Name: establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment
Uploaded: 2015-06-05T15:00:00.000+00:00
Duration: 7 min 53 s
Description: Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

This work was supported by National Institutes of Health Grant to A.S.M. (EY021784).

The following protocol complies with the Thomas Jefferson University Institutional Animal Care and Use Committee guidelines and with the ARVO Statement for the Use of Animals in Vision Research.
1. Setup and Preparation of Lenses for Ex&#160;Vivo Wound Culture
<ol>
	<li>Place three 100 mm petri dishes in a sterile, laminar flow hood. Fill two of the petri dishes halfway with Tris/Dextrose buffer (TD buffer; 140 mM NaCl, 5 mM KCl, 0.7 mM Na2PO4, 5 mM D-glucose, 8.25 mM Tris Base, pH to 7.4 with HCl) at RT, leaving the third empty. Pre-warm culture media (Media 199 supplemented with, 1% L-glutamine and 1% penicillin/streptomycin) to 37oC. 
	Note: The standard wound healing culture media is serum-free, as occurs in vivo; however, the wound-repair cultures can be grown successfully in defined media conditions that include serum or other factors.</li>
	<li>Remove fertile embryonic day 15 white leghorn chick egg from incubator (held at 37.7&#176; C with gentle rocking)</li>
	<li>Place selected egg in the laminar flow hood and clean outside of shell with 70% ethanol from a wash bottle. Conduct all procedures below under aseptic conditions in the laminar flow hood, using sterile solutions and instruments.</li>
	<li>Crack egg and place contents into the empty 100 mm petri dish. Decapitate embryo using standard forceps and fine scissors. Place the chick embryo head in a petri dish containing TD buffer and properly dispose of the remainder of the embryo. Optionally keep the chick embryo heads in TD buffer for a short period of time, no longer than 15 min.</li>
	<li>Place the chick embryo head on a petri dish lid. Using high precision forceps, remove the lens along with its attached vitreous humor from the eye in the following sequence. Pinch the back of the eye with the forceps to create a small opening in the back of the eye.</li>
	<li>Then, grasp the vitreous humor with forceps and gently tug on the vitreous with a rolling motion, the vitreous with the lens attached will be dislodged from the eye. Place lens/vitreous in the remaining petri dish containing TD buffer. Allow the lenses to remain in TD buffer for no longer than 30 min.</li>
	<li>Move the lens to a new petri dish lid under a dissecting microscope. From this point on perform all steps under a dissecting microscope. With high precision forceps carefully brush away any ciliary body (pigmented cells) that were dislodged with the lens using the edge of the forceps, taking caution not to damage the lens tissue. 
	Note: Removing the ciliary body ensures that cell types that are not endogenous to the lens are not included in the wound repair culture.</li>
	<li>Separate the lens from the vitreous humor with high precision forceps by pinching off the vitreous body from its association with the posterior lens capsule.</li>
	<li>Using high precision forceps transfer the lens into a small drop of TD buffer (about 200 &#181;l) in a 35mm tissue culture dish.</li>
</ol>
2. Performing Mock Cataract Surgery
<ol>
	<li>Orient the lens in the drop of TD buffer in the 35 mm dish with the anterior aspect of the lens facing up. 
	Note: The anterior of the lens is easily identified by the presence of a dense ring in the tissue that notes the border between the anterior and equatorial region of the lens epithelium. In contrast, there is an absence of markings on the posterior of the lens capsule to which the lens fiber cells are attached.</li>
	<li>Using two high precision forceps make a small incision (approximately 850&#181;m) in the center of the anterior lens capsule, the thick basement membrane that surrounds the lens tissue, and its associated anterior lens epithelium, by grasping the tissue with one forceps in each hand and gently tugging in opposing directions.</li>
	<li>Remove the fiber cell mass, which makes up the bulk of the lens tissue, dislodging it from its attachments to the lens epithelium and surrounding lens capsule by hydro-elution (an approach used in classic cataract surgery, modeled in Figure&#160;1A).</li>
	<li>Fill a 1 ml syringe with a 27.5 G needle tip with 300 &#181;l of TD buffer. Insert the needle tip into the incision made in the anterior lens capsule, and about halfway into the lens.</li>
	<li>Gently depress the syringe injecting the TD buffer into the lens fiber cell mass. Inject between 50 and 200 &#181;l TD, and never more than 300 &#181;l. Observe the fiber cell mass loosening itself from the epithelium and lens capsule.</li>
	<li>Using high precision forceps, remove the loosened fiber cell mass from the lens through the anterior incision site. 
	Note: This procedure leaves the posterior lens basement membrane capsule to which the fiber cells had been attached denuded of cells, and an injured lens epithelium just adjacent to this site.</li>
</ol>
3. Preparing the Wounded Lens for Ex Vivo Culture
<ol>
	<li>Flatten the lens capsular bag that results from the cataract surgery described above on the culture dish, cell side up, by making five cuts in the anterior aspect of the capsular bag.</li>
	<li>Cut perpendicular to the original incision site through to the equator of the lens. Flatten the resultant five &#8220;flaps&#8221; of lens capsule with attached epithelium on the culture dish capsule side down, cell side up. Note the ex vivo wounded lens now should take on a star or flowerlike shape (see Figure&#160;1B).</li>
	<li>To secure the capsule to the dish, press softly down with the forceps at each point of the star. This will make a small indentation at the five most outside tips of the explant and result in sustained attachment to the dish. 
	Note: It is possible to damage the capsule during this procedure and so it is important to secure the capsule to the dish as close to the tips of the flaps as possible, as well as to make the least amount of securing points as possible (generally two, maximum of three per flap).</li>
	<li>Remove the TD buffer from the 35mm dish and replace it with 1.5 ml&#160;of pre-warmed media. Cover the 35mm dish with its lid and place in the incubator (37&#176;C, 5% CO2).</li>
</ol>
4. Separation of the Central Migration Zone (CMZ), where Re-epithelialization of the Wounded Area of the Posterior Capsule Occurs, from the Original Attachment Zone (OAZ) of Lens Epithelial Cells, for Quantitative Analysis.
Note: The cells begin to move into the CMZ region immediately in response to injury. By day one in culture enough cells are migrating across the CMZ for molecular and biochemical analysis, following separation of the CMZ and the OAZ by micro-dissection23. This protocol involves removal of one flap (OAZ) at a time from the wounded area of the capsule.
<ol>
	<li>Observe a demarcation, clearly visible under the dissecting microscope, between the OAZ and CMZ (see Figure&#160;2A). Using two high precision forceps, grasp with both forceps at the edge of the OAZ/CMZ line, one just adjacent to the other, on either side of the line (see Figure&#160;2A, B, arrow).</li>
	<li>Using one hand/one forceps on the CMZ side continue to hold onto the wounded culture, while with the other hand/forceps, gently pull the OAZ along the OAZ/CMZ line. The CMZ easily separates from the OAZ along this line. Continue along this line around the entire culture until the two regions are completely separated.</li>
	<li>Study the separated OAZ and CMZ fractions for molecular analysis such as RNA-Seq 24 or biochemical analysis such as western blot or co-immunoprecipitation 23,25.</li>
</ol>

