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 100 &mu;g/ml Poly-D-Lysine and 10 &mu;g/ml Laminin working solution is added onto each coverslip and the culture plate is transferred into a 37 &deg;C incubator. After 1-2 hr, the coating solution is removed and the coverslips are washed 3 times with sterile 1X PBS.</li>
	<li>To prepare the culture medium, the Minimum Essential Medium (MEM) is supplemented with 5% fetal bovine serum (FBS), 1X Penicillin-Streptomycin solution (500 units of penicillin and 500 &mu;g of streptomycin), 1X GlutaMAX-I supplement, and the antimitotic reagents containing 20 &mu;M 5-fluoro-2-deoxyuridine and 20 &mu;M uridine (FDU/R). For the serum-free medium, the FBS is replaced with the supplement B27.</li>
	<li>The collagenase A solution is prepared by dissolving collagenase A powder with MEM to make a working concentration of 1 mg/ml. For the trypsin, the 1X TrypLE Express is used.</li>
</ol>
2. Dissection and Harvest of Adult Mouse DRG Neurons
<ol>
	<li>After euthanizing the 6- to 10-weeks old adult mouse, remove the dorsal skin and cut off the whole spinal column. Wash the removed spinal column with 1 X PBS 2-3 times.</li>
	<li>Pin the removed spinal column to the dissecting plate with ventral side up, and carefully removes muscles to expose the sensory nerves under a dissecting microscope. The nerves connected to DRGs at the lumber level are the thickest and easy to locate. Therefore, for most experiments only the lumbar DRGs are harvested.</li>
	<li>Cut through each vertebra along the centerline with small scissors and carefully remove the intervertebral discs. Using forceps to split each vertebra to expose the spinal cord.</li>
	<li>To dissect out the lumber DRGs, lift up each lumber nerve with forceps and trace toward the spinal cord to locate the associated lumbar DRGs (from L1 to L6).</li>
	<li>Cut off each DRG from attached peripheral nerve, dorsal and ventral roots with spring scissors, and store it in microfuge tube with MEM medium placed on ice. More DRGs at different spinal levels (e.g. thoracic DRGs) can be dissected out in a similar way when necessary.</li>
</ol>
3. Digestion and Dissociation of Adult Mouse DRG Neurons
<ol>
	<li>After collecting all the dissected DRGs, replace the MEM medium with 1 ml collagenase A solution and incubate the microfuge tube at 37 &deg;C for 90 min.</li>
	<li>Replace collagenase A solution with 500 &mu;l 1X TrypLE Express solution and incubate at 37 &deg;C for 15-20 min.</li>
	<li>Remove the TrypLE Express solution and wash the DRGs with 1 ml prepared culture medium (containing 5% FBS) for 3 times.</li>
	<li>Add 600 &mu;l culture medium and gently pipette up and down to triturate the tissues for 20-30 times using a 1 ml blue, graduated pipette tip.</li>
	<li>After trituration, allow the non-dissociated tissues to settle down to the bottom of the microfuge and transfer the cell suspension to a 10 ml sterile tube. Add another 600 &mu;l culture medium and repeat the trituration step until most tissues are dissociated. The obtained cell suspension contains both neurons and non-neuronal cells. In most cases, cells from 6 DRGs (~5 x104) are used for one electroporation reaction.</li>
</ol>
4. Genetic Manipulation of Neurons via Electroporation
<ol>
	<li>Prepare the transfection solution by mixing the Amaxa nucleofection solution for mouse neurons with DNA plasmids (~10 &mu;g) or siRNA oligos (~0.2 nmol) to make a final volume of 100 &mu;l for each transfection.</li>
	<li>Centrifuge the dissociated cells at 680 rpm for 7 min at room temperature, and discard supernatant as much as possible. Add the prepared transfection solution and gently pipette up and down 3-4 times with 200 &mu;l pipette tips to re-suspend the cells.</li>
	<li>Transfer the cell suspension mixed with the transfection solution to the electroporation cuvette and electroporate the cells using the Amaxa Nucleofector system program G-013.</li>
	<li>After electroporation, immediately add 500 &mu;l of pre-warmed (37 &deg;C) culture medium containing FBS to the cuvette and transfer all the solution (~600 &mu;l) into the coated culture plate at desired cell densities. For experiment that requires re-suspension and re-plating, neurons are cultured directly on the plastic culture dish at high density (10000-20000 cells/well). For experiment that directly examines axon growth, neurons are plated onto coated glass coverslips at lower density (3000-5000 cells/well). Place the culture plate into the incubator (37 &deg;C, 5% CO2).</li>
	<li>Four hr after plating when neurons have attached to the substrates, gently replace the culture medium (which contains the Amaxa nucleofection solution) with 500 &mu;l fresh and pre-warmed culture medium, and return the plate to the incubator for additional culture (37 &deg;C, 5% CO2). Both culture medium containing FBS or the serum-free medium can be used at this step.</li>
</ol>
5. Culturing Adult DRG Neurons for Axon Growth Analysis
<ol>
	<li>For neurons transfected with DNA plasmids, the expression of gene-of-interest (e.g. EGFP) can be observed as early as a few hr after electroporation. For neurons transfected with siRNAs, we usually wait 3-4 days to allow sufficient depletion of the endogenous proteins. The cultured neurons can be either directly fixed for axon growth analysis at various time points (1-4 days after electroporation) or re-suspended and re-plated to analyze axon regrowth (see below).</li>
