XVII. Tagung über Pferdekrankheiten im Rahmen der EQUITANA

Ausgabe 6·2006 Vorschau wissenschaftliches Programm/Scientific Program Forecast kongressspiegel 3
Potential use of micro-array technology (bio-puce) in
the diagnosis of inflammatory disorders in the horse
Pierre Lekeux, Aurélie Thomas, Eve Ramery, Tatiana Art, Rodrigue Closset, Fabrice Bureau, Liege, Belgium
The performance of a competition
horse is based on genetic potential,
excellent health and optimal
training. Nowadays, two ways to evaluate
performances exist: performance
control itself and veterinary
monitoring. The latter classically
consists of a clinical check-up and
complementary analyses, as for
example blood analysis, radiography,
or ultrasound. In spite of the regularity
of veterinary check-ups, in
many situations health problems
cannot be prevented or controlled in
time, which has a negative influence
on the horses’ performances. So for
instance pathological conditions are
detected when they have already
induced irreversible lesions, e.g.
when joint inflammation has already
started to damage the joint cartilage.
It also occurs when the pathology is
not determined with sufficient accuracy:
the proposed treatment is then
non-specific and often not very efficient.
The perfect example here is
inflammation caused by mechanical
stress, allergy or infection: the symptoms
are often similar, but the three
aetiologies require specific treatment.
New technology „DNA chip“
In order to propose solutions for
the limiting factors of medical monitoring,
a tool, born from modern
technologies, is being developed in
the equine species. This is the microarray
or bio-chip, also called a „DNA
chip“ in English literature. This new
technology should permit:
• the detection of certain conditions
earlier, i.e. before they induce
lesions and disrupt performances;
• the more specific determination of
the origin of the conditions, in order
to use a better targeted, and thus,
more efficient therapy;
• the detection of horse’s predisposition
to certain conditions by genetic
profiles, in order to set up appropriate
preventive care.
Micro-arrays technology permits
a quick determination of the level of
gene expression by analysing a sample
of the horse’s cells. Genes can be
normally-, but also under- or overexpressed.
In the ideal situation, each
gene should be expressed in the function
of the horse’s needs in each
moment. If the gene is underexpressed,
too little of the corresponding
proteins will be synthesised.
On the contrary, if the gene is
over-expressed, too much of the corresponding
proteins will be synthesised.
Proteins are only fully efficient
when synthesised in the exact
amounts needed, at the right
moment and at the right place. Too
high levels of proteins are therefore
just as bad as too low levels.
Instant image of cell gene
expression
The middle term development of
micro-arrays applied to sport horse’
veterinary medicine is motivated by
the many advantages offered by this
new technology.
Nowadays, the genetic molecular
methods can only analyse one gene
at a time. The new micro-arrays,
thanks to miniaturization techniques,
are able to analyse several
thousands of genes on only one glass
strip a few centimetres wide. Thus it
is now possible to understand interactions
between different genes,
many of which had been unknown
until today. This makes the complete
understanding of the horse’s pathology
much easier for the veterinary
practitioner.
Micro-arrays give an instant
image of the exact way each cell gene
is expressed. This ensures that underor
over-expressions of genes are easily
detectable. The gene expression
level can thus be measured in different
situations like, for instance,
before and after effort.
The basis of the approach
By studying the expression level
of each gene, biological micro-arrays
allow the diagnosis and the characterization
of a pathological condition
at its immediate source. The information
obtained brings a very precise
diagnosis because it is considering
every gene. This is nevertheless not
The use of scintigraphic examination
in sport horses with unexplained lameness
Sue Dyson, Suffolk, United Kingdom
Nuclear scintigraphy can be used
in a variety of different clinical situations
to facilitate the diagnosis of
lameness and poor performance in
sports horses:
• Pain localised to a region, but no
detectable radiographic abnormality,
• Partial improvement to local analgesic
techniques, suspected bone
injury, but unable to identify lesion,
• For the determination of the likely
significance of subtle radiographic
abnormalities,
• If there are no localising clinical
signs and there is no or little
response to local analgesia,
• Clinical signs suggestive of fracture
but no localising clinical signs,
• Poor performance but no localising
clinical signs,
• Episodic lameness that cannot be
reproduced.
It is however crucial to recognise
that increased radiopharmaceutical
uptake (IRU) is not necessarily synonymous
with pain causing lame-
ness. Recognition of common sites of
asymptomatic incidental regions of
IRU is crucial for accurate diagnosis.
