This work was supported by grants to F. Z. from NIH (R01NS064288) and the Craig H. Neilsen Foundation.

1. Preparation of Coverslips, Culture Medium, and Digestion Enzymes
<ol>
	<li>The 12-mm round #1 glass coverslips are used for the neuronal culture. The coverslips are cleaned with 10% HCL overnight followed by ultrasonic wash with distilled and deionized water for 3 times (20 min/time). The cleaned coverslips are stored in 70% ethanol for future use. Before each experiments, the coverslips are air dried and placed into the culture plate.</li>
	<li>To coat the dried coverslips, 100 &mu;l of coating solution containing ...

<ol>
	<li>Yiu, G., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+inhibition+of+CNS+axon+regeneration.">Glial inhibition of CNS axon regeneration.</a> Nat. Rev. Neurosci. 7, 617-627 (2006).</li><li>Liu, K., Tedeschi, A., Park, K. K., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Intrinsic+Mechanisms+of+Axon+Regeneration.">Neuronal Intrinsic Mechanisms of Axon Regeneration.</a> Annu. Rev. Neurosci. , (2010).</li><li>Zhou, F. Q., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+control+of+developmental+and+regenerative+axon+growth.">Intracellular control of developmental and regenerative axon growth.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 361, 1575-1592 (2006).</li><li>Neumann, S., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+dorsal+column+fibers+into+and+beyond+the+lesion+site+following+adult+spinal+cord+injury.">Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury.</a> Neuron. 23, 83-91 (1999).</li><li>Liu, R. Y., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+signaling+pathways+mediate+regenerative+versus+developmental+sensory+axon+growth.">Different signaling pathways mediate regenerative versus developmental sensory axon growth.</a> J. Neurosci. 21, RC164 (2001).</li><li>Zhou, F. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+support+regenerative+axon+assembly+over+CSPGs+by+an+ECM-integrin-independent+mechanism.">Neurotrophins support regenerative axon assembly over CSPGs by an ECM-integrin-independent mechanism.</a> J. Cell Sci. 119, 2787-2796 (2006).</li><li>Zou, H., Ho, C., Wong, K., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axotomy-induced+Smad1+activation+promotes+axonal+growth+in+adult+sensory+neurons.">Axotomy-induced Smad1 activation promotes axonal growth in adult sensory neurons.</a> J. Neurosci. 29, 7116-7123 (2009).</li><li>Zhou, F. Q., Zhou, J., Dedhar, S., Wu, Y. H., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF-induced+axon+growth+is+mediated+by+localized+inactivation+of+GSK-3beta+and+functions+of+the+microtubule+plus+end+binding+protein+APC.">NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC.</a> Neuron. 42, 897-912 (2004).</li><li>Saijilafu, E. M., Hur, F. Q., Zhou, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+axon+regeneration+via+in+vivo+electroporation+of+adult+mouse+sensory+neurons.">Genetic dissection of axon regeneration via in vivo electroporation of adult mouse sensory neurons.</a> Nat. Commun. 2, 543-54 (2011).</li><li>Costigan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicate+high-density+rat+genome+oligonucleotide+microarrays+reveal+hundreds+of+regulated+genes+in+the+dorsal+root+ganglion+after+peripheral+nerve+injury.">Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury.</a> BMC Neurosci. 3, 16 (2002).</li><li>Xiao, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+gene+expression+profile+of+dorsal+root+ganglion+in+the+rat+peripheral+axotomy+model+of+neuropathic+pain.">Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 8360-8365 (2002).</li><li>Michaelevski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+to+transcription+networks+in+the+neuronal+retrograde+injury+response.">Signaling to transcription networks in the neuronal retrograde injury response.</a> Sci. Signal. 3, ra53 (2010).</li></ol>

Adult DRG neurons regenerate their axons robustly after peripheral nerve injury in vivo and in vitro, thus providing a useful system to study axon regeneration in adult animals. In vitro culture of adult DRG neurons is becoming a widely used method to investigate the molecular mechanisms by which axon regeneration is regulated. The in vitro procedure of culturing adult mouse DRG neurons presented here enables rapid and effective genetic study of regenerative axon growth. The re-suspens...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>MEM</td>
 <td>Invitrogen</td>
 <td>11090-081</td>
 </tr>
 <tr>
 <td>Poly-D-Lysine hydrobromide </td>
 <td>Sigma -Aldrich</td>
 <td>P6407</td>
 </tr>
 <tr>
 <td>Laminin </td>
 <td>Invitrogen</td>
 <td>23017-015</td>
 </tr>
 <tr>
 <td>5-fluoro-2-deoxyuridine</td>
 <td>Sigma -Aldrich</td>
 <td>F0503</td>
 </tr>
 <tr>
 <td>Uridine </td>
 <td>Sigma -Aldrich</td>
 <td>U3003</td>
 </tr>
 <tr>
 <td>Collagenase A </td>
 <td>Roche </td>
 <td>10103578001</td>
 </tr>
 <tr>
 <td>TrypLE Express </td>
 <td>Invitrogen</td>
 <td>12604-013</td>
 </tr>
 <tr>
 <td>Fetal bovine serum </td>
 <td>Invitrogen</td>
 <td>10270-098</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin (100X)</td>
 <td>Invitrogen</td>
 <td>15140-122</td>
 </tr>
 <tr>
 <td>GlutaMAX-I (100X) </td>
 <td>Invitrogen</td>
 <td>35050-038</td>
 </tr>
 <tr>
 <td>Glass coverslips (#1)</td>
 <td>Electron Microscopy sciences</td>
 <td>72196-12</td>
 </tr>
 <tr>
 <td>24 well cell culture plate</td>
 <td>Becton Dickinson</td>
 <td>35-3047</td>
 </tr>
 <tr>
 <td>1X PBS</td>
 <td>Mediatech</td>
 <td>21-040-CV</td>
 </tr>
 <tr>
 <td>Sterile, distilled and deionized water </td>
 <td>Mediatech</td>
 <td>25-055-CV</td>
 </tr>
 <tr>
 <td>Nucleofector and electroporation Kits for Mouse Neurons</td>
 <td>Lonza</td>
 <td>VPG-1001</td>
 </tr>
 <tr>
 <td>ON-TARGETplus siRNA against c-Jun</td>
 <td>Dharmacon</td>
 <td>L-043776</td>
 </tr>
 <tr>
 <td>Anti--&beta;III tubulin antibody (Tuj-1) </td>
 <td>Covance</td>
 <td>MMS-435P </td>
 </tr>
 <tr>
 <td>ProLong Gold Antifade mounting solution</td>
 <td>Invitrogen</td>
 <td>P36930</td>
 </tr>
 </tbody>
</table>

genetic study of axon regeneration with cultured adult dorsal root ganglion neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

Watch this Scientific Journal Video about Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons at JoVE.com

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

genetic-study-axon-regeneration-with-cultured-adult-dorsal-root

Research

JoVE Journal

Neuroscience

26.4K visualizações.  Johns Hopkins University School of Medicine. Um In vitro Modelo para o estudo genético de regeneração do axônio usando cultivadas adultos do mouse dorsal neurônios do gânglio da raiz é descrito. O método inclui um passo re-suspension/re-plating para permitir re-crescimento axonal a partir de neurónios submetidos a manipulação genética. Esta abordagem é especialmente útil para a perda de função de estudos de regeneração axonal utilizando RNAi baseado knockdown proteína.