Application Note

Neuroscience: Peripheral Nerve Imaging

Introduction This application note describes: The establishment of a mouse model of nerve degeneration and regeneration induced by a crush injury to the saphenous nerve. The use of Cellvizios novel imaging technology of Fibered Confocal Fluorescence Microscopy, which enables a minimally invasive and longitudinal monitoring of the axonal degeneration and regeneration processes. Materials and Methods In this application, Cellvizio was used to study the neuronal degeneration and regeneration processes in live, anaesthetized, adult Thy1-YFP transgenic mice. A small 2 mm incision was first made in the skin, through which a handheld microprobe of 650 m diameter was directly inserted. The saphenous nerve was then imaged through the perineurium, allowing repeated measurements to be made without nerve damage. This novel technology marked to in vitro imaging with a traditional fluorescence microscope. The Cellvizio LAB is a complete imaging system based on a fibered technology for fluorescence confocal imaging of the living animal. It acquires high resolution image sequences, displays them in real-time, enables live measurements and stores the image sequences. The ProFlex Microprobe is a highly advanced optical imaging tool incorporating proprietary fiber optic objective lens technology.
Application Note: Peripheral Nerve Imaging

Each microprobe comprises tens of thousands of individual fiber optics encased within a single probe. ProFlex Microprobes are available in a range of diameters from 4.2 mm down to 300 m. The small size and flexibility of the microprobes enable direct access to a region of interest within a living animal either externally, endoscopically or via a minimally invasive procedure. Coupled to the Laser Scanning Unit (LSU) and ImageCell, the image processing software, the system renders realtime dynamic image sequences with a lateral resolution as fine as 1.4 m and at 12 frames per second (with capabilities up to 200 frames per second). In Vivo Imaging of Peripheral Nervous System The Cellvizio has already proven its suitability for live imaging of the peripheral nervous system. Using transgenic mice strains with YFP-positive nervous system, the Cellvizio images cellular bodies (Figure A), axon bundles (Figure B) and single axons (Figure C), which could be followed over long distances with the ProFlex. In addition, images of small nervous structures such as dendritic endings (Figure D), axonal endings (Figure E) and neuromuscular junctions (Figure F) are readily accessible with ease and minimal invasiveness. Steady image sequences can be acquired using the handheld ProFlex or by securing the ProFlex into an appropriate holding device. The images shown represent single frames extracted from image sequences obtained by following the structures over long distances and time.

The suitability of the Cellvizio for high resolution imaging of live structures will provide scientists the first real opportunity to perform unique biomedical research studies such as: The measurement of regenerative nerve outgrowth The evaluation of fiber density in tissue reinnervation The analysis of the formation and the number of nerve endings to evaluate the functional recovery of neurotransmission

CELLULAR BODIES

Figure A - Cellular bodies in the dorsal root ganglia

AXON BUNDLE

Figure B - Sciatic nerve imaged at 5 m lateral resolution, permitting visualization of single axons 1

ISOLATED FIBER

Crush Injury of the Saphenous Nerve A mouse model of nerve regeneration induced by crush injury of the saphenous nerve, which includes both motor and sensory fibers, was used. The saphenous nerve, located at the anterior face of the posterior leg (Figure 1), was selected for its superficial location providing easy access through a two millimeter incision of the skin. The crush induces the degeneration of the distal nerve fragments prior to their disappearing following Wallerian degeneration. This process is slow and takes several days. In the meantime, nerve fibers begin to regenerate from the injury site along the initial path towards the distal stump. The goal was to provide a direct and rapid monitoring of the axon degeneration and regeneration processes, in a live animal without tissue sampling. Images and measurements obtained with the Cellvizio were bench-marked against those obtained using standard fluorescence microscopy.

An epifluorescence microscope was used, with a 10x/0.30 objective. Images acquired using both techniques are shown. The image of the explanted and fixed nerve, marked by a schematic microscope (Figure 2), was obtained using a tabletop epifluorescence microscope. The explanted nerve was fixed uncut in formaldehyde for one hour and then observed. These images were compared to images of the saphenous nerve acquired in vivo and in situ using a Cellvizio. Figure 3 shows the axon bundle before (top) and after (bottom) the crush. It is important to note that the nerve is being viewed through the perineurium, without damaging the nerve tissue, which made it possible to monitor the axon regeneration process repetitively over several days. Experimental Setup Adult male Thy1-YFP transgenic mice (ref.: B6.Cg-Tg (Thy1YFP)16Jrs/J, Jackson Laboratories; Feng et al., 2000) were anesthetized with intra-peritoneal injections of ketamine.

