eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of ihOEG (Ts12 and Ts14) for the assay
NOTE: This step must be done 24 h before RGN dissection and coculture.
<ol>
	<li>Treat 12 mm &#216; coverslips with 10 &#181;g/mL poly-L-lysine (PLL) for 1 h. 
	NOTE: The coverslips can be left overnight in the PLL solution.</li>
	<li>Wash the coverslips with 1x phosphate buffer saline (PBS) three times.</li>
	<li>Detach Ts12 and Ts14 ihOEG cells from the p60 cell culture dish.
	<ol>
		<li>Add 4 mL of DMEM/F12 (basal medium)-FBS (fetal bovine serum) culture medium to a 15 mL conical tube. Warm at 37 &#176;C in a clean water bath.</li>
		<li>Remove the medium from the plates and wash the cells with 1 mL of 1x PBS (phosphate-buffered saline)-EDTA (ethylene diamine tetraacetic acid) once.</li>
		<li>Add 1 mL of trypsin-EDTA to the OEG cells and incubate for 3&#8211;5 min at 37 &#176;C, 5% CO2.</li>
		<li>Collect cells with a p1000 pipette and transfer them to the medium prepared in step 2.1.</li>
		<li>Centrifuge for 5 min at 200 x g.</li>
		<li>Aspire the supernatant.</li>
		<li>Add 1 mL of ME10 medium and resuspend the pellet.</li>
		<li>Count the cell number in a hemocytometer.</li>
	</ol>
	</li>
	<li>Seed 80,000 Ts14 cells or 100,000 Ts12 cells/well onto the coverslips in 24-well plates in 500 &#181;L of ME10 medium.</li>
	<li>Culture cells at 37 &#176;C in 5% CO2 for 24 h.</li>
</ol>
2. Retinal tissue dissection
NOTE: 2-month-old male Wistar rats are used as RGN source. Two retinas (one rat) for 20 wells of a 24-well cell dish. Autoclave surgical material before use. Papain dissociation kit is commercially purchased (Table of Materials). Follow the provider&#180;s instructions for reconstitution. Reconstitute D, L-2-amino-5-phosphonovaleric acid (APV) in 5 mM stock and prepare the aliquots.
<ol>
	<li>On the day of the assay, prepare the following media.
	<ol>
		<li>Prepare a p60 cell culture dish with 5 mL of cold EBSS (Earle&#39;s balanced salt solution) (vial 1 of the papain dissociation kit).</li>
		<li>Prepare a p60 cell culture dish with reconstituted vial 2 (papain) of the papain dissociation kit plus 50 &#181;L of APV. Then, add 250 &#181;L of reconstituted vial 3 (DNase plus 5 &#181;L of APV).</li>
		<li>In a sterile tube, mix 2.7 mL of vial 1 with 300 &#181;L of vial 4 (albumin-ovomucoid protease inhibitor). Add 150 &#181;L of vial 3 (DNase) plus 30 &#181;L of APV.</li>
		<li>Prepare 20 mL of Neurobasal-B27 medium (NB-B27).</li>
	</ol>
	</li>
	<li>Sacrifice a rat by asphyxiation with CO2.</li>
	<li>Remove the head by decapitation with a guillotine; place it in a 100 mm Petri dish and spray the head with ethanol 70% before placing it in a laminar flow hood.</li>
	<li>Cut the rat&#180;s whiskers with scissors so they do not interfere with the eye manipulation.</li>
	<li>Grip the optic nerve with forceps to pull out the eyeball enough to be able to make an incision across the eye with a scalpel.</li>
	<li>Remove the lens and vitreous humor and pull out the retina (orange-like tissue) while the remaining layers of the eye (including the pigment epithelial layer) stay inside.</li>
	<li>Place the retina in the p60 cell culture dish prepared in step 2.1.1.</li>
	<li>Transfer the retina to the p60 cell culture dish prepared in step 2.1.2 and cut it with the scalpel into small pieces of approximate size &#60; 1 mm.</li>
	<li>Transfer to a 15 mL plastic tube.</li>
	<li>Incubate the tissue for 30 min, in a humidified incubator at 37 &#176;C under 5% CO2, with agitation every 10 min.</li>
	<li>Dissociate cell clumps by pipetting up and down with a glass Pasteur pipette.</li>
	<li>Centrifuge the cell suspension at 200 x g for 5 min.</li>
	<li>Discard the supernatant. To inactivate papain, resuspend the cell pellet in the solution prepared in step 2.1.3 (1.5 mL for 2 eyes).</li>
	<li>Carefully pipet this cell suspension into 5 mL of reconstituted vial 4.</li>
	<li>Centrifuge at 200 x g for 5 min.</li>
	<li>While centrifuging, completely remove the ME-10 medium from the OEG 24 well cell plate (previously prepared in step 1) and replace it with 500 &#181;L of NB-B27 medium per well.</li>
	<li>Discard the supernatant and resuspend the cells in 2 mL of NB-B27 medium.</li>
	<li>Plate 100 &#181;L of retinal cell suspension, per well of the m24 plate, onto PLL-treated or OEG monolayers-coverslips.</li>
	<li>Maintain cultures at 37 &#176;C with 5% CO2 for 96 h in NB-B27 medium.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>D,L-2-amino-5-phosphonovaleric acid</td>
			<td>Sigma</td>
			<td>283967</td>
			<td>NMDA receptor inhibitor</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>11573397</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>FBS-Hyclone</td>
			<td>Fisher Scientific</td>
			<td>16291082</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605F</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Neuronal cells culture medium</td>
		</tr>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td>For use in neural cell isolation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-EDTA</td>
			<td>Lonza</td>
			<td>H3BE02-017F</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Amphotericin B</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>Bacteriostatic and bactericidal</td>
		</tr>
		<tr>
			<td>Pituitary extract</td>
			<td>Gibco</td>
			<td>13028014</td>
			<td>Bovine pituitary extract</td>
		</tr>
		<tr>
			<td>Poly -L- lysine (PLL)</td>
			<td>Sigma</td>
			<td>A-003-M</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculturing of retinal ganglion neurons with olfactory ensheathing glia

