Name: Anterior Eye Segment Dissection: A Method to Isolate Cornea Bearing Anterior Segment from Porcine Eye
Uploaded: 2023-04-30T14:52:27
Description: Source: Boice, E. N., Snider, E. J. Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance. J. Vis. Exp. ...

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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 conju...

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.

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Research

JoVE Encyclopedia of Experiments

Biology

Large Animal Models

Porcine

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.

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

JoVE Journal

Medicine

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.

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 ...

Neuroscience

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.

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

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