Name: ex vivo corneal organ culture model for wound healing studies
Uploaded: 2019-02-15T12:30:00
Description: Watch this Scientific Journal Video about Ex Vivo Corneal Organ Culture Model for Wound Healing Studies at JoVE.com

This work was supported by NIH-NEI R01 EY024942, Research to Prevent Blindness, Upstate Medical University Unrestricted Research Funds, and Lions District 20-Y. Microscopy and image analysis of paraffin sections were performed at the Microscopy CORE and histological slide preparation was performed at the Biorepository and Pathology CORE at the Icahn School of Medicine at Mount Sinai.

1. Organ Culture
<ol>
	<li>Preparations
	<ol>
		<li>Prepare agar solution as follows. In a small flask, prepare 1% agar and 1 mg/mL bovine collagen in DMEM-F12 up to 20 mL. Bring to boil on a hot plate. Put the solution into a 50 mL conical tube. Place tube in a water bath on a hot plate to keep the solution from solidifying.</li>
		<li>Prepare supplemented serum-free media (SSFM) as per the composition provided in the Table of Materials. 
		NOTE: The necessary am...

This protocol describes a model for studying wound healing in a natural stratified 3D environment. Use of organ culture as an intermediate between cell culture and in vivo studies significantly reduces costs as well as reducing procedures on live animals. Other 3D models have been of great benefit to the field including self-synthesizing collagen gels made from primary human corneal fibroblasts2 or these same cells embedded in gels made from animal-derived collagens31. The ...