Source: Portela-Lomba, M., et al. <a href="https://app.jove.com/v/61863">Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to isolate retinal ganglion neurons (RGNs) from a rat&#39;s retina and coculture them with olfactory ensheathing glia (OEG) cells. This method allows for studying the neuro-regenerative potential of OEGs after neural injury.

Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take a rat&#39;s retina, a light-sensitive layer of the eye containing retinal ganglion neurons, or RGNs.
Place the retina in a cold-balanced salt solution to prevent tissue damage.
Transfer the retina to a solution containing protease and DNase and cut it into smaller pieces.
Transfer the contents to a tube and incubate.
The protease breaks down the extracellular matrix and loosens the cells, while DNase degrades any contaminating DNA.
Pipette up and down to disperse cell clumps.
Add an inhibitor solution to stop the protease action.
Centrifuge the cells to settle them. Remove the supernatant and resuspend the cells in a suitable growth medium.
Add this cell suspension to the monolayer of olfactory ensheathing glia or OEG cells, a type of glial cell, and incubate.
The RGNs integrate into the OEG monolayer, establishing the coculture model for assessing the neuroregenerative potential of OEGs.

coculturing-retinal-ganglion-neurons-with-olfactory-ensheathing

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

29 visualizações. Fonte: Portela-Lomba, M., et al. Cocultura de neurônios ganglionares retinianos de ratos axotomizados com glia de revestimento olfativo, como um modelo in vitro de regeneração axonal adulta. J. Vis. Exp. (2020) Este vídeo demonstra o procedimento para isolar os neurônios ganglionares da retina (RGNs) da retina de um rato e cocultivá-los com células da glia de revestimento olfativo (OEG). Este método permite estudar o potencial neuro-regenerativo dos OEGs após lesão neural. 

Vídeo: Cocultura de neurônios ganglionares da retina com glia de revestimento olfativo - Conceito

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Isolamento de células de gânglio retiniano murino primário (RGCs) por citometria de fluxo

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

JoVE Journal

Bioengenharia

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

A indireta Neuron-astrócitos Coculture Ensaio: An<em&gt; In Vitro</em&gt; Set-up para a investigação detalhada da Relação Neuron-glia

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Neurociência

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Visualizando mudanças no Circuito Neuron-Glia com a Técnica de Imagem de Cálcio

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia

Transcrição

Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia