Name: investigating mammalian axon regeneration: in vivo electroporation of adult mouse dorsal root ganglion
Uploaded: 2018-09-01T14:30:00
Description: Watch this Scientific Journal Video about Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion at JoVE.com

The study was funded (awarded to F-Q.Z.) by NIH (R01NS064288, R01NS085176, R01GM111514, R01EY027347), the Craig H. Neilsen Foundation and the BrightFocus Foundation.

All animal experiments were performed in accordance with the animal protocol approved by the Johns Hopkins Institutional Animal Care and Use Committee.
1. Materials and Reagents
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	<li>Animals
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		<li>Use six-week-old female CF1 mice weighing 30&#8211;35 g for the experiments. 
		NOTE: The mice were group-housed (5 mice per cage) in individually ventilated sterilized cages with 1/4&#8221; corn cob bedding and 2&#8221; square nestlets for nesting. The cages were m...

<ol>
	<li>Saijilafu, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K-GSK3+signalling+regulates+mammalian+axon+regeneration+by+inducing+the+expression+of+Smad1.">PI3K-GSK3 signalling regulates mammalian axon regeneration by inducing the expression of Smad1.</a> Nature Communications. 4, 2690 (2013).</li><li>Zhang, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt-independent+GSK3+inactivation+downstream+of+PI3K+signaling+regulates+mammalian+axon+regeneration.">Akt-independent GSK3 inactivation downstream of PI3K signaling regulates mammalian axon regeneration.</a> Biochemical and Biophysical Research Communications. 443 (2), 743-748 (2014).</li><li>Jiang, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-26a+supports+mammalian+axon+regeneration+in+vivo+by+suppressing+GSK3beta+expression.">MicroRNA-26a supports mammalian axon regeneration in vivo by suppressing GSK3beta expression.</a> Cell Death &amp; Disease. 6, 1865 (2015).</li><li>Krames, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+dorsal+root+ganglion+in+the+development+of+neuropathic+pain.">The role of the dorsal root ganglion in the development of neuropathic pain.</a> Pain Medicine. 15 (10), 1669-1685 (2014).</li><li>Salimzadeh, L., Jaberipour, M., Hosseini, A., Ghaderi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-viral+transfection+methods+optimized+for+gene+delivery+to+a+lung+cancer+cell+line.">Non-viral transfection methods optimized for gene delivery to a lung cancer cell line.</a> Avicenna Journal of Medical Biotechnology. 5 (2), 68-77 (2013).</li><li>Keeler, A. M., ElMallah, M. K., Flotte, T. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-138+and+SIRT1+form+a+mutual+negative+feedback+loop+to+regulate+mammalian+axon+regeneration.">MicroRNA-138 and SIRT1 form a mutual negative feedback loop to regulate mammalian axon regeneration.</a> Genes &amp; Development. 27 (13), 1473-1483 (2013).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240 (1), 237-246 (2001).</li><li>Saijilafu, E. M., Hur, F. 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Several surgical steps require particular attention. The L4 and L5 DRGs (location of somas), which dominate the sciatic nerve, need to be correctly identified and injected with gene constructs. Otherwise, the GFP-labeling will be absent in sciatic nerve axons. The iliac crests can be viewed as useful anatomical landmarks to pinpoint L4 and L5 DRGs. In most mice, the facet joint between L5 and L6 vertebrae is proximate to iliac crests12. Alternatively, L3 DRG can be chosen instead of L5, especially...