Scarring in the cornea resulting from injury, trauma, or infection can lead to debilitating opacities and permanent vision loss. Thus, there is a critical need to identify pathways that can be targeted for therapeutic intervention. Current treatment options are limited and consist primarily of corneal transplantations, which are not accessible to patients across the world. Both human (Figure 1) and animal corneas can be utilized for 2D and 3D cell culture studies1,2. Human cadaver corneas not suitable for transplant can be obtained from eye banks or centralized tissue banks (National Disease Research Interchange (NDRI)), and animal eyes can be obtained from an abattoir. Primary corneal epithelial cells, stromal fibroblasts, and more recently, endothelial cells, can be isolated and cultured from these tissues for wound healing and toxicology studies3,4,5. In addition to the importance of understanding the molecular basis of blinding eye disease, the accessibility of tissue and the ability to culture primary cells has made the cornea an important model system for study. The cornea is ideal for testing the effects of agents on scarring as the normal cornea is transparent and certain types of wounds create opacities or fibrotic scars (reviewed in6). Several in vivo corneal wound healing models have also been extensively utilized for scarring studies1. Less utilized has been the ex vivo corneal wound healing model7,8 that is describe in detail here. The goal of this method is to quantify scarring outcomes characterized by fibrotic makers in a 3D multicellular corneal ex vivo model system.
Corneal epithelial wounding that does not breach the epithelial basement membrane normally closes within 24-72 h9. Soon after wounding, the cells at the edge of the epithelium start spreading and migrating into the epithelial free surface, to reestablish epithelial barrier function. This activity is sequentially followed by activation of corneal basal cell proliferation first and, in a later stage, of precursor cells located at the outer limbal zone to achieve recovery of epithelial cell mass10,11. These wounds often heal without scarring. However, a wound that penetrates the basement membrane into the stroma often results in scar formation1. After corneal stromal wounding, the stroma is populated with cells of multiple origins including differentiated resident stromal cells as well as bone marrow-derived fibrocytes12,13,14. Fibrotic scarring is characterized by the persistence of myofibroblasts in a healing wound. These pathological myofibroblasts demonstrate increased adhesion through the accumulation of integrins in focal adhesions, contractile &#945;-smooth muscle actin (&#945;-SMA) stress fibers, and local activation of extracellular matrix (ECM)-sequestered latent-transforming growth factor-beta (TGF&#946;). The differentiation of epithelial-derived cells, known as epithelial to mesenchymal transition (EMT), may also contribute to scar formation6.
There is a delicate balance between cell differentiation and apoptosis after wounding. Because of the breach in the basement membrane, growth factors such as platelet-derived growth factor (PDGF) and TGF&#946; from tears and the epithelium bathe the stroma, inducing myofibroblast differentiation, a sustained autocrine loop of TGF&#946;&#160;activation, and the secretion of disorganized fibrotic ECM15,16. The persistence of myofibroblasts in the healed wound promotes haze and scarring in the cornea (Figure 2). However, in regeneratively healed wound although myofibroblasts develop, they apoptose and thus are absent or significantly reduced in number in the healed tissue (reviewed in reference6,10). Thus, research on fibrotic scarring has focused at least in part on targeting molecules that prevent excessive myofibroblast development or myofibroblast persistence17,18. Because myofibroblast persistence characterizes both scarring and fibrotic disease in all tissues19, the cornea may be useful as a model system to study general cellular mechanisms of fibrosis.
In our model system, the cornea is wounded with a cylindrical blade called a trephine while still in the globe. Human and pig corneas can be wounded with either a 6 or 7 mm trephine; for rabbit corneas a 6 mm trephine is preferred. The pig cornea is similar in size to the human cornea. Because they are cost effective and readily available in large numbers, pig corneas are routinely used for organ culture. Furthermore, antibodies and siRNAs made to react with human have consistently cross-reacted with pig7. After wounding, corneas are cut from the globe with the limbus intact and mounted on an agar/collagen base. The corneas are cultured in serum-free media plus stabilized vitamin C to simulate fibroblast proliferation and ECM deposition20. Neither the addition of serum nor growth factors are needed to induce myofibroblast formation7. Corneas are routinely fixed and processed for histology after two weeks of culture. For gene knockdown, or to test the effects of an agent on wound healing, the wound can be treated with siRNA in the wound after wounding7 or a soluble agent can be added to the media, respectively8.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10-010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep&#160;</td>
			<td>MP Biomedicals</td>
			<td>91670049</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Collagen Solution</td>
			<td>Advance Biomatrix</td>
			<td>5005</td>
			<td></td>
		</tr>
		<tr>
			<td>Pig eyes with lids attached&#160;</td>
			<td>Pel-freeze, Arkansas</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>6.0 mm trephine&#160;</td>
