We thank Dr. Alan Tessler for comments and editorial help. This work was supported by NIH NS062320.

1. Microscope set up and imaging preparation
<ol>
 <li>Our imaging set up consists of a Leica MZ16 fluorescent stereomicroscope with a fast shutter and a cooled CCD camera controlled by Metamorph software.</li>
 <li>Prepare a thermostatically controlled heating pad and adjust output to 32.5&deg;C to maintain the animal's body temperature during and following surgery.</li>
 <li>Warm sterile Ringer's solution or artificial cerebrospinal fluid (ACSF) to 32.5&deg;C in advance for irrigation of spinal cord during surgery.</...

<ol>
	<li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+the+neuromuscular+junction.">Watching the neuromuscular junction.</a> J Neurocytol. 32, 767-775 (2003).</li><li>Bishop, D. L., Misgeld, T., Walsh, M. K., Gan, W. B., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+branch+removal+at+developing+synapses+by+axosome+shedding.">Axon branch removal at developing synapses by axosome shedding.</a> Neuron. 44, 651-661 (2004).</li><li>Balice-Gordon, R. J., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+visualization+of+the+growth+of+pre-+and+postsynaptic+elements+of+neuromuscular+junctions+in+the+mouse.">in vivo visualization of the growth of pre- and postsynaptic elements of neuromuscular junctions in the mouse.</a> J Neurosci. 10, 894-908 (1990).</li><li>Trachtenberg, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+imaging+of+experience-dependent+synaptic+plasticity+in+adult+cortex.">Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.</a> Nature. 420, 788-794 (2002).</li><li>Pan, F., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+dendritic+spine+development+in+the+mouse+cortex.">Two-photon imaging of dendritic spine development in the mouse cortex.</a> Dev Neurobiol. 68, 771-778 (2008).</li><li>Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+synaptic+plasticity+and+pathology+in+the+living+mouse+brain.">Two-photon imaging of synaptic plasticity and pathology in the living mouse brain.</a> NeuroRx. 3, 489-496 (2006).</li><li>Kerschensteiner, M., Schwab, M. E., Lichtman, J. W., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+axonal+degeneration+and+regeneration+in+the+injured+spinal+cord.">in vivo imaging of axonal degeneration and regeneration in the injured spinal cord.</a> Nat Med. 11, 572-577 (2005).</li><li>Misgeld, T., Nikic, I., Kerschensteiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+single+axons+in+the+mouse+spinal+cord.">in vivo imaging of single axons in the mouse spinal cord.</a> Nat Protoc. 2, 263-268 (2007).</li><li>Davalos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+in+vivo+imaging+of+densely+populated+glia,+axons+and+blood+vessels+in+the+mouse+spinal+cord+using+two-photon+microscopy.">Stable in vivo imaging of densely populated glia, axons and blood vessels in the mouse spinal cord using two-photon microscopy.</a> J Neurosci Methods. 169, 1-7 (2008).</li><li>Maio, D. D. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+dorsal+root+regeneration:+Rapid+immobilization+and+presynaptic+differentiation+at+the+CNS/PNS+border.">in vivo imaging of dorsal root regeneration: Rapid immobilization and presynaptic differentiation at the CNS/PNS border.</a> Journal of Neuroscience. 31, 4569-4582 (2011).</li></ol>

No conflicts of interest declared.

Imaging DR regeneration directly in living mice is particularly challenging because it requires a substantial dorsal laminectomy to monitor axon growth over a wide area followed by multiple invasive surgical and anesthetic procedures in subsequent imaging sessions. Several strategies helped to overcome these challenges. First, successful imaging required reducing mouse mortality (approximately 25%) by minimizing the duration of anesthesia and bleeding, and by meticulous post-op care. The mortality was also reduced by usi...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>H line thy1-YFP (2-4 months old, either sex) </td>
			<td>Jackson Laboratory (Bar Harbor, ME)</td>
			<td>003782</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine (AnaSed injection, sterile solution) </td>
			<td>Lloyd Laboratories, (Shenandoah, LA)</td>
			<td>4811</td>
			<td>8 mg/kg</td>
		</tr>
		<tr>
			<td>Ketamine (Ketamine hydrochloride injection, USP) </td>
			<td>Hospira, Inc. (Lake Forest, IL)</td>
			<td>2051</td>
			<td>120 mg/kg</td>
		</tr>
		<tr>
			<td>Buprenorphine (Buprenex injectable) (0.05 mg/kg)</td>
			<td>Reckitt Benckiser Pharmaceuticals Inc.(Richmond, VA)</td>
			<td>7571</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Small animal hair clippers </td>
			<td>Oster Professional, (McMinnville, TN)</td>
			<td>76059-030</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hair removal lotion</td>
			<td>Church &amp; Dwight Co (Princeton, NJ)</td>
			<td>NAIR with Baby Oil</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gauze sponges</td>
			<td>Fisher Scientific, (Pittsburgh, PA)</td>
			<td>22-362-173</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cotton-tipped swabs</td>
			<td>Fisher Scientific, (Pittsburgh, PA)</td>
			<td>14-960-3Q</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 mL syringes</td>
			<td>Becton, Dickson and Company Franklin Lakes, NJ)</td>
			<td>309602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Subcutaneous (Sub-Q) needles, 26ga.</td>
			<td>Becton, Dickson and Company (Franklin Lakes, NJ)</td>
			<td>305115</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spring scissors and forceps</td>
			<td>Fine Science Tools, (Foster City, CA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2.5-mm curved rongeurs </td>
			<td>Fine Science Tools, (Foster City, CA)</td>
			<td>16221-14</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lactated Ringer's Injection USP</td>
			<td>B. Braun Medical Inc., (Irvine, CA)</td>
			<td>BBR-L7502</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile saline solution</td>
			<td>APP Pharmaceuticals, (Schaumburg, IL)</td>
			<td>918610</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thin synthetic matrix membrane (Biobrane)</td>
			<td>Bertek Pharmaceuticals, (Morgantown, WV)</td>
			<td>62794-096-251</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Artificial dura</td>
			<td>Gore Preclude MVP Dura Substitute, W.L. Gore and Associates, (Flagstaff, AZ)</td>
			<td>1MVP40</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5-0 silk sutures</td>
			<td>Ethicon, Inc. (Somerville, NJ)</td>
			<td>K-580</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Wound clips</td>
			<td>Perfect &#x2013; Ets Bruneau, (Burnea, France)</td>
			<td>A75</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescent stereomicroscope</td>
			<td>Leica Microsystems, (Wetzlar, Germany)</td>
			<td>MZ16</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Hamamatsu, (Bridgewater, NJ)</td>
			<td>ORCA-Rx2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>World Precision Instruments (Sarasota, FL)</td>
			<td>ATC 1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Metamorph software</td>
			<td>Molecular Devices, (Sunnyvale, CA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Photoshop</td>
			<td>Adobe Systems, San Jose, CA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

live imaging of dorsal root axons after rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

Watch this Scientific Journal Video about Live Imaging of Dorsal Root Axons after Rhizotomy at JoVE.com

Live Imaging of Dorsal Root Axons after Rhizotomy

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

live-imaging-of-dorsal-root-axons-after-rhizotomy

Research

JoVE Journal

Neuroscience

13.7K ビュー.  Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology. アン in vivoでイメージングプロトコルが記述されています。手順は、広視野蛍光顕微鏡とTHY1 - YFPトランスジェニックマウスを利用し、CNSのインタフェースを持つPNSと軸索の相互作用が4cm以上の軸索再生の繰り返しイメージングを可能にする。