	<li>For RNAi-mediated loss-of-function studies, the cultured neurons can be re-suspended and re-plated to allow axons to regrow from neurons, in which the targeted proteins are already depleted. To do so, replace the old culture medium of the high-density cultured neurons with 1 ml pre-warmed fresh medium and pipette gently up and down for 6-10 times to re-suspend the attached neurons from the culture plate. Because non-neuronal cells (e.g. Schwann cells) attach much tighter to the culture dish than neurons, the re-suspended cells are mostly neurons.</li>
	<li>Transfer the re-suspended neurons into a microfuge tube and gently triturate 10-15 times to re-dissociate the cell clumps into the single cell suspension.</li>
	<li>Re-plate the re-dissociated neurons onto newly prepared coverslips at low density (3000-5000 cells/well) and culture overnight (16-24 hr).</li>
</ol>
6. Fixation, Immuno-staining, and Fluorescence Imaging
<ol>
	<li>Aspirate the medium and add 4% paraformaldehyde (PFA) (200 &mu;l/well) to fix the cells for 15-20 min at room temperature. The fixed cells are then washed with 1X PBS for 3 times.</li>
	<li>Aspirate the PBS and add the blocking solution (1% bovine serum albumin, 0.1% Triton X-100, and 2.0% normal goat serum in 1X PBS) to the fixed neurons for 60 min at room temperature.</li>
	<li>To label axons, the neurons are immuno-stained with anti-neurofilaments or anti-&beta;III tubulin antibody. To do so, place a 30&mu;l drop of primary antibody solution (1:1200 dilution for the -&beta;III tubulin antibody) on a parafilm for each coverslip. Invert the coverslips and place them onto the primary antibody solution for 60 min at room temperature.</li>
	<li>Return coverslips to the original culture plate and wash them with 1X PBS for 3 times.</li>
	<li>Repeat the same procedure for the secondary antibody.</li>
	<li>Wash the coverslips with distilled water for 3 times, and then mount coverslips on glass slides with the mounting solution (e.g. ProLong Gold Antifade).</li>
	<li>The stained neurons can be imaged with any fluorescence microscopy system equipped with a digital camera. The axon lengths are measured and analyzed with imaging analysis software.</li>
</ol>
7. Representative Results
In the absence of any added extracellular growth factors, the adult DRG neurons usually start to grow axons 48 hr after first plating. The axons often show branched morphologies (Figure 1). In contrast, the re-plated neurons start to extend axons only a few hr after plating, and the axons elongate with much reduced branching (Figure 2). These results suggest that re-plated neurons share similar properties to those of conditioning lesioned neurons. By using this approach, we have recently performed loss-of-function studies to examine the role of axon regeneration associated transcription factor c-Jun in axon growth from adult DRG neurons in vitro. The results showed that electroporation of a group of 4 different siRNAs targeting different regions of c-Jun (ON-TARGETplus) markedly reduced the protein levels of c-Jun in adult DRG neurons 3 days after transfection (Figure 3)9. When the neurons were re-plated and cultured overnight, axon growth from c-Jun knockdown neurons was significantly reduced (Control: 348.37&plusmn; 16.21mm; si-c-Jun: 262.32&plusmn;15.69 &mu;m, Figure 3)9. These results indicate that cultured adult DRG neurons provide a useful model system to study axon growth from adult neurons.
<img alt="Figure 1" src="/files/ftp_upload/4141/4141fig1.jpg" /> 
Figure 1. Adult mouse DRG neurons cultured at low density for 3 days. The neurons were stained with anti--&beta;III tubulin antibody. Note that most axons show branched morphologies. Scale bar: 125 &mu;m.
<img alt="Figure 2" src="/files/ftp_upload/4141/4141fig2.jpg" /> 
Figure 2. Re-plating adult mouse DRG neurons after 3 days in culture. (A) Adult DRG neurons cultured at high density for 3 days. (B) Re-plated adult DRG neurons were cultured at low density for overnight. Note that most axon show elongated morphologies with little axon branching. Scale bar: 250 &mu;m in A and 125 &mu;m in B.
<img alt="Figure 3" src="/files/ftp_upload/4141/4141fig3.jpg" /> 
Figure 3. Role of c-Jun in axon growth from adult DRG neurons in vitro. (A) Western blot analysis of c-Jun in adult mouse DRG neurons after electroporation of c-Jun siRNAs. The result shows markedly reduced level of c-Jun. (B) Control neurons transfected with EGFP grew long axons after re-plating and overnight culture. (C) Co-transfection of c-Jun siRNAs and EGFP impaired axon growth from adult DRG neurons after re-plating and overnight culture. Red: Tuj-1 staining; Green: EGFP. Scale bar: 125 &mu;m. These results have been published in Saijilafu et al.9.

<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>

No conflicts of interest declared.

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

26.5K Görüntüleme.  Johns Hopkins University School of Medicine. Bir In vitro modeli açıklanmıştır. Metodu genetik manipülasyon geçiren nöronlarla akson yeniden büyümesini izin vermek için bir re-suspension/re-plating adım içerir. Bu yaklaşım, RNAi bazlı protein Knockdown kullanarak akson rejenerasyonu kaybı fonksiyon-çalışmaları için özellikle yararlıdır.

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

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...