Application Note - Neuroscience: Peripheral Nerve Imaging

Application Note

Neuroscience: Peripheral Nerve Imaging

Introduction Each microprobe comprises tens The suitability of the Cellvizio for
of thousands of individual fiber high resolution imaging of live
This application note describes: optics encased within a single structures will provide scientists
probe. ProFlex Microprobes are the first real opportunity to perform
• The establishment of a mouse available in a range of diameters unique biomedical research studies
model of nerve degeneration and from 4.2 mm down to 300 µm. such as:
regeneration induced by a crush The small size and flexibility of the
injury to the saphenous nerve. microprobes enable direct access • The measurement of
to a region of interest within a
• The use of Cellvizio’s novel regenerative nerve outgrowth
living animal either externally,
imaging technology of Fibered • The evaluation of fiber density in
endoscopically or via a minimally
Confocal Fluorescence tissue reinnervation
invasive procedure.
Microscopy, which enables
a minimally invasive and • The analysis of the formation and
Coupled to the Laser Scanning Unit
longitudinal monitoring of the number of nerve endings to
(LSU) and ImageCell, the image
the axonal degeneration and evaluate the functional recovery
processing software, the system
regeneration processes. of neurotransmission
renders realtime dynamic image
sequences with a lateral resolution
Materials and Methods
as fine as 1.4 µm and at 12 frames
In this application, Cellvizio per second (with capabilities up to
was used to study the neuronal 200 frames per second).
degeneration and regeneration
In Vivo Imaging of Peripheral CELLULAR BODIES
processes in live, anaesthetized,
Nervous System
adult Thy1-YFP transgenic mice.
The Cellvizio has already proven
A small 2 mm incision was first its suitability for live imaging of
made in the skin, through which a the peripheral nervous system.
handheld microprobe of Using transgenic mice strains with
650 µm diameter was directly YFP-positive nervous system, the
inserted. The saphenous nerve Cellvizio images cellular bodies
was then imaged through the (Figure A), axon bundles (Figure
perineurium, allowing repeated B) and single axons (Figure C),
measurements to be made which could be followed over long
without nerve damage. This distances with the ProFlex. Figure A - Cellular bodies in the dorsal
novel technology marked to in root ganglia
vitro imaging with a traditional In addition, images of small
fluorescence microscope. nervous structures such as
dendritic endings (Figure D), AXON BUNDLE
The Cellvizio® LAB is a complete axonal endings (Figure E) and
imaging system based on a fibered neuromuscular junctions (Figure
technology for fluorescence F) are readily accessible with ease
confocal imaging of the living and minimal invasiveness. Steady
animal. It acquires high resolution image sequences can be acquired
image sequences, displays using the handheld ProFlex or
them in real-time, enables live by securing the ProFlex into an
measurements and stores the appropriate holding device.
image sequences.
The images shown represent single
The ProFlex™ Microprobe is a frames extracted from image
highly advanced optical imaging sequences obtained by following Figure B - Sciatic nerve imaged at
tool incorporating proprietary fiber the structures over long distances 5 µm lateral resolution, permitting
optic objective lens technology. and time. visualization of single axons

Application Note: Peripheral Nerve Imaging 1

ISOLATED FIBER Crush Injury of the Saphenous An epifluorescence microscope was
Nerve used, with a 10x/0.30 objective.

A mouse model of nerve Images acquired using both
regeneration induced by crush techniques are shown. The image
injury of the saphenous nerve, of the explanted and fixed nerve,
which includes both motor and marked by a schematic microscope
sensory fibers, was used. (Figure 2), was obtained using
The saphenous nerve, located at a tabletop epifluorescence
the anterior face of the posterior microscope. The explanted nerve
leg (Figure 1), was selected for its was fixed uncut in formaldehyde
superficial location providing easy for one hour and then observed.
Figure C - Single nerve fiber of the
cutaneous sensory network, which can access through a two millimeter
incision of the skin. These images were compared to
be followed over several millimeters
images of the saphenous nerve
DENDRITIC ENDINGS The crush induces the degeneration acquired in vivo and in situ using a
of the distal nerve fragments prior Cellvizio.
to their disappearing following
Wallerian degeneration. Figure 3 shows the axon bundle
This process is slow and takes before (top) and after (bottom) the
several days. In the meantime, crush. It is important to note that
nerve fibers begin to regenerate the nerve is being viewed through
from the injury site along the initial the perineurium, without damaging
path towards the distal stump. the nerve tissue, which made
it possible to monitor the axon
The goal was to provide a direct regeneration process repetitively
and rapid monitoring of the axon over several days.
Figure D - Terminals of a sensory fiber degeneration and regeneration
imaged under skin processes, in a live animal without Experimental Setup
tissue sampling.
AXONAL ENDINGS Adult male Thy1-YFP transgenic
Images and measurements mice (ref.: B6.Cg-Tg (Thy1-
obtained with the Cellvizio YFP)16Jrs/J, Jackson Laboratories;
were bench-marked against Feng et al., 2000) were
those obtained using standard anesthetized with intra-peritoneal
fluorescence microscopy. injections of ketamine.

