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. Hippocampal Dissection
<ol>
	<li>Spray the postnatal day 7 (P7) Sprague Dawley rat pup with 70% ethanol outside of the laminar flow clean bench and quickly decapitate the animal using large sterile surgical scissors inside the laminar flow bench. Let the head drop into an ice-cold dissecting solution in one of the Petri dishes.</li>
	<li>In the Petri dish, rinse off the blood and quickly transfer the head to sterilized filter paper, ventral side down.</li>
	<li>Using the scalpel, cut along the dorsal surface in the sagittal plane to expose the underlying skull. Cut through the skin, but not the underlying bone, which is soft and easily penetrable in rats of this age. Set aside this &#34;dirty&#34; scalpel and do not use it on brain tissue.</li>
	<li>Using the small dissector scissors (angled to the side) and forceps, cut open the skull along the sagittal suture of the skull to bregma, the anatomical point on the skull where the coronal suture is intersected perpendicularly by the sagittal suture. Use forceps to pull skull flaps up and away from the midline of the skull.</li>
	<li>Place the micro spoon on the underside of the brain, beneath the brain stem, to gently lift the brain out of the skull. Lift the brain to expose the optic nerves and olfactory bulb on the basal surface of the brain. Cut these structures with small scissors to fully detach the brain from the skull. Remove and transfer the intact brain to the other large Petri dish containing an ice-cold dissecting solution.</li>
	<li>Using the micro spoon, transfer the brain to a small Petri-dish lid containing sterile filter paper. With a sterile Pasteur pipette, rinse the brain with a few drops of dissecting solution to moisten the tissue.</li>
	<li>Using a &#34;clean&#34; scalpel blade, cut the two hemispheres apart. Transfer the left hemisphere back to a large Petri dish with a micro spoon and place the hemisphere pia side down in an ice-cold dissecting solution for subsequent use.</li>
	<li>View the medial face of the right hemisphere and identify the edge fornix, a prominent band of white matter along the medial edge of the hippocampus. Using a sterile scalpel, make a sagittal cut through the fornix, but take care because only 0.5 cm of the scalpel tip will be sufficient to cut the fornix.</li>
	<li>Using two micro-spatulas, remove the first hippocampus from the right hemisphere by placing the right-hand-spatula on the brain stem and lifting the overlying cortex with the left-hand spatula. Gently lift the cortex to reveal the lateral ventricle and medial surface of the hippocampus. A white curved line, the fimbria, should now be visible.</li>
	<li>Align the curvature of the spatula with the curvature of the fimbria and gently press the spatula under the fimbria. Slide the spatula left and ride along the rostral-caudal axis, and then lift the spatula in the dorsal direction to remove the hippocampus.</li>
	<li>Transfer the hippocampus to a 2nd&#160;small Petri dish with an ice-cold dissecting solution. Repeat the same procedure on the left hemisphere to remove the left hippocampus.</li>
	<li>Using a micro spatula, carefully transfer the hippocampi to the tissue chopper stage. Arrange them adjacent and parallel to each other and perpendicular to the axis of the chopper blade. Use a paintbrush to position the tissue and add a few drops of the dissecting solution on top of the hippocampi.</li>
	<li>Cut the tissue into 400 &#956;m slices without pausing to remove individual slices (usually, they will not adhere to the blade). After the whole hippocampus has been cut, use the paintbrush to gently transfer the sections to a 2nd&#160;small Petri dish with the dissection solution.</li>
	<li>Under a dissecting microscope, carefully separate the floating slices using a micro-spatula and paintbrush.</li>
	<li>Remove the pre-prepared culture plate with culture insert from the incubator and place it in a laminar flow hood.</li>
	<li>Using a fire-polished Pasteur pipette, draw 4-5 slices into the pipette and transfer slices to the apical surface of the culture insert membrane. Next, adjust the positioning with a paintbrush and leave space between individual sections and the border of the culture insert.</li>
	<li>Using a sterile Pasteur pipette, remove the excess dissecting solution from the apical surface of the membrane. 
	NOTE: While removing the solution, avoid drawing tissue sections into the pipette. Alternatively, use a regular pipette (P200 or P1000) with sterilized pipette tips to slowly remove the solution.</li>
	<li>Place the culture plate with serum-containing culture medium and the hippocampal slices back into the incubator at 35 &#176;C and 5% CO2. 
	NOTE: If the experiment calls for generating cultures from multiple animals, thoroughly clean the laminar flow clean bench between dissections. Return instruments to alcohol solution and replace all Petri dishes, filter papers, and sterile razor blades before the second dissection.</li>
</ol>
2. Feeding and Maintaining Organotypic Slices
<ol>
	<li>Feed cultures in a sterile laminar flow clean bench.</li>
	<li>Perform the first feeding of the cultured sections two days post-dissection. Aspirate old culture medium using the sterilized glass pipette.</li>
	<li>Use the sterile 5 ml serological pipette to add 1 ml of fresh, sterile, serum-containing medium to the wells.</li>
	<li>Gently replace the culture insert and remove any air bubbles that may have formed underneath the membrane surface.</li>
	<li>After the first feeding, change the medium every other day.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture inserts, 30 mm diameter, 0.4 &#181;m pore size</td>
			<td>Thermo scientific&#160;</td>
			<td>140660</td>
			<td>Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td>
		</tr>
		<tr>
			<td>Conical Centrifuge tubes (sterile)</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissector scissors (angled to side)</td>
			<td>Fine Science Tools&#160;</td>
			<td>14082-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Gibco</td>
			<td>11095; liquid</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS) (500 ml)</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Horse Serum Heat Inactivated (500 ml)</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td>Make 50 ml aliquots and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish (100 mm x 15 mm) and (60 mm x 15 mm)</td>
			<td>Fisher Brand</td>
			<td>FB0875712 and FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades #11</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle #3</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Sorfa Medical Plastic Co.</td>
			<td>P8050</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile vacuum filter</td>
			<td>Thermo-Scientific</td>
			<td>565-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>14054-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe driven filter unit</td>
			<td>Millipore-Millex</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue chopper with moveable stage</td>
			<td>Stoelting&#160;</td>
			<td>51425</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine tip paintbrush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of organotypic slice cultures from a rat pup&#39;s hippocampus