			<td>Katena</td>
			<td>K28014</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blade&#160;</td>
			<td>Personna</td>
			<td>0.009</td>
			<td></td>
		</tr>
		<tr>
			<td>Small scissor</td>
			<td>Fisher</td>
			<td>895110</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher</td>
			<td>08953-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Kim Wipes&#160;</td>
			<td>Kimberly-Clark&#8482; 34120</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dishes&#160;</td>
			<td>Falcon</td>
			<td>08-772B</td>
			<td></td>
		</tr>
		<tr>
			<td>Supplemented Serum- Free media (SSFM)</td>
			<td></td>
			<td></td>
			<td>Add all of the following components to DMEM/F-12:&#160; ITS, RPMI, Glutathione, L-Glutamine, MEM Non essential amino acids, MEM Sodium Pyruvate, ABAM, Gentamicin, Vitamin C.&#160;</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>11330</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS Liquid Media Supplement&#160;</td>
			<td>Sigma</td>
			<td>I3146</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>RPMI 1640 Vitamins Solution&#160;</td>
			<td>Sigma</td>
			<td>R7256</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Glutathione</td>
			<td>Sigma</td>
			<td>G6013</td>
			<td>Use at 1 &#181;g/mL. Freeze aliquots; do not reuse after thawing.</td>
		</tr>
		<tr>
			<td>1% L-glutamine solution&#160;</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution&#160;</td>
			<td>Gibco</td>
			<td>11140</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>MEM Sodium pyruvate solution&#160;</td>
			<td>Gibco</td>
			<td>11360</td>
			<td>1 M Stocks (1000X) and freeze in single use aliquits.&#160; Use from freezer each time media is made.</td>
		</tr>
		<tr>
			<td>ABAM&#160;</td>
			<td>Sigma</td>
			<td>A7292</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Gentamicin&#160;</td>
			<td>Sigma</td>
			<td>30-005-CR</td>
			<td>200X</td>
		</tr>
		<tr>
			<td>Vitamin C&#160;</td>
			<td>Wako</td>
			<td>070-0483</td>
			<td>2-0-aD Glucopyranosyl-Ascorbic Acid. 1 mM stocks (1000x)</td>
		</tr>
		<tr>
			<td>10% Iodine&#160;</td>
			<td>Fisher Chemical</td>
			<td>SI86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Path Cassettes&#160;</td>
			<td>Fisher</td>
			<td>22-272416</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td>Jackson Immuno Research</td>
			<td>005-000-121</td>
			<td>We use 3% NGS</td>
		</tr>
		<tr>
			<td>Mounting Media&#160;</td>
			<td>Thermo Scientific</td>
			<td>TA-030-FM</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe Clear&#160;</td>
			<td>Fisher</td>
			<td>314-629</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol</td>
			<td>Ultra Pure</td>
			<td>200CSGP</td>
			<td>200 Proof, diluted at 100%, 70%, 50%)&#160;</td>
		</tr>
		<tr>
			<td>Sodium citrate&#160;</td>
			<td>Fisher</td>
			<td>BP327</td>
			<td>10mM, pH 6.4</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>EMD Millipore</td>
			<td>M10742500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bluing agent&#160;</td>
			<td>Ricca Chemical Company</td>
			<td>220-106</td>
			<td></td>
		</tr>
		<tr>
			<td>1% Triton X-100</td>
			<td>Fisher</td>
			<td>9002-93-1</td>
			<td>Diluted in PBS</td>
		</tr>
		<tr>
			<td>0.1% Tween 20&#160;</td>
			<td>Fisher</td>
			<td>BP337</td>
			<td>Diluted in PBS</td>
		</tr>
		<tr>
			<td>3% Hydrogen Peroxide&#160;</td>
			<td>Fisher</td>
			<td>H324</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB Kit&#160;</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar&#160;</td>
			<td>Fisher&#160;</td>
			<td>BP1423-500</td>
			<td>Agar solution: prepare 1% agar and 1 mg/mL bovine collagen in DMEM-F12 up to 20 mL</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bermis</td>
			<td>13-374-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Moist Chamber</td>
			<td></td>
			<td></td>
			<td>Use any chamber, cover it with wet Wipe Tissue and then put a layer of Parafilm over it.</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen RNAprotect Cell Reagent</td>
			<td>Qiagen</td>
			<td>&#160;76104</td>
			<td></td>
		</tr>
		<tr>
			<td>Ambion PureLink RNA Mini Kit</td>
			<td>Thermo Scientific</td>
			<td>12183018A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Fibronectin-EDA Antibody</td>
			<td>Sigma</td>
			<td>F6140</td>
			<td>1:200 Diluted in&#160; 3% normal goat serum</td>
		</tr>
		<tr>
			<td>Anti-alpha smooth muscle actin Antibody</td>
			<td>Sigma</td>
			<td>A2547 or C6198 (cy3 conjugated)</td>
			<td>1:200 Diluted in 3% normal goat serum</td>
		</tr>
		<tr>
			<td>Permafluor&#160;</td>
			<td>Thermo Scientific</td>
			<td>TA-030-FM</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI&#160;</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Gt anti -MS IgG (H+L) Secondary Antibody, HRP&#160;</td>
			<td>Invitrogen</td>
			<td>62-6520</td>
			<td>1:100 diluted in 3% normal goat serum (for a-SMA, DAB staining)</td>
		</tr>
		<tr>
			<td>Gt anti -MS IgM (H+L) Secondary Antibody, HRP&#160;</td>
			<td>Thermo Scientific</td>
			<td>PA1-85999</td>
			<td>1:100 diluted in 3% normal goat serum (for FN-EDA, DAB staining)</td>
		</tr>
		<tr>
			<td>Gt anti -MS IgG (H+L) Secondary Antibody, Cy3&#160;</td>
			<td>Jackson Immuno Research</td>
			<td>115-165-146</td>
			<td>1:200 Diluted in&#160; 3% normal goat serum (for a-SMA, Fluorescence staining)</td>
		</tr>
		<tr>
			<td>&#160;Zeiss Axioplan2&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>SPOT-2</td>
			<td colspan="2">Diagnostic Instruments, Sterling Heights, Michigan</td>
			<td>CCD camera</td>
		</tr>
	</tbody>
</table>

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.

Immunohistochemistry is the primary assay utilized to analyze the success of the ex vivo wound healing experiment. Figure 4 depicts the epithelium and anterior stroma in control tissue (Figure 4A, 4B). Six hours after wounding, the epithelium was absent (Figure 4C, 4D). Six days after wounding as expected, the epithelium had regrown (Figure 4E</s...

Watch this Scientific Journal Video about Ex Vivo Corneal Organ Culture Model for Wound Healing Studies at JoVE.com

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

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 ...

This paper described a simple method for assaying wounding in a three-dimensional, multi-cellular organ culture model system. We do this by using pig eyes, but even if you are not familiar with eyes, you can do this assay. As an overview, we received porcine eyes, cut out the globes, make a circular wound in the cornea that goes through the epithelium and about one third of the stroma.We cut out the cornea, and mount it on an Agar base, and put this construct in the incubator. The tissue will fill in creating a scar in the cornea. You don't need to add any growth factors, or even serum.This is done in serum-free media. Although this is performed in the cornea, it can be used as a model system for fibrotic healing in general. As many of the cellular pathways that are activated during scarring are similar between systems.In a biosafety hood, use a straight edge surgical blade to remove the globe from the lid on an ethanol-cleaned chopping board, and remove the excess fatty tissue from each eye. Holding the cleaned globe posteriorly with forceps immediately dip the eye in PBS followed by three quick dips in 10%iodine, and two quick dips in PBS. Then wrap the eye circumferentially with a clean lab tissue to wrap with enough pressure to achieve a taut corneal surface without contacting the cornea.Using a six millimeter trephine, penetrate the epithelium in the anterior stroma at the center of the cornea without making a full thickness wound through the entire cornea, and rotate the trephine 180 degrees clockwise and counterclockwise five times while applying light pressure to deepen the wound. When the wound is deep enough to allow a tissue flap to be lifted with forceps, use a surgical blade to cut the flap parallel to the globe while continuing to lift the anterior cornea within the wound margin. When the entire flap has been removed, a circular wound should be located at the center of the cornea.To harvest the cornea grasp the eye with the lab tissue, and use a surgical blade to make a small incision one millimeter away from the edge of the cornea to include the limbus and tissue collection. Using small, sharp scissors continue the incision around the globe keeping a millimeter margin across the cornea to keep the limbus in tact. Then place the cornea, wound-side down, in a 60 millimeter, or 100 millimeter dish containing one milliliter of PBS.To mount the cornea, use two pairs of forceps to hold the cornea epithelial-side up to create a cup, and use a sterile transfer pipette to add warmed agar solution into the cornea until the cup is full. After the agar hardens, carefully place the cornea in a new 60 millimeter plate agar-side down and cover the plate with a lid. Then add four milliliters of supplemented serum-free medium to each plate of corneas maintaining the corneas at an air-liquid interface at the limbal border in a cell culture incubator at 37 degrees celsius in 5%carbon dioxide, and wetting the corneal surfaces once daily with one drop of supplemented serum-free medium from the conditioned medium in the dish to keep the tissues hydrated.For gene knockdown, mix five microliters of small interfering or siRNa with 50 microliters of reduced serum minimum essential medium and two microliters of transfection reagent. With 50 microliters of reduced serum medium per cornea. After five minutes, mix the two mixtures together, and add 200 microliters of reduced serum minimum medium to each siRNA mixture.Then pipette the siRNA solutions drop wise onto each wound. Then put the corneas into the incubator. After three hours, use the medium in each dish to wash the siRNA from the corneal surfaces, and replace the conditioned medium in each dish with fresh supplemented serum free medium plus antibiotics.Then return the corneal cultures to the cell culture incubator, and incubate for two weeks. Six hours after wounding, the corneal epithelium is absent. Six days after wounding, the epithelium regrows.Fluorescence amino staining with alpha smooth muscle actin reveals a gradient of active myofibroblasts along the wound margin from the anterior to posterior stroma. Immuno-histochemical staining for alpha smooth muscle actin reveals a dramatic increase in alpha smooth muscle actin protein expression in wounded plus control siRNA corneas. Compared to unwounded and target protein siRNA treated, wounded corneas.Similarly, fibronectin extra domain A is upregulated in siRNA treated wounded corneas compared to unwounded and target protein siRNA treated wounded corneas. Demonstrating a successful knock down of the target protein. Further, treatment of the wounded corneas with the toxin that nonspecifically targets the target protein prevents re-epithelialization and results in qualitative cell death, a disorganized matrix, and stromal vacuoles suggesting that the toxin does not promote healing at the concentration assayed.In conclusion, while attempting this procedure it's important to remember to keep the blade parallel to the tissue while cutting, so that it doesn't penetrate the globe. Different wounding techniques can be employed with this assay, including chemical burn or epithelial scrape. It can also be used for drug toxicity assays to determine if the epithelium heals and at what rate.This is a cost-savings ex vivo, organ culture model system that can precede your in vivo wound healing studies.

ex-vivo-corneal-organ-culture-model-for-wound-healing-studies

Research

JoVE Journal

Medicine

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

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 ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

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, ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

A Human Corneal Organ Culture Model of Descemet&#039;s Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...