Adult THY1-YFP Mouse

Figure E - Motor nerve terminals of a
b
neuromuscular junction

NEUROMUSCULAR JUNCTIONS a

Figure 1 - (a) Saphenous nerves on the underside of the posterior legs,
chosen for their superficial location (b) ProFlex™ probe allowing easy access
Figure F - Neuromuscular junctions, through a minimally-invasive incision of the skin
showing both nerve and muscle fibers.
Visualization of the muscle fiber made
possible with Syto 13


In vitro explanted and fixed nerve Post-Crush Outgrowth Measurement

The tiled image of a fixed explanted nerve viewed under a standard
fluorescence microscope, four days after the crush (top of Figure 4), shows
the regeneration of axons from the crush site, the front of progression
and the remaining degenerative fragments. The crush site presents no
staining, probably due to the loss of the fluorescent agent (YFP is soluble)
during the manipulation for tissue sampling. The fiber ends of the front of
progression are visible in the debris from Wallerian degeneration (see the
high magnification images on Figure 4).
Figure 2 - Explanted, fixed and
uncut saphenous nerve acquired with In the corresponding images acquired using the Cellvizio, we can clearly
an epifluorescence microscope identify the zone of degeneration, the zone of regeneration (bottom left of
Figure 4) and the front of progression (bottom right of Figure 4) despite
the lower contrast caused by imaging through the perineurium. In dynamic
In vivo and in situ dynamic acquisition sequences, the front of progression is even more clearly visible.

It is therefore possible to visualize nerve regeneration and to measure the
length of outgrowth using a graduated wire applied along the nerve, both
without tissue biopsy.
Crush Regenerative Axons Front of Progression Degenerative Fragments

1 mm

Epifluorescence
Microscope

Figure 3 - Saphenous nerve acquired
in vivo and in situ with the Cellvizio
both, before (top) and after (bottom)
the crush. The crush induces a
rapid loss of fluorescence at the
sight of injury, probably due to the Cellvizio® LAB
solubilization of the YFP-protein.
Figure 4: Four Days After Crush - Top: Tiled image of the saphenous nerve from the
crush site to the degenerative fragments, as well as high magnification images of
the regenerative segments and the front of progression, all from an epifluorescence
Each 2 posterior leg was shaved microscope. Bottom: Cellvizio Images of the regenerative segments and the front of
over a 0.5 cm area, in order to progression with ends of regenerative nerves clearly visible. The bottom right image
visualize the saphenous vein, which was constructed by tiling images from a dynamic sequence acquired with Cellvizio.
runs along the saphenous nerve.
Fibered Confocal Fluorescence Microscopy
A 2 mm cut was made above the
vein. The model consists in the Figure 5 - Length
production of a crush injury to the of outgrowth
saphenous nerve with a ligature measured, on a
maintained for two minutes. total of 30 mice,
after a crush of
The degeneration and regeneration the saphenous
processes can then be monitored nerve, both with
over multiple days by opening and an epifluorescence
microscope (yellow)
suturing the small cut as needed.
and the Cellvizio
(blue).


Axonal outgrowth was measured Crush Degenerative Fragments

in three groups of ten mice using
both a standard fluorescence
microscope and a Cellvizio. The
graph in Figure 5 displays the
results.
1 mm
• Both methodologies show that
the length of the outgrowth
Epifluorescence
increases from Day 3 to Day 5 Microscope
after the crush, as reported by
Pan et al; 2003

• In both cases, this approach has
a high reproducibility, as seen
from the low standard deviations

• The measurements of the axonal Cellvizio®
outgrowth using a Cellvizio LAB
reveals a very high correlation
Figure 6 - Four Days After Crush with vincristine administration - Top: Tiled image
with those obtained from a and high resolution images of the saphenous nerve obtained with epifluorescence
microscope microscope, depicting the crush site and no regenerative segments within the debris
of Wallerian degeneration. Bottom: Visualization of same sections using the Cellvizio
• However, the actual lengths of
the outgrowth were 30% greater, To quantify the effects of vincristine The measurements taken from
on average, when measured on nerve regeneration, four mice images acquired by the Cellvizio
using a Cellvizio. The reduced were administered a one-time dose show that the vincristine
length of the sampled nerve of vincristine on Day 1 after the transiently inhibits the regeneration
observed under a standard crush and another four mice were of axons from Day 1 to Day 6
fluorescence microscope is administered an injection of saline after the crush, as reported in
probably a result of the retraction on Day 1 after the crush. the literature (Ruigt et al., 1995;
of the nerve segment due to the Shiraishi et al., 1985; Nakamura et
section and the immersion in a The Cellvizio was used to analyze, al., 2001; Paydarfar JA and Paniello
fixative solution measure and compare the RC, 2001). Regrowth then occurs
outgrowth length over fifteen days to reach maximal length by Day
Effect of Vincristine on Nerve (Figure 7). 15.
Regeneration After a Crush

The next step in the development Fibered Confocal Fluorescence Microscopy
of this model was to test the
administration of a neurotoxic
drug, such as vincristine.
Vincristine, a chemotherapeutic
molecule, was administered at
0.5 mg/kg in a one-shot intra-
peritoneal injection on Day 1 after
the crush. High doses of vincristine
are known to induce peripheral
neuropathy and transiently block
nerve regeneration.