Examples
Pain localised to a region, but no
detectable radiographic abnormality.
E.g., foot pain:
• identification of focal IRU in lateral
pool phase images in the region of
the deep digital flexor tendon
(DDFT),
• identification of IRU at the site of
insertion of the DDFT on the distal
phalanx; solar images are usually
most sensitive,
• identification of focal IRU at the
insertion of a collateral ligament of
the distal interphalangeal joint on
the distal phalanx; solar images are
usually most sensitive,
• identification of focal or diffuse
IRU in the navicular bone.
E.g., fetlock region pain:
• identification of focal IRU in the
proximal epiphyseal region of the
proximal phalanx probably repre-
possible in a simple blood sample, as
only the molecules actually present in
the blood are usable.
As the diagnosis is extremely precise,
the treatment is also. The
approach consists of pharmacologically
stimulating the expression of
the under-expressed genes, and, conversely,
provoking the inhibition of
those that are over-expressed. This
way only the proteins implicated in
the pathology are concerned by the
treatment. The micro-arrays thus
permit a fast and specific treatment
of the horse in question. Thanks to
their quick and adequate intervention,
micro-arrays encourage the animal’s
healing, minimizing the risk of
undesired side-effects.
Micro-arrays diagnosis is not
based on a detection of symptoms,
but on a modification of the gene
expression. This makes pathology
detection possible even before the
onset of its clinical manifestation.
The physical entity of the horse is protected
and its performances are less
affected.
Two limiting factors still exist
It is indeed possible to imagine a
micro-arrays analysis on a regular
base before and after training, in
order to verify the high-level sport
horse’s health and to determine its
potential predispositions to develop
pathological conditions. The preventive
measures can be adapted according
to the characteristics and specific
needs of every horse.
Up to now, two limiting factors
exist in the development of the biological
micro-arrays technology. The
horse’s genome is not yet completely
decoded. This means not all equine
genes are currently understood.
Nevertheless, research teams discover
new genes every day. The
equine genome will thus be completely
decoded in the near future.
The cost of a biological micro-array is
still substantial and its use is still limited
to scientific research. However,
the price will drop dramatically in due
course and the use of micro-arrays
will certainly be affordable for moni-
senting an incomplete sagittal fracture
• identification of focal IRU in a
condyle of the third metacarpal bone
reflecting traumatically induced or
stress-induced osseous pathology.
E.g., metacarpal region:
• identification of focal IRU in the
proximal palmar cortex of the third
metacarpal bone, reflecting a palmar
cortical stress fracture,
• identification of focal IRU in the
proximal plantar aspect of the third
metatarsal bone reflecting entheseous
injury at the origin of the suspensory
ligament.
E.g., hock pain:
• improvement in lameness after
fibular & tibial nerve blocks but no
response to intra-articular analgesia
of the tarsocrural, centrodistal
and tarsometatarsal joints; identification
of IRU in the talus reflecting
osteoarthritis, an incomplete
parasagittal fracture of the talus or
a developing osseous cyst-like
lesion (OCLL).
toring racing horses after its commercialisation.
Examples to be seen
The analysis is carried out on
horse cells. These can come from
blood, joint liquid puncture, tracheobronchial
wash or uterine wash. The
genetic material is extracted and put
on the micro-arrays in the laboratory,
in order to be treated. Thanks to a very
sophisticated technique, a characteristic
picture of the studied genes’
expression is obtained. This picture
is then compared to one obtained
from a known horse on the same
micro-array. Every colour spot represents
a specific gene. In the lecture
we will show an example of two
abnormally expressed genes.
Applications
Most applications of microarrays
with sport horses are based on
the analysis of the gene expression
level in order to detect under- and
over-expression. It then becomes
possible to evaluate the adaptation of
the horse to its training. For instance,
genes corresponding to anti-oxidative
enzymes such as glutathione peroxidase
(GPx) can be analysed. The
synthesis of this protein should be
proportional to the horse’s needs
before and after effort. Under-expression
of these genes produces oxidative
stress and can lead to effortlinked
muscular dystrophy. If such
an under-expression is detected, it
could be compensated pharmacologically
by specifically stimulating the
deficient gene’s expression. On the
other hand, it is also possible to complement
the animal with anti-oxidants
in its food on a daily basis, for
instance. The horse should reach its
best performances again, and recover
its optimum performance.
According to the same principle,
micro-arrays can also diagnose
inflammation very precisely, by
analysing joint liquid, for example.
The evidence of the over expression
of genes corresponding to proinflammatory
mediators can indeed
permit the characterisation of the
exact type of inflammation (acute or
chronic; mechanical, allergic or
infectious;…). The immediate concern
is that the active principle to stop
the inflammation process can be
quickly identified. This way time is
Partial improvement to local
analgesic techniques, suspected bone
injury, but unable to identify lesion.
E.g., identification of IRU in the palmar
aspect of the diaphyseal region
of the third metacarpal bone after
partial improvement to subcarpal
nerve blocks of the palmar and palmar
metacarpal nerves.
E.g., identification of focal IRU in
the distal row of carpal bones after
partial response to subcarpal analgesia
with or without a positive
response to median and ulnar nerve
blocks.
For the determination of
the likely significance of subtle
radiographic abnormalities.
E.g., subtle loss of opacity of the flexor
cortex of the navicular bone, close to
the sagittal ridge:
• Significance of an OCLL,
• Significance of focal modelling of
an ossified cartilage of the foot.
If there are no localising clinical
signs and there is no or little response
to local analgesia.
E.g., identification of IRU in the
humeral tubercles or the deltoid
tuberosity of the humerus – subsequent
radiographic and ultrasono-
won and the transformation of an
acute into a chronic inflammation,
which would have diminished short
term performances and provoked
median term irreversible lesions, can
be avoided. Targeting the treatment
increases its efficiency and decreases
its secondary effects.
Other applications
for micro-arrays exist.
For instance, it can be
interesting to follow
genetic profiles in a
champion’s lineage.
These profiles can be
associated with posi- Pierre Lekeux
tive characteristics or excellent performances.
On the other hand, a
champion’s profile selection could
favour the outbreak of certain pathological
conditions if preventive measures
are not taken soon enough.
Finally, micro-array technology can
also be used to accurately identify
pathogens like bacteria or viruses,
and this has positive effects on the
spread of veterinary zoonoses.
Biological micro-arrays are molecular
tools that should quickly find
their place in veterinary medicine to
complement current diagnostic techniques.
Thanks to progress in
research into equine genome decoding
and molecular technology, reliable
and cheap micro-arrays should
be commercialised in the near future.
This should contribute to the
increase of the well-being, health and
performances of our horses.
Further readings
1. Liew CC, Ma J, Tang HC, Zheng R,
Dempsey AA. (2006) The peripheral blood
transcriptome dynamically reflects system
wide biology: a potential diagnostic tool. J
Lab Clin Med. 147:126-32.
2. Lu W, Bertone AL. (2004) Generation and
performance of an equine-specific largescale
gene expression microarray.; Am J Vet
Res. 65:1664-73.
3. Thomas A., Closset R., Bureau F., Lekeux
P. (2005) : Principes des microdamiers à
ADN et applications potentielles en sciences
vétérinaires. Ann. Méd. Vét. , 149, 93-
116.
4. Venkatasubbarao S. (2004) Microarrays –
status and prospects. Trends Biotechnol.,
22, 630-637.
5. Weeraratna AT, Nagel JE, de Mello-Coelho
V, Taub DD. (2004) Gene expression profiling:
from microarrays to medicine. J Clin
Immuno; 24:213-24.
Lecture/Vortrag
Friday March 16 2007, 05:20 p.m./
Freitag, 16.03.2007, 17:20 Uhr
graphic evaluation
may identify a lesion.
E.g., Identification of
IRU in the first rib,
consistent with a fracture.
E.g., Identification
of IRU in the third
trochanter of the Sue Dyson
femur or a tuber ischii, reflecting a
fracture or entheseous injury.
Clinical signs suggestive of
fracture, but no localising clinical
signs.
E.g., incomplete dorsal sagittal fracture
of the proximal aspect of the
proximal phalanx.
Poor performance but no
localising clinical signs.
E.g., identification of linear streaks of
IRU in the hindlimb musculature
consistent with recurrent exertional
rhabdomyolysis.
Episodic lameness that cannot be
reproduced
E.g., identification of focal IRU in
the third carpal bone of an endurance
horse only lame after 40 miles.
Lecture/Vortrag
Friday March 16 2007, 09:00 a.m./
Freitag, 16.03.2007, 09:00 Uhr