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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Collect Lumbar DRG from Experimental Rats
<ol>
	<li>Use 2- to 3-week-old Sprague-Dawley (SD) rats for lumbar DRG collection. 
	NOTE: DRG neurons collected from rats over 4 weeks of age do not grow well under the culture conditions described herein.</li>
	<li>Sterilize all surgical instruments in an autoclave.</li>
	<li>Anesthetize the rat with a 1:1 mixture of tiletamine and zolazepam (20 mg/kg; intraperitoneal injection (IP)) and wait until the animal shows no foot-withdrawal response in a toe-pinch test. 
	NOTE: Different anesthesia strategies can be used successfully in this protocol.</li>
	<li>Sacrifice the rat by decapitation with a commercial guillotine.</li>
	<li>Use the guillotine to isolate the body trunk of the rat between the forelimb and femur. See Figure 1A for a diagram of the region to be collected. 
	NOTE: The caudal cut line should be just rostral to the femur. The lumbar L6 DRG will be excised if the cut site is too high in the spinal column.</li>
	<li>Cut along the sternum and remove all organs/tissues with dissection scissors (Figure 2A-a).</li>
	<li>Cut along the side of trunk to collect the dorsal part of the rat and remove the skin. See Figure 1B for a photograph of the dissected dorsal trunk.</li>
	<li>Prepare the tissue on ice before collecting DRG. Clean the fur and blood from gloves and sterilize them with 75% ethanol before proceeding to the next step.</li>
	<li>Remove the muscles covering the lumbar spine. First, make two cuts along the sides of the spinal column (left and right) and one lateral cut to mark the rostral extent of the lumbar spine. Then, remove the dorsal muscles of the spine with bone cutting forceps (Figure 2A-b).</li>
	<li>Remove the dorsal portion of the vertebrae with bone cutting forceps and expose the spinal cord.</li>
	<li>Remove the spinal cord with dissection scissors (Figure 2A-c) and forceps (Figure 2A-d).</li>
	<li>Identify the lumbar DRG by counting vertebrae from the last rib (Thoracic Vertebra 13). See Figure 1C for a diagram of the vertebrae positions.</li>
	<li>Collect the bilateral lumbar DRG (L1-L6) with micro-scissors (Figure 2A-f) into a 35-mm culture dish with 2 mL ice-cold serum-free medium. Remove the neuronal fibers (as indicated in Figure 1C) from connecting DRG, then transfer it into the culture dish to improve the purity of the cultures. 
	NOTE: The collected DRG can be kept in medium on ice for about 1 h. Meanwhile, multiple rats can be euthanized to create a larger pool of DRG.</li>
</ol>
2. Primary Culture of Rat Lumber DRG
NOTE: The following steps should be performed in a laminar flow hood.
<ol>
	<li>Prepare culture medium containing 10% fetal bovine serum, 100 mM sodium pyruvate, and 1x penicillin/streptomycin in 1x DMEM-F12.</li>
	<li>Coat the cell-culture treated 24-well plate with 200 &#181;g/mL poly-L-lysine for 2 h then wash with sterilized water.</li>
	<li>Pre-incubate the culture dish with 1 mL culture medium in a 37 &#176;C CO2 incubator before use for least 30 min.</li>
	<li>Transfer the DRG-containing 35 mm dish into a laminar hood and wash the DRG with serum-free medium 3 times by pipette. 
	NOTE: The outside of the dish should be cleaned with 75% ethanol before transferring into the hood. The 35 mm dish can contain DRG from a number of rats (this will depend on the demands of the experimental design).</li>
	<li>Move the DRG (from a single rat or combined from multiple rats) to a new 35 mm culture dish, which contains 2 mL of collagenase type IA (1 mg/mL in serum-free medium) with sterile tweezers (Figure 2A-e). 
	NOTE: The collagenase solution should be sterilized by passing it through a 0.22 &#181;m syringe filter.</li>
	<li>Digest the DRG in the collagenase solution in a 37 &#176;C CO2 incubator for 30 min.</li>
	<li>Remove the collagenase solution and wash the DRG 3 times in 2 mL Hank&#39;s balanced salt solution (HBSS). 
	NOTE: There may be residual fibers or tissues that come off the DRG into the solution. Remove them by pipette with the washing solution.</li>
	<li>Add 2 mL pre-warmed 0.05% trypsin-EDTA into the DRG-containing 35 mm dish and digest the DRG in a 37 &#176;C CO2 incubator for 30 min.</li>
	<li>Transfer the 2 mL of DRG-containing solution to a 15 mL centrifuge tube by glass pipette. 
	NOTE: The DRG might stick to the glass pipette so this step should be performed with care. DRG loss can be avoided by keeping the DRG-containing solution in the tapered end of a glass pipette (about 0.5 mL) and transferring the solution into the centrifuge tube slowly but without pause.</li>
	<li>Centrifuge the solution at 290 x g for 5 min at 4 &#176;C. Remove the supernatant and add another 2-mL serum-free medium to resuspend the DRG.</li>
	<li>Repeat step 2.10 2 times but change the serum-free medium to pre-warmed culture medium on the last time.</li>
	<li>Manually triturate the DRG approximately 60 times using a flame-polished Pasteur pipette (length 230 mm and tip head inner diameter 1 mm). See Figure 2B for a photograph comparing the orifice of a flame-polished Pasteur pipette to a non-polished pipette. 
	NOTE: The inside diameter of the flame-polished Pasteur pipette is approximately 10% smaller than the control pipette and the inside of the tapered end should be smoother. Be careful not to create bubbles when triturating the cells.</li>
	<li>Remove the poly-L-lysine-coated dish from the CO2 incubator. Aspirate the incubated culture medium from the dish and seed the dissociated cells onto the coated dish.</li>
	<li>Seed the DRG cells from one rat (bilateral collection from L1-L6, for 12 total DRG) into four wells of a 24-well plate; there are approximately 5 x 104 cells in one well of a 24-well plate. 
	NOTE: This density is suitable for immunostaining. For Western blot or RNA extraction, seed the DRG cells from one rat (bilateral L1-L6) into one well of a 6-well plate.</li>
	<li>Replace the culture medium on the following day with the addition of 10 &#181;M cytarabine (Ara-C) and 100 ng/mL NGF and refresh the medium every two days thereafter. 
	NOTE: The thoracic DRG also can also be cultured by this protocol, if they have been collected from the thoracic spine.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mixture of tiletamine and zolazepam (Zoletil)</td>
			<td>Virbac</td>
			<td>Zoletil 50</td>
			<td>anaesthetic</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Biological Industries</td>
			<td>04-001-1</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Biological Industries</td>
			<td>03-033-1</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Invitrogen</td>
			<td>12400024</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>Poly-I-lysine</td>
			<td>Sigma</td>
			<td>P9011</td>
			<td>Coating dish</td>
		</tr>
		<tr>
			<td>Collagenase IA</td>
			<td>Sigma</td>
			<td>9001-12-1</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Invitrogen</td>
			<td>14170-112</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Biological Industries</td>
			<td>03-051-5</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Hilgenberg</td>
			<td>3150102</td>
			<td>Cell trituration</td>
		</tr>
		<tr>
			<td>Cytarabine (Ara-C)</td>
			<td>Sigma</td>
			<td>C6645</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>Millipore</td>
			<td>NC011</td>
			<td>Culture Medium</td>
		</tr>
	</tbody>
</table>

