Application Note - Neuroscience: Peripheral Nerve Imaging


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In this application, Cellvizio was used to study the neuronal degeneration and regeneration processes in live, anaesthetized, adult Thy1-YFP transgenic mice.

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Application Note - Neuroscience: Peripheral Nerve Imaging

  1. 1. Application Note Neuroscience: Peripheral Nerve ImagingIntroduction Each microprobe comprises tens The suitability of the Cellvizio for of thousands of individual fiber high resolution imaging of liveThis 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 resolutionMaterials and Methods as fine as 1.4 µm and at 12 framesIn this application, Cellvizio per second (with capabilities up towas used to study the neuronal 200 frames per second).degeneration and regeneration In Vivo Imaging of Peripheral CELLULAR BODIESprocesses in live, anaesthetized, Nervous Systemadult Thy1-YFP transgenic mice. The Cellvizio has already provenA small 2 mm incision was first its suitability for live imaging ofmade in the skin, through which a the peripheral nervous system.handheld microprobe of Using transgenic mice strains with650 µm diameter was directly YFP-positive nervous system, theinserted. The saphenous nerve Cellvizio images cellular bodieswas then imaged through the (Figure A), axon bundles (Figureperineurium, allowing repeated B) and single axons (Figure C),measurements to be made which could be followed over longwithout nerve damage. This distances with the ProFlex. Figure A - Cellular bodies in the dorsalnovel technology marked to in root gangliavitro imaging with a traditional In addition, images of smallfluorescence microscope. nervous structures such as dendritic endings (Figure D), AXON BUNDLEThe Cellvizio® LAB is a complete axonal endings (Figure E) andimaging system based on a fibered neuromuscular junctions (Figuretechnology for fluorescence F) are readily accessible with easeconfocal imaging of the living and minimal invasiveness. Steadyanimal. It acquires high resolution image sequences can be acquiredimage sequences, displays using the handheld ProFlex orthem in real-time, enables live by securing the ProFlex into anmeasurements and stores the appropriate holding device.image sequences. The images shown represent singleThe ProFlex™ Microprobe is a frames extracted from imagehighly advanced optical imaging sequences obtained by following Figure B - Sciatic nerve imaged attool incorporating proprietary fiber the structures over long distances 5 µm lateral resolution, permittingoptic objective lens technology. and time. visualization of single axonsApplication Note: Peripheral Nerve Imaging 1
  2. 2. 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 thecutaneous sensory network, which can access through a two millimeter incision of the skin. These images were compared tobe followed over several millimeters images of the saphenous nerveDENDRITIC 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 regenerationimaged 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 MouseFigure E - Motor nerve terminals of a bneuromuscular junctionNEUROMUSCULAR JUNCTIONS a Figure 1 - (a) Saphenous nerves on the underside of the posterior legs, chosen for their superficial location (b) ProFlex™ probe allowing easy accessFigure F - Neuromuscular junctions, through a minimally-invasive incision of the skinshowing both nerve and muscle fibers.Visualization of the muscle fiber madepossible with Syto 13Application Note: Peripheral Nerve Imaging 2
  3. 3. 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 epifluorescenceEach 2 posterior leg was shaved microscope. Bottom: Cellvizio Images of the regenerative segments and the front ofover a 0.5 cm area, in order to progression with ends of regenerative nerves clearly visible. The bottom right imagevisualize the saphenous vein, which was constructed by tiling images from a dynamic sequence acquired with Cellvizio.runs along the saphenous nerve. Fibered Confocal Fluorescence MicroscopyA 2 mm cut was made above thevein. The model consists in the Figure 5 - Lengthproduction of a crush injury to the of outgrowthsaphenous nerve with a ligature measured, on amaintained for two minutes. total of 30 mice, after a crush ofThe degeneration and regeneration the saphenousprocesses can then be monitored nerve, both withover multiple days by opening and an epifluorescence microscope (yellow)suturing the small cut as needed. and the Cellvizio (blue).Application Note: Peripheral Nerve Imaging 3
  4. 4. Axonal outgrowth was measured Crush Degenerative Fragmentsin three groups of ten mice usingboth a standard fluorescencemicroscope and a Cellvizio. Thegraph in Figure 5 displays theresults. 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 DayEffect of Vincristine on Nerve (Figure 7). 15.Regeneration After a CrushThe next step in the development Fibered Confocal Fluorescence Microscopyof this model was to test theadministration of a neurotoxicdrug, such as vincristine.Vincristine, a chemotherapeuticmolecule, was administered at0.5 mg/kg in a one-shot intra-peritoneal injection on Day 1 afterthe crush. High doses of vincristineare known to induce peripheralneuropathy and transiently blocknerve regeneration.As depicted in both imagingmodalities (Figure 6) at Day 4after the crush, vincristine blocksthe regeneration process. Boththe Cellvizio and the standardfluorescence microscope show Figure 7 - Cellvizio measurement of the effect of vincristine on nerve regenerationnerve debris of degenerating axons after crush. Pink: Test group of four mice receiving an intra-peritoneal injection ofand no regrowing fibers. 0.5 mg/kg of vincristine at Day 1 after the crush. Orange: Control group of four mice receiving only saline.Application Note: Peripheral Nerve Imaging 4
  5. 5. 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 measurementAs demonstrated the images acquired using a Cellvizio provide a reliable approach to the imaging of theperipheral nervous system as validated by comparison with studies using standard fluorescence microscopy.Repetitive MeasurementsThe minimally invasive access in a living animal allows repetitive measurements in time, as opposed to a singlemeasurement session from one sacrificed mouse in regular microscopy, and a follow-up analysis of regenerationon the same animal.Time of MeasurementsIt 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 permeasurement 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.Application Note: Peripheral Nerve Imaging 5
  6. 6. Credits and ReferencesThis work was published in: Pierre Vincent, Uwe Maskos,Igor Charvet, Laurence Bourgeais, Luc Stoppini, NathalieLeresche, Jean-Pierre Changeux, Régis Lambert, PaoloMeda, Danièle Paupardin-Tritsch. “Live imaging ofneural structure and function by fibered fluorescencemicroscopy.” (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- 114882. 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):41-513. 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):530-54. 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):9-175. 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: 5-86. Paydarfar JA, Paniello RC (2001) Functional study of four neurotoxins as inhibitors of post-traumatic nerve regeneration. Laryngoscope 111: 844-850 VisualSonics Inc. T.1.416.484.5000 Toll Free (North America) 1.866.416.4636 Toll Free (Europe) +800.0751.2020 E. VisualSonics®, Vevo®, MicroMarker™, VevoStrain™, DEPO®, SoniGene™, RMV™, EKV® and Insight through In Vivo Imaging are trademarks™ of VisualSonics Inc.Application Note: Peripheral Nerve Imaging 6