and drug
treatments as strategies to defeat disease among flocks, has
used monitoring of the respiratory system of the developing
chicken in safety pharmacology testing of new vaccines. For
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Microtubule-associated protein-2
Relevance to Human Disease
The Chicken Model for Nonclinical Safety Studies of Drugs
model to study the actions of steroid hormones on the fetal
lung. This study used growth, cellular proliferation, and rate
of synthesis of the major surfactant phospholipids as parameters of lung development and function. Further, effects on
lung development of in ovo hypophysectomy and treatment
with corticosterone or pituitary transplantation were assessed
(Hylka and Doneen, 1983). Antenatal corticosteroid treatment
is also associated with impaired cerebellar development in
humans, a finding that has been reproduced in the chicken
embryo (Aden et al., 2008, 2011). Thus, the chicken embryo
model can be used advantageously to study the impact on two
organ systems (Fig. 1) of pharmaceuticals clinically administered for a major human neonatal condition.
Fetal hypoxia is another important concern in neonatal
medicine, and a hypoxia model has been established in
chickens. In a study from 2010, fertilized eggs were incubated
at various levels of oxygenation to support the concept that
fetal oxygen tension is a key signal in the regulation of the
surfactant system. Disaturated phospholipids and vascular
endothelial growth factor were used as parameters of lung
function at different stages of chicken embryo development,
and a critical window of vulnerability to oxygen tension was
established (Been et al., 2010). Another relevant example is
surfactant protein B, which is expressed in chicken lungs from
TABLE 2
The expanded toolbox available for the chicken embryo model
Tools and Methods
Ex ovo incubation
Imaging
Fluorescent tracers
MRI
PET
Chicken stem cells
Stem cells available from
several organs
Chimeric models
Quail-chick chimeras
Reference to Method or Review
Auerbach et al., 1974
Imaging and microsurgery (Yalcin et al., 2010)
Technical aspects described in Dohle et al. 2009
Kulesa et al., 2013
Clarke, 2008
Axon tracing (Glover et al., 1986)
Altered projections in the spinal cord caused by
retinoids (Forehand et al., 1998)
Hogers et al., 2009
Monitoring of chicken brain development (Zhou et al., 2015)
Walking-like brain function in embryos
(Balaban et al., 2012)
The chicken embryo for evaluation of radiopharmaceuticals
(Haller et al., 2015)
Intarapat and Stern, 2013
Neuronal differentiation of pluripotent stem-like cells in
chickens (Dai et al., 2014)
Developed by Le Douarin and Renaud, 1969
Fate-mapping studies to investigate developmental roles
of the neural crest (Le Douarin, 2004)
Studies of cerebellar development (Hallonet et al., 1990)
Study of neuronal migration (Kharazi, Levy et al., 2013)
Reviewed in Teillet et al., 2008
Xenotransplantation of human Glover et al., 2009
stem cells into the chicken
Boulland et al., 2010
Disease-prone strains
Fayoumi strain
A model for epilepsy; described in
Nunoya et al., 1983
Obese strain of chicken
Gain and loss of function
Gain of function
In ovo electroporation
Examples of Applications
Studies of autoimmune thyroiditis as
described in Wick, Sundick et al., 1974
and Dietrich et al., 1999
Sauka-Spengler and Barembaum, 2008
Krull, 2004
Nakamura et al., 2004
Retroviral vector
Loss of function
RNAi gene silencing
Sato, 2004
Morpholinos
Kos et al., 2003
Andermatt and Stoeckli, 2014
Evaluation of anticonvulsant drugs (Loscher, 1984;
Johnson et al., 1985) and how seizures alter the
development of the brain (Gong et al., 2003)
Neuroendocrine alterations caused by thyroiditis
(Schauenstein et al., 1987)
Overexpression of genes in Purkinje cells (Luo and
Redies, 2004)
Tbx4 and Fgf10 in lung bud formation
(Sakiyama et al., 2003)
Wnt5a in pulmonary development (Loscertales et al., 2008)
Axonin/TAG-1 in cerebellar development (Baeriswyl
and Stoeckli, 2008)
Transcription factor FoxD3 in the development of the
neural crest (Kos et al., 2001)
MRI, magnetic resonance imaging; PET, positron emission tomography; RNAi, RNA interference.
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example, effects on distal lung maturation with insufficient
development of air capillaries have been found in an in ovo
trial testing a vaccine against Newcastle disease virus
(Dilaveris et al., 2007). Safety studies of respiratory function in adult and juvenile animals are usually monitored in
head-out or whole-body plethysmographs. Whereas measuring respiratory function in a mammalian fetus requires
invasive or indirect electrophysiology or imaging techniques (Greer, 2012), the same monitoring in the chicken
fetus can be performed using noninvasive barometric methods, a real advantage of the chicken embryo model system
(Szdzuy and Mortola, 2007).
Although not yet widely implemented in nonclinical pharmaceutical studies, the respiratory system of the chicken
embryo has been used as a model for research purposes.
Antenatal corticosteroids are routinely given to pregnant
women when there is a threat of premature birth to accelerate
lung maturation and prevent respiratory distress. However,
after several decades, there is still no international consensus
on an optimal regimen of corticosteroid treatment. This is still
a highly relevant clinical problem, and experimental embryo
animal model systems in which the effects of different
corticosteroids on lung development can be monitored are
crucial. Hylka and Doneen (1983) used the chicken embryo
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Bjørnstad et al.
Summary
Many features of the chicken embryo, including low cost, selfcontained development, short incubation time, conveniently
large size, accessibility, and availability of a large number of
techniques for experimental manipulation and characterization, make it a desirable laboratory animal model. The chicken
embryo has traditionally been used to address mechanisms of
development and neonatal function, but has an expanding
potential as an efficient and inexpensive screening model for
drug development. The development of the CNS and the
cardiovascular and respiratory systems is well characterized,
as are the main differences from human development. We
therefore propose that the chicken embryo should be included
as a mainstay model for early nonclinical safety assessment.
Acknowledgments
The authors thank Anne Soleng at the Norwegian Medicines
Agency for valuable discussion.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: Bjørnstad,
Austdal, Roald, Glover, Paulsen.
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