Source: Mosa, A. J. et al., <a href="https://app.jove.com/v/52353">Organotypic Slice Cultures for Studies of Postnatal Neurogenesis.</a> J. Vis. Exp. (2015).
This video demonstrates a method for preparing organotypic hippocampal slice cultures from the brain of a rat pup. It outlines the steps involved in the dissection of the hippocampus, slicing, and culture conditions required to promote cell viability and growth in the hippocampal slices, ultimately generating organotypic slice cultures.

Preparation of Organotypic Slice Cultures from a Rat Pup&#39;s Hippocampus

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 pup&#39;s brain and separate the brain hemispheres.
View the medial face of one brain hemisphere.&#160;
Identify the fornix, a white matter band located at the medial edge of the hippocampus, and cut through it.
Separate the surrounding tissue, exposing the hippocampus and fimbria, a curved structure of white matter tracts.
Slide a spatula under the fimbria, lift the hippocampus, and transfer it to an ice-cold buffer.
Place the hippocampi from both hemispheres on a tissue chopper and slice them.
Transfer the slices to an insert membrane placed within a culture plate containing media.
Adjust the slice positions to allow nutrient and oxygen diffusion. Remove excess buffer to prevent dilution of the culture media.
Incubate. The insert membrane coating facilitates the adhesion of the slices, reducing tissue damage.
Change the media regularly to replenish nutrients, facilitating hippocampal neurogenesis and the generation of organotypic slice cultures.

preparation-of-organotypic-slice-cultures-from-a-rat-pup-s-hippocampus

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

76 vues. Source : Mosa, A. J. et al., Cultures de tranches organotypiques pour les études de neurogenèse postnatale. J. Vis. Exp. (2015). Cette vidéo montre une méthode de préparation de cultures de tranches d’hippocampe organotypiques à partir du cerveau d’un ratillon. Il décrit les étapes impliquées dans la dissection de l’hippocampe, le tranchage et les conditions de culture nécessaires pour favoriser la viabilité cellulaire et la croissance dans les tranches de l’hippocampe, gén?...

Vidéo: Préparation de cultures de tranches organotypiques à partir de l'hippocampe d'un petit rat - Concept

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Une méthode simplifiée pour les ultra-basse densité, à long terme primaire hippocampique Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Recherche

JoVE Journal

Neurosciences

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Hippocampe organotypiques comme modèle d’étude Neuroprotection et son caractère invasif des cellules tumorales

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Électroporation unicellulaire à travers différentes cultures de tranches organotypiques de neurones excitateurs excitateurs de l’hippocampe de souris et de neurones inhibiteurs spécifiques à une classe

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

Preparation of Organotypic Slice Cultures from a Rat Pup's Hippocampus

Transcription

Preparation of Organotypic Slice Cultures from a Rat Pup's Hippocampus