Name: anterior eye segment dissection: a method to isolate cornea bearing anterior segment from porcine eye
Uploaded: 2023-04-30T14:52:27.000+00:00
Duration: 1 min 58 s
Description: Source: Boice, E. N., Snider, E. J. Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance. J. Vis. Exp. ...

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. Dissection of tissue
<ol>
	<li>Prepare the porcine tissue using non-sterile dissection workspace.
	<ol>
		<li>Procure enucleated porcine eyes from a local abattoir, animal studies, or vendor. Maintain the eyes on ice submerged in PBS during delivery and use immediately upon receiving.</li>
		<li>Cut away the extraorbital tissue and trim the conjunctiva leaving only the corneoscleral shell and optic nerve. Perform the dissection under non-sterile conditions with large surgical scissors and forceps in a gross dissection tool pack.</li>
		<li>Place the eyes back into fresh PBS on ice until all eyes required for experimental setup have been preliminary/gross dissected.</li>
		<li>Submerge the eyes in 10% betadine solution for 2 min in closed containers and transfer aseptically to BSC II cabinet for microdissection (cabinet #1). Perform all the subsequent works under sterile conditions to minimize contaminations during setup.</li>
	</ol>
	</li>
	<li>Sterilely dissect anterior segments.
	<ol>
		<li>After 2 min in betadine solution, transfer the eyes into three serial washes of sterile 1x Antibiotic-Antimycotic-PBS (AA-PBS) to remove excess betadine solution from the ocular surface while maintaining sterility of the ocular tissue. After three washes, maintain the tissue in AA-PBS until further use.</li>
		<li>Hemisect the eye using a razor blade/scalpel and curved scissors. Place the eye on an AA-PBS-soaked gauze and create an incision with a sterile razor blade or scalpel near the equator of the eye (60/40 split with 40 on the anterior side). Using curved surgical scissors, hemisect the eye to isolate the anterior eye (corneal half). 
		NOTE: The cut around the anterior segment needs to be continuous to prevent jagged, rough edges in the sclera that will create fluid leaks after setup in the organ culture.</li>
		<li>Use microscissors as a shovel to scoop vitreous humor from the anterior segment. Remove the lens from the anterior segment using microscissors. Leave the anterior segments in AA-PBS until further dissection steps. 
		NOTE: All the eyes that will be dissected can be held at this step and one by one taken through the remainder of the dissection process.</li>
		<li>With a dissecting microscope, cut back iris to iris root gradually, radially until trabecular meshwork (TM) is visible. The TM is a pigmented tissue that comprises fibers circumferentially oriented around the corneoscleral shell. Careful cuts into the iris toward the iris root will expose the depth of the TM under the tissue.</li>
		<li>Cut 360&#176; around the iris at the same depth as the initial cut into the tissue to expose the entire TM region. Clean up any remaining residual iris covering TM as is necessary.</li>
		<li>Trim the ciliary body remnants posterior to the TM, leaving only a thin band of tissue posterior to the TM region (approximately 1 mm).</li>
		<li>Place the dissected anterior segment (AS) in media until further setup in anterior segment organ culture (ASOC) in dish assembly BSC II cabinet (cabinet #2). 
		NOTE: All eyes can be held at this point prior to ASOC setup if a single user is performing dissection and organ culture dish assembly.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>Anti-Anti</td><td>Gibco</td><td>15240-096</td><td></td></tr><tr><td>Betadine</td><td>Fisher Scientific</td><td>NC1696484</td><td></td></tr><tr><td>Cotton swabs</td><td>Puritan</td><td>25-8061WC</td><td></td></tr><tr><td>Fine forceps</td><td>World Precision Instruments</td><td>15914</td><td></td></tr><tr><td>Gauze</td><td>Covidien</td><td>8044</td><td></td></tr><tr><td>Large forceps</td><td>World Precision Instruments</td><td>500365</td><td></td></tr><tr><td>Large surgical scissors</td><td>World Precision Instruments</td><td>503261</td><td></td></tr><tr><td>Medium toothed forceps</td><td>World Precision Instruments</td><td>501217</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Petri dish (100 mm)</td><td>Fisher</td><td>FB0875713</td><td></td></tr><tr><td>Razor blade</td><td>Fisher</td><td>12-640</td><td></td></tr><tr><td>Sterile 100 mL beakers with lids</td><td>VWR</td><td>15704-092</td><td></td></tr><tr><td>Vannas&amp;nbsp;scissors</td><td>World Precision Instruments</td><td>WP5070</td><td></td></tr></tbody></table>

anterior eye segment dissection: a method to isolate cornea bearing anterior segment from porcine eye