Injuries in the nervous system caused by neural trauma or various neurodegenerative diseases usually result in defects in motor, sensory and cognitive functions. Recently, much effort has been devoted to regenerative potency re-establishment in adult neurons to restore the physiological functionsof injured neurons1,2,3. Sensory neurons in the DRG are a cluster of nerve cells that convey different sensory stimuli, such as pain, temperature, touch, or body posture, to the brain. Each of these neurons is pseudo-unipolar and contains a single axon that bifurcates with one branch extending toward the periphery and the other branch heading toward the spinal cord4. The adult sensory neurons in DRGs are among a few mature mammalian neurons known to regenerate their axons actively after injury. Hence, injuries of sensory axons have been extensively employed as a crucial model to study the mechanisms of axonal regeneration in vivo.
In vivo gene transfection techniques, which are usually less time-consuming to set up and more flexible than using transgenic animals, have been playing essential roles in studying the functions of genes and signaling pathways in the nervous system. The main techniques can be categorized into two approaches: instrument-based and virus-based5. Viral-based in vivo gene delivery in adult neurons can provide precise spatiotemporal manipulation of gene expression6. However, labor-intensive processes are involved in viral-based methods, such as the production and purification of viral particles containing the desired gene. In addition, many viral vectors could activate the immune system of the host, which may interfere with the data acquisition, data analysis and possibly mislead the interpretation of experimental results. Electroporation, a typical instrument-based transfection approach, uses an electrical pulse to increase the permeability of cell and nuclear membranes transiently, which favors the influx of gene vectors or small RNA oligos from space outside of the cells7. In vitro electroporation is widely recognized as a transient but highly efficient strategy for manipulating targeted gene expression in many cell types. Although in vivo electroporation only leads to transient gene expression with low transfecting efficiency compared to viral vectors, it has various advantages over viral approaches. For instance, it can be applied to almost all tissues and cells7,8,9. In addition, either plasmids encoding genes-of-interest or small RNA oligos (e.g., siRNAs, microRNAs) against certain transcripts can be injected into the target tissue directly and then electrically pulsed, which make the procedure less labor- and time- consuming. Moreover, transfecting multiple plasmids and RNA oligos simultaneously with single electroporation is possible.
We have established an in vivo electroporation approach to manipulate gene expression in adult mouse sensory neurons and successfully applied and validated such approach in numerous pioneer studies1,2,3,8,10. Here, we present a detailed protocol to facilitate the usage of this approach for future studies of mammalian axon regeneration.

<table border="0" cellpadding="0" cellspacing="0" style="width:1039px;" width="1037">
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	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:335px;">ECM 830 Square wave electroporation system</td>
			<td style="width:232px;">BTX Harvard Apparatus</td>
			<td style="width:121px;">45-0052</td>
			<td style="width:351px;">For in vivo electroporation</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:335px;">Tweezertrodes electrodes</td>
			<td style="width:232px;">BTX Harvard Apparatus</td>
			<td style="width:121px;">45-0524</td>
			<td>For in vivo electroporation, 1 mm flat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Picospritzer III</td>
			<td style="width:232px;">Parker Instrumentation</td>
			<td style="width:121px;">1096</td>
			<td>Intracellular Microinjection Dispense Systems</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass Capillary Puller</td>
			<td style="width:232px;">NARISHIGE</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments, Inc.</td>
			<td style="width:121px;">1902328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Stereo Dissection Microscope</td>
			<td style="width:232px;">Leica</td>
			<td style="width:121px;">M80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Microsurgery Rongeur</td>
			<td style="width:232px;">F.S.T</td>
			<td style="width:121px;">16221-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Microsurgery Forceps</td>
			<td style="width:232px;">FST by DUMONT, Switzerland</td>
			<td style="width:121px;">11255-20</td>
			<td>Only for sciatic nerve crush</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass Capillary</td>
			<td style="width:232px;">World Precision Instruments, Inc.</td>
			<td style="width:121px;">TW100-4</td>
			<td>10 cm, standard wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Tape</td>
			<td style="width:232px;">Fisherbrand</td>
			<td style="width:121px;">15-901-30</td>
			<td>For fixing the mouse on the corkboard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2, 2, 2-Tribromoethanol (Avertin)</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">T48402</td>
			<td>Avertin stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-methyl-2-butanol</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">152463</td>
			<td>Avertin stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">siRNA Fluorescent Universal Negative Control #1</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">SIC003</td>
			<td>Non-target siRNA with fluorescence</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:335px;">microRNA Mimic Transfection Control with Dy547</td>
			<td style="width:232px;">Dharmacon</td>
			<td style="width:121px;">CP-004500-01-05</td>
			<td>Non-target microRNA with fluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Plasmids preparation kit</td>
			<td style="width:232px;">Invitrogen Purelink</td>
			<td style="width:121px;">K210016</td>
			<td>GFP-coding plasmid preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fast Green Dye</td>
			<td style="width:232px;">Millipore-Sigma</td>
			<td style="width:121px;">F7252</td>
			<td>For better visualization of the DRG outline during injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Ketamine</td>
			<td style="width:232px;">Putney, Inc</td>
			<td style="width:121px;">NDC 26637-731-51</td>
			<td>Anesthesia induction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Xylazine</td>
			<td style="width:232px;">AnaSed</td>
			<td style="width:121px;">NDC 59399-110-20</td>
			<td>Anesthesia induction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Acetaminophen</td>
			<td style="width:232px;">McNeil Consumer Healthcare</td>
			<td style="width:121px;">NDC 50580-449-36</td>
			<td>Post-surgical pain relief</td>
		</tr>
	</tbody>
</table>

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.

