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. Retinal cup dissection 
<ol>
	<li>Euthanize a mouse by CO2&#160;asphyxiation following institutional guidelines on euthanasia. 
	NOTE: Do not leave the animal in a CO2&#160;chamber longer than the time needed for euthanasia.</li>
	<li>Enucleate eyes and place into ice-old 1x PBS buffer in a petri-dish and then place it under a dissection microscope.</li>
	<li>Carefully remove, by cutting with microscissors, the extra rectus muscles attached outside the eyeball and cut off the optic nerve.</li>
	<li>Use a 30 G needle to punch a hole at the edge of the cornea (limbus); this serves as the insertion site for the microscissors. Then, use a fine dissection microscissors to make a circular cut along the edge of the cornea, separating it from the posterior eye cup.</li>
	<li>Use sharp dissection forceps to remove the cornea, lens, and the vitreous humor away from the eye cup.</li>
	<li>Use fine dissection microscissors to make several small cuts on the scleral layer at the rim of eye cup. Avoid cutting the retina layer. Use two sharp dissection forceps to hold on to the scleral tissue at each side of the cut and very carefully pull on the scleral layer to remove it from the neural retina. Repeat this around the eye cup until all sclera is removed and an intact retinal cup is obtained.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1X PBS</td>
			<td>Thermo Fisher</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>30-gauge needle</td>
			<td>BD Precision Glide</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy puncher, 1 mm</td>
			<td>Integra Miltex</td>
			<td>33-31AA</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 asphyxiation chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection forceps-Dumont #5</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>Stright tip</td>
		</tr>
		<tr>
			<td>Dissection forceps-Dumont #7</td>
			<td>Fine Science Tools</td>
			<td>11274-20</td>
			<td>Curved tip</td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe forceps</td>
			<td>Fine Science Tools</td>
			<td>11051-10</td>
			<td>Curved, Serrated tip</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Fine Science Tools</td>
			<td>15004-08</td>
			<td>Curved tip</td>
		</tr>
	</tbody>
</table>

retinal cup extraction from mouse: a surgical procedure to obtain intact retinal cup from mouse eye

Source: Jiang, K. et al. <a href="https://www.jove.com/v/62914">Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples.</a> J. Vis. Exp (2021)
This video describes a protocol to isolate the retinal cup without the outer covering layers from the eyeball of a mouse. The extracted retinal tissue can help study various metabolic pathways occurring in the retinal cells.

Retinal Cup Extraction from Mouse: A Surgical Procedure to Obtain Intact Retinal Cup from Mouse Eye

Source: Jiang, K. et al. Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples. J. Vis. Exp (2021)
This video ...

The murine eye is covered by three layers: the outermost fibrous sclera, the middle choroid coat, and the innermost retina. The cup-shaped cavity between the retina and eye lens contains vitreous humor &#8212; a gelatinous medium that helps transmit light to the retina.&#160; In turn, the retina passes the resulting visual information via the optic nerve to the brain.
To extract the retinal cup, take a euthanized mouse and enucleate its eye. Submerge the eye in a Petri dish containing an appropriate ice-cold buffer to prevent tissue degradation. Place the Petri dish under a dissection microscope.
Snip off the remnants of the optic nerve from the base of the eyeball. Make a small incision at the junction of the retina and the cornea&#160; &#8212; a transparent covering around the eye lens. Using this opening, make a complete circular cut along the retina-cornea junction to detach the lens and the vitreous humor gel from the eye.
Remove the lens and vitreous humor to obtain a semi-circular eye cup containing the retina lined by choroid and sclera. Create multiple superficial cuts throughout the scleral portion of the eye cup. Use these cuts to separate the outer eye layers for obtaining an intact retinal cup for downstream applications.

retinal-cup-extraction-from-mouse-surgical-procedure-to-obtain-intact

Research

JoVE Encyclopedia of Experiments

Biology

Rodent Models

Adult

2.3K Views. Source: Jiang, K. et al. Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples. J. Vis. Exp (2021) This video describes a protocol to isolate the retinal cup without the outer covering layers from the eyeball of a mouse. The extracted retinal tissue can help study various metabolic pathways occurring in the retinal cells. 

Video: Retinal Cup Extraction from Mouse: A Surgical Procedure to Obtain Intact Retinal Cup from Mouse Eye - Concept

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

JoVE Journal

Neuroscience

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Retinal Cup Extraction from Mouse: A Surgical Procedure to Obtain Intact Retinal Cup from Mouse Eye

Transcript