eoe sub articles

EoE Sub Articles

1. Explant culture of DRG from Adult Mouse
<ol>
	<li>Place the collected DRG in a 35 mm Petri dish containing 3 mL of ice-cold serum-free media (SFM).</li>
	<li>Transfer each individual DRG in a dry glass Petri dish and, under a surgical microscope, clean and trim off excess fibers and connective tissue still attached to the DRG using a blade. The DRG is easily identifiable as a bulgy transparent structure along the white spinal nerve/root. Blood vessels often are found surrounding the DRG.</li>
	<li>P...

generating dorsal root ganglion explant and dissociated cell culture

Source: Fornaro, M. et al., <a href="https://app.jove.com/v/56757">Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection.</a> J. Vis. Exp. (2018).
This video demonstrates a method to develop dorsal root ganglia or DRG explant culture and DRG-dissociated cell models using isolated mouse DRG tissues. These models help to study various mechanisms with neuron-glial interactions in a controlled environment.

Generating Dorsal Root Ganglion Explant and Dissociated Cell Culture

1. Explant culture of DRG from Adult Mouse

	Place the collected DRG in a 35 mm Petri dish containing 3 mL of ice-cold serum-free media (SFM).
	Transfer ...

Start with dorsal root ganglion or DRG tissues in a serum-free medium. The DRG tissue contains sensory neurons and satellite glial cells embedded in the extracellular matrix, ECM.&#160;
Seed them onto a culture dish coated with a gelatinous protein mixture to enhance tissue adhesion.
Over time, glial cells release neurotrophic growth factors that allow neuronal extensions, establishing a DRG explant culture.
To develop a dissociated cell culture, take these DRG explants in a tube. Treat them with collagenase to degrade ECM and then with trypsin, which disrupts the cell connections, releasing neurons and glial cells.
Add a medium enriched with serum to stop the enzymatic reaction.
Pipette the contents repeatedly to create a single-cell suspension and filter it to remove debris.
Centrifuge this cell suspension and remove the enzyme-containing supernatant.
Resuspend the cells in a neurobasal medium and transfer them onto a laminin-coated plate.
The satellite glial cells and sensory neurons adhere to the laminin-coated plate, establishing a dissociated cell culture.

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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

JoVE Journal

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

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, ...

Generating Dorsal Root Ganglion Explant and Dissociated Cell Culture

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