Figure C - Single nerve fiber of the cutaneous sensory network, which can be followed over several millimeters DENDRITIC ENDINGS

Figure D - Terminals of a sensory fiber imaged under skin AXONAL ENDINGS

Adult THY1-YFP Mouse

Figure E - Motor nerve terminals of a neuromuscular junction NEUROMUSCULAR JUNCTIONS

b a

Figure F - Neuromuscular junctions, showing both nerve and muscle fibers. Visualization of the muscle fiber made possible with Syto 13 Application Note: Peripheral Nerve Imaging

Figure 1 - (a) Saphenous nerves on the underside of the posterior legs, chosen for their superficial location (b) ProFlex probe allowing easy access through a minimally-invasive incision of the skin

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 an epifluorescence microscope In vivo and in situ dynamic acquisition

In the corresponding images acquired using the Cellvizio, we can clearly 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 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 solubilization of the YFP-protein.

Cellvizio LAB

Each 2 posterior leg was shaved over a 0.5 cm area, in order to visualize the saphenous vein, which runs along the saphenous nerve. A 2 mm cut was made above the vein. The model consists in the production of a crush injury to the saphenous nerve with a ligature maintained for two minutes. The degeneration and regeneration processes can then be monitored over multiple days by opening and suturing the small cut as needed.

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 microscope. Bottom: Cellvizio Images of the regenerative segments and the front of progression with ends of regenerative nerves clearly visible. The bottom right image was constructed by tiling images from a dynamic sequence acquired with Cellvizio.

Fibered Confocal Fluorescence Microscopy

Figure 5 - Length of outgrowth measured, on a total of 30 mice, after a crush of the saphenous nerve, both with an epifluorescence microscope (yellow) and the Cellvizio (blue).

Application Note: Peripheral Nerve Imaging

Axonal outgrowth was measured in three groups of ten mice using both a standard fluorescence microscope and a Cellvizio. The graph in Figure 5 displays the results. Both methodologies show that the length of the outgrowth increases from Day 3 to Day 5 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 outgrowth using a Cellvizio reveals a very high correlation with those obtained from a microscope However, the actual lengths of the outgrowth were 30% greater, on average, when measured using a Cellvizio. The reduced length of the sampled nerve observed under a standard fluorescence microscope is probably a result of the retraction of the nerve segment due to the section and the immersion in a fixative solution Effect of Vincristine on Nerve Regeneration After a Crush The next step in the development 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 intraperitoneal 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 nerve debris of degenerating axons and no regrowing fibers.
Application Note: Peripheral Nerve Imaging

Crush

Degenerative Fragments

1 mm

Epifluorescence Microscope

Cellvizio LAB Figure 6 - Four Days After Crush with vincristine administration - Top: Tiled image and high resolution images of the saphenous nerve obtained with epifluorescence microscope, depicting the crush site and no regenerative segments within the debris of Wallerian degeneration. Bottom: Visualization of same sections using the Cellvizio

To quantify the effects of vincristine on nerve regeneration, four mice were administered a one-time dose of vincristine on Day 1 after the crush and another four mice were administered an injection of saline on Day 1 after the crush. The Cellvizio was used to analyze, measure and compare the outgrowth length over fifteen days (Figure 7).

The measurements taken from images acquired by the Cellvizio show that the vincristine transiently inhibits the regeneration of axons from Day 1 to Day 6 after the crush, as reported in the literature (Ruigt et al., 1995; Shiraishi et al., 1985; Nakamura et al., 2001; Paydarfar JA and Paniello RC, 2001). Regrowth then occurs to reach maximal length by Day 15.

Fibered Confocal Fluorescence Microscopy

Figure 7 - Cellvizio measurement of the effect of vincristine on nerve regeneration after crush. Pink: Test group of four mice receiving an intra-peritoneal injection of 0.5 mg/kg of vincristine at Day 1 after the crush. Orange: Control group of four mice receiving only saline. 4

Tabletop Fluorescence Microscopy

Sacrificed animal Explanted and fixed nerve One mouse per measurement 50 minutes per measurement

Fibered Confocal Fluorescence Microscopy

Live, anesthetized animal In vivo and in situ imaging Repeated measurement on the same mouse 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 degeneration and regeneration in situ, in a living animal, has many significant advantages as compared to traditional fluorescence microscopy. It enables longitudinal monitoring of the degeneration and regeneration processes, as well as the measurement of the length of the nerve outgrowth. Furthermore, it significantly reduces the time necessary for measurement by a factor of ten. The 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.

Application Note: Peripheral Nerve Imaging

Credits and References This work was published in: Pierre Vincent, Uwe Maskos, Igor Charvet, Laurence Bourgeais, Luc Stoppini, Nathalie Leresche, Jean-Pierre Changeux, Rgis Lambert, Paolo Meda, Danile Paupardin-Tritsch. Live imaging of neural structure and function by fibered fluorescence microscopy. (2006) EMBO Reports 7, 11, 11541161 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):1147911488 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 electrophysiological 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: Peripheral Nerve Imaging