To quantify the cytotoxicity of the current protocol and to validate that transfection rate of in vivo DRG electroporation is high enough, we injected and electroporated fluorescently-tagged microRNA or siRNA into L4 and L5 DRGs. The detached DRGs were processed through cryo-sectioning and immunohistochemistry (Figure 1A-B). When estimating the cell survival rate after injection and electroporation, the intact DRGs from L4 and L5 wer...

Watch this Scientific Journal Video about Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion at JoVE.com

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.

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 ...

This method provides an in-vivo gene transfection technique to manipulate gene expression in adult mouse sensory neurons for future studies of mammalian exo-regeneration. The main advantage of this technique is that it's less labor and time consuming, and can both overexpress and knock down the target genes simultaneously and separately. To begin this procedure, mark both sides of the iliac crest of an anesthetized mouse with a fine marker.Draw a line connecting two points of the iliac crest to facilitate the identification of L5 DRGs positions. Then, make a three centimeter incision along the midline of the lower back with micro scissors. Afterward, detach the paraspinous muscles, such as the musculus multifidus and the musculus longissimus lumborum, from the L3 to S1 spinous processes, and expose the fascia joint of the L4-5 and L5-6.Next, use a micro rongeur to remove the fascia joints of the L4-5 and L5-6. Subsequently, remove the left neural arch of L4 and L5, to expose the dorsal side of the DRGs. For DRG injection, load the DNA plasmids or RNA oligos into the glass capillary pipette.Then, insert the tip of the capillary glass pipette, carefully, into the DRG, and gradually inject one microliter solution of DNA plasmids or RNA oligos using the intracellular micro injection dispense system. For electroporation, pinch the target DRG with the electrodes gently, and apply five square electric pulses with the electroporation system. Subsequently, close the muscle, then the skin layers with nylon sutures.Two or three days after the DRG electroporation, anesthetize the mouse intraperitoneally, tape its limbs on the cork board, and make a one centimeter incision 0.5 centimeters to the left side along the midline. Next, cut the muscles, such as gluteus maximus muscle and piriformis muscle, longitudinally. Then, expose the segment of the sciatic nerve between the greater sciatic foramen, and the sciatic notch.Crush the nerve with microsurgery forceps for 12 seconds, and mark the crush site with a nylon epineural suture. Afterward, close the muscle and skin layers with nylon suture. After profusion, separate the DRGs together with the nerve roots and sciatic nerve, carefully, with micro scissors and micro forceps under the dissection microscope.Then, transfer the sciatic nerve directly in 4%PFA overnight at four degrees celsius. To image and measure the fluorescence labeled sensory axons under the dissection microscope, strip off the attached tissue and membrane on the fixed sciatic nerve with micro scissors and micro forceps, carefully. Then, wash the nerve with PVS three times.Next, place the sciatic nerve on a slide and keep it straight. Add 80 microliters of anti-fade solution around the nerve, then lay a cover slip on it. Flatten the whole mounted tissue with pressure.Afterward, place the flattened tissue under the inverted epifluorescence microscope, equipped with an accessory for mosaic acquisition and image processing. When measuring the length of regenerated axons, trace all identifiable fluorescently labeled axons in the sciatic nerve from the crush site to the distal axon ends. When applying the current, in-vivo electroporation method to study axonal regeneration, the sciatic nerve was flattened and imaged.Every regenerated axon with distinctive trajectory and recognizable distal axon end, can be traced from the crush site indicated by the suture knot. The white arrowhead, arrow, and star, indicate three distinctive axon ends, all of which extend from the crush site. Additionally, the DRGs were electroporated with EGFP plasmids, and the spinal cord was harvested.The EGFP labeled, dorsal column axons, in the spinal cord, were imaged and identified in both the longitudinal and sagittal projecting images. Here is a detailed view of axons in the dorsal column, and here is a detailed view of axons in the the dorsal root entry zone. This image shows the sagittal projection of the EGFP labeled axons within the dorsal column.Alternatively, the confocal images can be processed with microscopy visualization software to reconstruct a 3D image. While attempting this procedure, it's important to remember to position the L4 and the L5 DRGs correctly, and to make a knot on the dural membrane without impaling the sciatic nerve, while labeling the crush site with a suture knot. After its development, this technique pave the way for researchers in the field of axon regeneration to explore genes that are required for PNS, natural axon regeneration, or genes that can further promote regeneration in adult mouse in-vivo.

Research

JoVE Journal

Neuroscience

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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 ...

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 ...

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 ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

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 ...

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

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 ...

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...