Source: Boice, E. N., Snider, E. J. <a href="https://www.jove.com/v/62649">Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance.</a> J. Vis. Exp. (2021).
In this video, we demonstrate the dissection of the cornea-bearing anterior segment of the porcine eye. The isolated segment can be used for organ culturing to study the pathophysiology of eye disorders.

Anterior Eye Segment Dissection: A Method to Isolate Cornea Bearing Anterior Segment from Porcine Eye

Source: Boice, E. N., Snider, E. J. Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance. J. Vis. Exp. ...

The vertebrate eye is divided into the anterior segment - situated ahead of the lens - and the posterior segment - situated behind the lens. The anterior segment contains the cornea, aqueous humor,&#160; iris, ciliary body, and lens; while the posterior segment contains the vitreous humor, retina, choroid, and the optic nerve. The anterior segment can be cultured to study pathophysiology of ocular disorders.
To dissect the anterior segment from an animal model, begin with a freshly isolated porcine eye. Remove the extra-orbital tissue from the eye and wash with antibiotic-supplemented buffer. Place the eye over a sterile, antibiotic-soaked gauze to prevent contamination of the eye.
Using a scalpel, incise the eye near the equator region to open the corneoscleral shell - fibrous and vascular layer. Use curved scissors to isolate the anterior segment from the posterior eye portion. Carefully flip the anterior eye segment. Then, scoop out the vitreous humor and remove the lens.
Excise the iris radially towards the iris roots to reveal the trabecular meshwork - a pigmented tissue through which aqueous humor drains out of the eye. Remove the traces of the ciliary body, leaving a thin tissue band. Wipe the cornea with a wet cotton swab to remove the residual pigment. The anterior eye segment can now be cultured for further experiments.

anterior-eye-segment-dissection-method-to-isolate-cornea-bearing

Research

JoVE Encyclopedia of Experiments

Biology

Large Animal Models

Porcine

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视频: 眼前节清扫术:一种从猪眼中分离带有角膜的眼前节的方法 - 概念

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

科研

JoVE Journal

医学

Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular pressure (IOP) which&#160;can identify various pathogenic paradigms related to the glaucoma pathogenesis. Ex vivo human anterior segment perfusion organ culture models have previously been successfully utilized and applied as effective technologies for the discovery of glaucoma pathogenesis and testing of therapeutics. Preclinical drug screening and research performed on ex vivo human organ systems can be more translatable to clinical research. This article describes in detail the generation and operation of a novel ex vivo human translaminar pressure model called the translaminar autonomous system (TAS). The TAS model can independently regulate ICP and IOP using human donor posterior segments. The model allows for studying pathogenesis in a preclinical manner. It can reduce the use of living animals in ophthalmic research. In contrast to in vitro experimental models, optic nerve head (ONH) tissue structure, complexity, and integrity can also be maintained within the ex vivo TAS model.

We describe and detail the use of the translaminar autonomous system. This system utilizes the human posterior segment to independently regulate the pressure inside the segment (intraocular) and surrounding the optic nerve (intracranial) to generate a translaminar pressure gradient that mimics features of glaucomatous optic neuropathy.

用于调节人类供体后段眼内压和颅内压的跨层自主系统模型

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular ...

神经科学

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

新型人类组织特征视网膜培养技术的特征

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Anterior Eye Segment Dissection: A Method to Isolate Cornea Bearing Anterior Segment from Porcine Eye

成績單

Anterior Eye Segment Dissection: A Method to Isolate Cornea Bearing Anterior Segment from Porcine Eye