establishing a primary dorsal root ganglia cell culture from a rat spinal column

Source: Lin, Y., et al. <a href="https://app.jove.com/v/57569">Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for isolating and culturing dorsal root ganglia (DRG) cells from the rat spinal column. DRGs excised from the spinal column are treated with enzymes and mechanically dissociated to obtain a single-cell suspension. The cells are cultured in a medium containing inhibitors and growth factors that limit glial cell proliferation and promote neuronal growth, respectively.

<img alt="Figure 1" src="/files/ftp_upload/22628/22628_Figure1.jpg" /> 
Figure 1: Tissue processing diagrams. Lumbar DRG are collected from 3 week-old rats. (A) The positions where the guillotine should be used to cut the animal are indicated by dotted lines. (B) The dorsal trunk with skin removed and (C) the locations of lumbar DRG (from L1-L6) are shown. The insert represents the DRG and the connecting fibers (which are indicated b...

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Collect Lumbar DRG from ...

Take a rat spinal column. Make two cuts along its sides and one lateral cut.
Remove the dorsal muscles and the dorsal portion of the vertebrae.
Extract the spinal cord.
Identify the dorsal root ganglia, or DRGs, located bilaterally along the lumbar vertebrae.
The DRGs contain neurons surrounded by glial cells.
Excise the DRGs and collect them in a dish. Wash the tissue repeatedly with serum-free media.
Transfer the DRGs to a plate containing collagenase solution and incubate. Collagenase degrades the extracellular matrix.
Wash with buffer. Add trypsin-EDTA and incubate to disrupt cell-cell junctions, loosening the cells.
Transfer the DRGs to a tube, centrifuge, and discard the supernatant.
Resuspend the tissue in culture media and pipette repeatedly to release the cells.
Transfer the cells onto a polymer-coated culture plate well to facilitate attachment.
Remove the media and add fresh media containing inhibitors and growth factors.
The inhibitors limit glial cell proliferation, while the growth factors promote neuronal growth.

establishing-primary-dorsal-root-ganglia-cell-culture-from-rat-spinal

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

108 Views. Źródło: Lin, Y., et al. Izolacja zwojów korzenia grzbietowego i hodowla pierwotna w celu zbadania uwalniania neuroprzekaźników. J. Vis. Exp. (2018). Ten film demonstruje technikę izolowania i hodowli komórek zwojów korzenia grzbietowego (DRG) z kręgosłupa szczura. DRG wycięte z kręgosłupa są traktowane enzymami i mechanicznie dysocjowane w celu uzyskania zawiesiny jednokomórkowej. Komórki są hodowane w pożywce zawierającej inhibitory i czynniki wzrostu, które odpowiednio og...

Video: Ustanowienie pierwotnej hodowli komórek zwojów korzenia grzbietowego z kręgosłupa szczura - Concept

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 ...

JoVE Journal

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 ...

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, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

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

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column

Transkrypcja

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column