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The authors declare that they have no competing financial interests.

Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni...

The clinically relevant, mock cataract surgery, ex vivo epithelial wound healing model described here was developed to provide a tool for investigating the mechanisms that regulate repair of epithelial tissues in response to an injury. Key features that were aimed for in creating this model included 1) providing conditions that closely replicated the in vivo response to wounding in a culture setting, 2) ease of modulating the regulatory elements of repair, and 3) ability to image the repair process, in its entirety, in real time. The challenge, therefore, was to create a culture model in which it was possible to study, and manipulate, epithelial wound repair in the cells&#8217; native microenvironment. The availability of this wound-repair model opens new possibilities for identifying the endogenous signaling cues from matrix proteins, cytokines and chemokines that regulate the repair process. In addition, the model is ideal for examining how an epithelium is able to move as a collective sheet to re-epithelialize the wound area 2,3, and for determining the lineage of mesenchymal leader cells at the wound edge that function in directing the collective migration of the injured epithelium 4. This model also provides a platform with which to identify therapeutics that could promote effective wound healing and prevent aberrant wound repair5.
There are already a number of available wound-repair models, both in culture and in vivo, which have provided most of what is known about the wound repair process today. In animal injury models, such as cornea 6-12 and skin13-17, there is the opportunity to study the response of the tissue to wounding in the context of all the repair mediators that could be involved in the process, including contributions from the vasculature and nervous system. However, there are limitations to manipulating the experimental conditions in vivo, and it is not yet possible to conduct imaging studies of the repair response in vivo, continuously over time. In contrast, most in vitro wound-repair culture models, such as the scratch wound, can be easily manipulated and followed over time but lack the environmental context of studying wound healing in the in vivo tissue. While ex vivo models offer the advantage of studying the injury repair process continuously over time in the context of the cells&#8217; microenvironment coupled with the ability to modulate the molecular regulators of repair at any time point in the process, there are few models that fit these parameters.
Here is described a procedure to generate highly reproducible ex vivo epithelial wound healing cultures that reproduce an epithelial tissue&#8217;s response to a physiological wounding. Using the chick embryo lens as a tissue source, an ex vivo mock cataract surgery is performed. The lens is an ideal tissue to use for these studies since it is self-contained within a thick basement membrane capsule, avascular, not innervated, and free of any associated stroma18,19. In the human disease, cataract surgery addresses vision loss due to opacification of the lens, and involves removal of the lens fiber cell mass, which comprises the bulk of the lens. Following cataract surgery vision is restored through the insertion of an artificial intraocular lens. The cataract surgery procedure, through removal of fiber cells, induces an injury response in the adjacent lens epithelium, which responds by re-epithelialization of the posterior area of the lens capsule that had been occupied by the fiber cells. In cataract surgery, as in most wound repair responses, there sometimes occurs an aberrant fibrotic outcome to the wound healing response, associated with the emergence of myofibroblasts, which in the lens is known as Posterior Capsule Opacification20-22. To generate the cataract surgery wound healing model, a cataract surgery procedure is mimicked in lenses removed from the chick embryo eye to produce a physiological injury. Microsurgical removal of lens fiber cells results in a very consistent circular wound area surrounded by the lens epithelial cells. This cell population remains firmly attached to the lens basement membrane capsule and is injured by the surgical procedure. The epithelial cells migrate onto the denuded area of the endogenous basement membrane to heal the wound, led by a population of vimentin-rich mesenchymal cells known in the repair process as leader cells1. With this model the response of an epithelium to injury can be easily visualized and followed with time in the context of the cells&#8217; microenvironment. The cells are readily accessible to modifications of the expression or activation of molecules expected to play a role in wound repair. A powerful feature of this model is the ability to isolate and study migration-specific changes in the framework of wound healing. The ability to prepare large numbers of aged matched ex vivo wound healing cultures for studies is another advantage of this model. Thus, this model system provides a unique opportunity to tease apart mechanisms of wound repair and test therapeutics for their effect on the wound healing process. The ex vivo mock cataract surgery model is expected to have wide applicability, providing a critical resource for studying mechanisms of injury repair.

<table><tbody><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>S271-3</td><td>Use at 140 mM in TD Buffer</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>P217-500</td><td>Use at 5 mM in TD Buffer</td></tr><tr><td>Sodium Phosphate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>S0876</td><td>Use at 0.7 mM in TD Buffer</td></tr><tr><td>D-glucose (Dextrose)</td><td>Fisher Scientific</td><td>D16-500</td><td>Use at 0.5 mM in TD Buffer</td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-1</td><td>Use at 8.25 mM in TD Buffer</td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>A144-500</td><td>Use to pH TD buffer to 7.4</td></tr><tr><td>Media 199</td><td>GIBCO</td><td>11150-059</td><td></td></tr><tr><td>L-glutamine</td><td>Corning/CellGro</td><td>25-005-CI</td><td>Use at 1% in Media199</td></tr><tr><td>Penicillin/streptomycin</td><td>Corning/CellGro</td><td>30-002-CI</td><td>Use at 1% in Media199</td></tr><tr><td>100 mm petri dishes</td><td>Fisher Scientific</td><td>FB0875711Z</td><td></td></tr><tr><td>Stericup Filter Unit</td><td>Millipore</td><td>SCGPU01RE</td><td>Use to filter sterilize Media</td></tr><tr><td>Dumont #5 forceps (need 2)</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>35 mm Cell Culture Dish</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>27 G 1 ml SlipTip with precision glide needle</td><td>BD</td><td>309623</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>Standard Forceps</td><td>Fine Science Tools</td><td>91100-12</td><td></td></tr><tr><td>Other Items Needed: General dissection instruments,&amp;nbsp; fertile white leghorn chicken eggs, check egg incubator (humidified, 37.7&amp;deg;C), laminar flow hood, binocular stereovision dissecting microscope</td><td></td><td></td><td></td></tr></tbody></table>

establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Ex Vivo Model created to study the wound healing process in the cells&#8217; native microenvironment
To investigate mechanisms involved in regulating wound healing of an epithelium within the cells&#8217; native microenvironment, a clinically relevant ex vivo mock cataract surgery model was created. This model is created from lens tissue which offers many advantages due to its intrinsic properties: 1) the lens is a self-contained organ surrounded by a thick basement membrane ...

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Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

establishment-clinically-relevant-ex-vivo-mock-cataract-surgery-model

Research

JoVE Journal

Developmental Biology

6.5K Views.  Thomas Jefferson University. Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment. 

Video: Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Bioengineering

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation results in calcium mobilization events that propagate from cell to cell, which are essential for initiating cellular motility into the wound bed, promoting efficient wound healing. The Trinkaus-Randall lab has developed a methodology for imaging the corneal wound healing response in ex vivo murine globes in real time. This approach involves enucleating an intact globe from a mouse that has been euthanized per established protocols and immediately incubating the globe with a calcium indicator dye. A counterstain that stains other features of the cell can be applied at this stage to assist with imaging and show cellular landmarks. The protocol worked well with several different live cell dyes used for counterstaining, including SiR actin to stain actin and deep red plasma membrane stain to stain the cell membrane. To examine the response to a wound, the corneal epithelium is injured using a 25 G needle, and the globes are placed in a 3D printed holder. The dimensions of the 3D printed holder are calibrated to ensure immobilization of the globe throughout the duration of the experiment and can be modified to accommodate eyes of different sizes. Live cell imaging of the wound response is performed continuously at various depths throughout the tissue over time using confocal microscopy. This protocol allows us to generate high-resolution, publication-quality images using a 20x air objective on a confocal microscope. Other objectives can also be used for this protocol. It represents a significant improvement in the quality of live cell imaging in ex vivo murine globes and permits the identification of nerves and epithelium.

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

The present work describes a novel experimental protocol that utilizes a 3D printed holder to enable high-resolution live cell imaging of enucleated globes. Through this protocol, the cellular calcium signaling activity in wounded corneal epithelium from ex vivo globes can be observed in real time.

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation ...

Biochemistry

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Medicine

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched in epitheliotropic growth factors that are essential in corneal wound healing. Unlike AS, PRP is based on a differential centrifugation system, yielding more platelet-derived growth factors. Autologous conditioned serum (ACS) not only preserves the preparation of AS and PRP, but also focuses on immune-modulating properties, which are important in inflammatory diseases.
The lack of standardized protocols and high preparation costs are limitations for the clinical application of ACS. This video experiment demonstrates a standard operating procedure for preparing modified autologous conditioned serum (mACS) eye drops. First, glycerol was added into heparin syringes as the blood cell stabilizer during hypoxic incubation. To activate the blood cells, a 4 h incubation at 37 &#176;C was initiated. Then, the blood samples were centrifuged at 3,500 &#215; g for 10 min at room temperature. After filtration of the supernatant through a 0.22 &#181;m filter, the mACS eye drops were fully prepared.
A tentative try-out of the therapeutic effect of mACS showed that it may have competitive advantages over conventional AS in the corneal wound healing in ex vivo mouse eyes. The AS used in this study was prepared according to published studies and the clinical practice in our hospital. Therefore, the efficacy of mACS on ocular surface diseases could be evaluated in future research through in vivo animal studies and clinical trials.

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

This article describes a protocol to simplify the process and render the preparation of autologous conditioned serum (ACS) less expensive. No special syringes or surface-coated glass beads are needed. Moreover, the modified ACS (mACS) has competitive advantages over conventional autologous serum in the corneal wound healing of murine eyes ex vivo.

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched ...

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

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In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

An Epithelial Abrasion Model for Studying Corneal Wound Healing

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica