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Neurotrophin 4/5 (NT 4/5) belongs to a family of
molecules called neurotrophins that are known to have important
functional roles in the development and plasticity of the nervous
system. Two members of this family signal through the same membrane
receptor trKB. These are NT 4/5 and brain derived neurotrophic factor
(BDNF). Regeneration of axons in the peripheral nervous system is
enhanced by BDNF. The role of NT 4/5 in regeneration is not known.
We used a strain of mice which had been genetically modified such
that the axons in their peripheral nerves contained yellow fluorescent
protein (YFP). In heterozygous animals (YFP) a subset of axons using
fluorecence microscopy. We also used mice null for NT 4/5 to study
the role of this neurotrophin in axon regeneration. Two types of
experiments were performed. In one set of animals (Protocol 1) a
peripheral nerve injury in a wild type YFP mouse was repaired using
a graft from a NT 4/5 mouse. In the other experiment NT 4/5 mice
and YFP mice were bred to create animals that were both NT 4/5 and
YFP. In these animals (Protocol 2) peripheral nerve injuries were
repaired using grafts obtained either from wild type or knockout
mice. In animals in Protocol 1 wild type axons regenerating into
grafts from NT 4/5 mice grew poorly relative to those growing into
grafts from wild type mice. In animals of Protocol 2 NT 4/5 axons
regenerated well through grafts from wild type mice. However NT
4/5 axons also regenerated well through grafts from NT 4/5 mice.
Thus NT 4/5 is required for normal regeneration of axons in wild
type mice but in NT 4/5 mice something else must compensate for
the loss of NT 4/5.
Neurotrophins are a group of family-oriented peptides
that promotes neuronal growth and influences the neuron count in
both the peripheral and central nervous systems. Nerve growth factor
(NGF) brain-derived neurotrophic factor (BDNF) NT-4/5 and NT-3 are
four members of the neurotrophin family of molecules. The receptors
to these neurotrophins can be split into two classes: “the
common neurotrophin” receptor (p75) and tropomyosin receptor
kinases (trk). These receptors bind different members of the neurotrophin
family differentially. Nerve growth facts bind to trkA BDNF and
NT-4/5 bind to trkB and NT-3 binds to trkC. Complete regeneration
of peripheral neurons after nerve injury is rare. However there
are factors that scientists have at their disposal that can encourage
axon regeneration. Fibronectin and laminin are two molecules found
in the extracellular matrix (ECM) that promote the growth of neurons.
The development of axons in the peripheral nervous system (PNS)
can be attributed to the influential role that the molecule laminin
plays in particular. The neurotrophin BDNF acting through the trkB
receptor has been implicated in promoting axon regeneration whereas
the role of NT-4/5 (which also binds to the same receptor trkB)
is not well known. In preliminary experiments in Dr. English’s
Laboratory axons did not regenerate into nerve grafts that did not
contain NT-4/5. It is not known if the same is true if NT-4/5 is
reduced rather than eliminated. It also is not known if axons deprived
of NT-4/5 will regenerate if placed in an environment rich in neurotrophin.
Experiments will be conducted using 6 mice. Half of
these mice serve as experimental subjects. Half of the mice will
serve as graft donors. The thy-1-YFP-H strain will characterize
the transgenic mice that will make up the experimental subjects.
In these mice the yellow fluorescent protein (YFP) is expressed
under the control of the Thy-1 promoter. The YFP completely fills
a subset of the axons in a peripheral nerve allowing us to study
axon regeneration by fluorescence microscopy. Six experimental subjects
will carry a second genetic manipulation. The other six experimental
subject mice will be Thy-1-YFP-H mice that are wild type for NT-4/5
expression (NT-4/5+/+). These animals also will be null for the
gene for NT-4/5 (NT-4/5 -/-). These double transgenic mice were
created by crossing Thy-1-YFP-H mice andNT-4/5 -/- mice. Graft donor
mice will be either wild type (C57B6J) or NT-4/5 +/- mice. All procedures
will be performed bilaterally. Two animals one graft donor and one
experimental subject will be deeply anesthetized with 90 mg/kg of
pentobarbital injected intraperitoneally. In the graft donor the
branches of the sciatic nerve will be identified and the common
peroneal (CP) nerve the object of study we be isolated. A 3-5 mm
long segment of the donor’s CP nerve will be cut and used
as a graft. In the experimental subject mice the CP nerve will be
cut near its branch point from the sciatic nerve. The CP nerve in
the experimental subject will be repaired using the segment of nerve
from the graft donor mouse. The graft donor mouse will then be euthanized
with an overdose of pentobarbital. Once the grafts are aligned as
closely as possible to the stumps of the cut CP nerve of the experimental
subject they will be attached using fibrin glue.
Two experimental paradigms will be used:
- In Thy-1- YFP mice one cut CP nerve will be repaired with a
graft from a NT-4/5 +/- mouse. This side of the animal will be
considered the experimental side. The cut CP nerve on the opposite
side of the mice will be repaired using a graft from a wild type
graft donor mouse. This side of each mouse will be considered
a control. Thus any regenerating CP axons will grow into a graft
where NT-4/5 is presumed to be less available than normal.
- In experimental subjects that are Thy-1-YFP-H/NT-4/5-/- one
cut CP nerve will be repaired with a graft from a wild type mouse.
Thus any regenerating CP axons will grow from an environment devoid
of NT-4/5 into a graft rich in NT-4/5. The cut CP nerve on the
opposite side of these mice will be repaired using a graft from
an NT-4/5-/- mouse. This side of each mouse will be considered
the experimental side (Fig.1B). Animals will survive for a 2-week
period and will then be euthanized with an overdose of pentobarbital
(150mg/kg).
Branches of the sciatic nerve including repaired
CP nerves will be dissected removed from the animal and fixed in
paraformaldehyde for an hour at room temperature. Once this has
been completed the nerves will be washed in buffer solution and
placed on a microscope slide. Then the slides will be cover-slipped
with Aquamount and sealed in place with clear nail polish. The harvested
nerve will be viewed with a confocal microscope using filter settings
for YFP. Stacks of optical sections will be obtained a10-micrometer
spacing through entire depth of nerve at 10X magnification. Images
of these stacks will be stored on disc for later analysis. Images
of optical sections will be used to reconstruct the trajectories
of regenerating YFP-containing axons in the grafted CP nerves. Lengths
of these regenerating axon profiles will be measured using Image
Pro software from the proximal end of the graft to their distal-most
extent.
In animals in Protocol 1, wild type axons regenerating
into grafts from NT 4/5 mice grew poorly relative to those growing
into grafts from wild type mice. In animals of Protocol, 2 NT 4/5
axons regenerated well through grafts from wild type mice. However
NT 4/5 axons also regenerated well through grafts from NT 4/5 mice.
Neurotrophin 4/5 is extremely influential in peripheral axon regeneration.
Our future directions include measuring the actual lengths of the
axons to better determine the extent of growth.
I would like to acknowledge Arthur English and his lab team for
allowing me to work with them this summer. This material is based
upon work supported by Howard Hughes Medical Institute under Grant
No. 52003727.
The role of neurotrophin 4/5 in peripheral axon regeneration
will be examined. NT 4/5 belongs to a group of molecules that play
a role in the axons regenerating. Brain derived neurotrophic factor
(BDNF) a molecule that binds to the same receptor as NT 4/5 has
been studied and proven to enhance axonal regeneration. YFP will
be used to trace the complete filling of axon regeneration through
fluorescence microscopy. Graft donor mice of the wild type and neurotrophin
strain will be depicted. The CP nerve in the experimental subject
will be repaired using the segment of nerve from the graft donor.
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