As depicted in both imaging
modalities (Figure 6) at Day 4
after the crush, vincristine blocks
the regeneration process. Both
the Cellvizio and the standard
fluorescence microscope show Figure 7 - Cellvizio measurement of the effect of vincristine on nerve regeneration
nerve debris of degenerating axons after crush. Pink: Test group of four mice receiving an intra-peritoneal injection of
and no regrowing fibers. 0.5 mg/kg of vincristine at Day 1 after the crush. Orange: Control group of four
mice receiving only saline.


Tabletop Fluorescence Microscopy Fibered Confocal Fluorescence Microscopy

• Sacrificed animal • Live, anesthetized animal
• Explanted and fixed nerve • In vivo and in situ imaging
• Repeated measurement on the same
• One mouse per measurement mouse
• 50 minutes per measurement • 5 minutes per measurement

As demonstrated the images acquired using a Cellvizio provide a reliable approach to the imaging of the
peripheral nervous system as validated by comparison with studies using standard fluorescence microscopy.
Repetitive Measurements
The minimally invasive access in a living animal allows repetitive measurements in time, as opposed to a single
measurement session from one sacrificed mouse in regular microscopy, and a follow-up analysis of regeneration
on the same animal.
Time of Measurements
It takes about 50 minutes to measure one regenerating nerve with a microscope on account of tissue sampling,
fixation, mounting, and microscope and camera preparation. In comparison, the Cellvizio can reduce the time per
measurement to 5 minutes from incision to post-measurement suture.

In conclusion, imaging peripheral nerves with the Cellvizio provides reliable results which are in accordance to
published literature and have been benchmarked against standard fluorescence microscopy. The instrument
is easy to use. As access is only minimally invasive and there is no tissue sampling, the Cellvizio provides a
better and more time-efficient alternative for longitudinal monitoring of axonal degeneration and regeneration
processes, measurement of length of outgrowth and monitoring the effect of neurotoxic, neurotrophic and
protective molecules.

Summary
Viewing the neuronal It enables longitudinal monitoring of the
degeneration and degeneration and regeneration processes,
regeneration in situ, in a as well as the measurement of the length
living animal, has many of the nerve outgrowth. Furthermore, it
significant advantages as significantly reduces the time necessary
compared to traditional for measurement by a factor of ten. The
fluorescence microscopy. Cellvizio® LAB is the only system available
that enables in vivo and in situ molecular
imaging of peripheral nerves down to the
resloution of single axons.


Credits and References

This work was published in: Pierre Vincent, Uwe Maskos,
Igor Charvet, Laurence Bourgeais, Luc Stoppini, Nathalie
Leresche, Jean-Pierre Changeux, Régis Lambert, Paolo
Meda, Danièle Paupardin-Tritsch. “Live imaging of
neural structure and function by fibered fluorescence
microscopy.” (2006) EMBO Reports 7, 11, 1154–1161”

1. Y.Albert Pan, Thomas Misgeld, Jeff W. Lichtman,
and Joshua R. Sanes (2003) Effects of Neurotoxic
and Neuroprotective Agents on Peripheral Nerve
Regeneration Assayed by Time-Lapse Imaging In
Vivo. The Journal of Neuroscience 23(36):11479-
11488
2. Feng G, Mellor RH, Bernstein M, Keller-Peck C,
Nguyen QT, Wallace M, Nerbonne JM, Lichtman
JW, Sanes JR. (2000) Imaging neuronal subsets in
transgenic mice expressing multiple spectral variants
of GFP. Neuron. 28(1):41-51
3. Ruigt GS, den Brok MH. (1995) Retardation of rat
sciatic nerve regeneration after local application
of minute doses of vincristine. Cancer Chemother.
Pharmacol. 36(6):530-5
4. Shiraishi S, Le Quesne PM, Gajree T. (1985) The
effect of vincristine on nerve regeneration in the
rat. An electro¬physiological study. J Neurol. Sci.
71(1):9-17
5. Nakamura Y, Shimizu H, Nishijima C, Ueno M,
Arakawa Y. (2001) Delayed functional recovery by
vincristine after sciatic nerve crush injury: a mouse
model of vincristine neurotoxicity. Neurosci. Lett.
304: 5-8
6. Paydarfar JA, Paniello RC (2001) Functional study
of four neurotoxins as inhibitors of post-traumatic
nerve regeneration. Laryngoscope 111: 844-850

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Application Note - Neuroscience: Peripheral Nerve Imaging

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