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 amount of SSFM to be prepared depends on the number of corneas to be processed. Usually 30 mL is enough media for 4 corneas.</li>
	</ol>
	</li>
	<li>Dissection 
	NOTE: Perform this step in a dissection hood or a chemical hood. The eyes are shipped with lids still attached in individual bags to protect the globes.
	<ol>
		<li>Remove globes from lids with a straight-edge surgical blade on an ethanol-cleaned chopping board. Remove excess fatty tissue from the eye using either a blade or a small scissors (Figure 3A, 3B).</li>
		<li>After removing the globe from the lid, hold the globe posteriorly with forceps and immediately dip the eye in phosphate-buffered saline (PBS). Quickly dip it 3x in 10% iodine (in a 100 mL beaker). Quickly dip 2x in PBS (~100 mL in a beaker, change PBS frequently).</li>
		<li>Using a clean towel or tissue, wrap the eye circumferentially with enough pressure to have a taut corneal surface to cut with the trephine. 
		NOTE: Take care to prevent the towel or tissue from contacting the cornea.</li>
	</ol>
	</li>
	<li>Wounding
	<ol>
		<li>Use a 6 mm trephine to wound the center of the cornea. Penetrate the epithelium and anterior stroma without making a full-thickness wound through the entire cornea. 
		NOTE: If the endothelium is penetrated, a loss of pressure and leaking fluid will be seen. In this case the eye should be discarded.</li>
		<li>Place the trephine in the center of the cornea, rotate it 180&#176; clockwise and counter-clockwise 5x (each time the direction is changed it will count as one time) while applying light pressure to deepen the wound. 
		NOTE: The wound should now be deep enough to allow a tissue flap to be lifted using a pair of forceps. If this is not the case repeat step 1.3.2.</li>
		<li>Lift the flap from the edges. At the same time, either with the other hand or with a second person, use a blade, cutting parallel to the globe to cut away the tissue as the forceps continue to lift off the anterior cornea within the wound margin. At the conclusion of this step there should be a circular wound located at the center of the cornea (See Figure 3C, 3D).</li>
	</ol>
	</li>
	<li>Cutting and Removing the Cornea from the Globe
	<ol>
		<li>Holding the eye with the tissue, make a small incision 1 mm away from the edge of the cornea with a blade so that the limbus is included in the organ culture.</li>
		<li>Using small, sharp scissors access the incision created in the prior step to cut around the globe, keeping a millimeter margin throughout the cornea to keep the limbus intact.</li>
		<li>Place the cornea in a 60 mm dish with 1 mL of PBS, wound side down until mounting.</li>
	</ol>
	</li>
	<li>Mounting
	<ol>
		<li>Make sure the agar has come to a warm temperature (approximately 25 &#176;C).</li>
		<li>With two pairs of forceps, create a cup by holding two sides of the cornea with the endothelial side up. Add the warmed-up agar solution into the cornea using a sterile transfer pipette until it is full.</li>
		<li>After the agar hardens (usually about 30-45 s) carefully flip the cornea with the agar into 60 mm plate (Figure 3E). Cover with a lid.</li>
	</ol>
	</li>
	<li>Incubation
	<ol>
		<li>Add 4 mL of SSFM to the plate, maintaining corneas at an air-liquid interface at the limbal border in 5% CO2 at 37 &#176;C. Refresh media after 24 h and thereafter every other day. 
		NOTE: If performing a transfection into the wound, omit the antibiotics until after transfection.</li>
		<li>Wet the corneal surface once daily by adding 1 drop of SSFM from the conditioned media in the dish to maintain moisture. For this, take the dish out of the incubator, place it under the hood, remove the dish lid, wet the surface with media from dish using a sterile pipette, cover again and put it back at the incubator.</li>
		<li>For gene knockdown, treat the wound with gene-targeting or control siRNA that is complexed to a lipid-mediated carrier as per the supplier&#39;s instructions (see below).</li>
		<li>Mix 5 &#181;L (50 pmol) of siRNA into 50 &#181;L of reduced-serum minimum essential media (e.g., Opti-MEM). Mix 2 &#181;L of transfection reagent into 50 &#181;L of reduced-serum media. Let this sit for 5 min and then mix them.</li>
		<li>Add 200 &#181;L of reduced-serum minimum media to the reagent/siRNA mixture.</li>
		<li>Pipette dropwise onto the wound and incubate for 3 h.</li>
		<li>Wash out siRNA from corneal surface with media in the dish.Change the incubation media to SSFM + antibiotics (see the Table of Materials). Continue incubation as mentioned previously (steps 1.6.1-1.6.2).</li>
	</ol>
	</li>
</ol>
2. Histology: Paraffin Sections and Immunostaining
<ol>
	<li>Preparing the Tissue
	<ol>
		<li>After a two-week incubation, if using some of the tissue for quantitative real time polymerase chain reaction (qRT-PCR) analysis, before fixing, cut the cornea in half through the wound. Place this half or only &#188; (either is enough tissue) into stabilizing RNA-protect reagent.</li>
		<li>Using a standard isolation kit, isolate RNA and perform qRT-PCR. 
		NOTE: Alternatively, the wounded part only can be isolated and tested for gene expression.</li>
		<li>Place the other half of the cornea into tissue pathology cassettes and submerge in fixative (10% formalin) for 2-4 days at room temperature (RT).</li>
		<li>Paraffin embed this half of the wounded cornea using standard techniques. 
		NOTE: Orient the wounded cornea to ensure that the tissue sectioning will produce a cross-section of the cornea.</li>
	</ol>
	</li>
	<li>Immunostaining using 3,3&#39;-diaminobenzidine (DAB)
	<ol>
		<li>Day 1
		<ol>
			<li>Label slides properly using a pencil. De-paraffinize the tissue by placing slides into a jar with clearing agent (2 changes, 10 min each).</li>
			<li>Rehydrate the tissue by transferring the slides into ethanol at decreasing concentrations (100%, 100%, 70%, 50%, dH2O, dH20, 5 min for each change).</li>
			<li>Perform antigen retrieval by microwaving the slides in a plastic jar with citrate buffer (10 mM, pH 6.4) for 5 min. First cycle 5 min at 50% power. Refill the jar with citrate buffer and repeat. Cool down for 10 min.</li>
			<li>Wash 3x with PBS, 2 min each. Permeabilize tissue with 1% Triton X-100 in PBS 10 min at RT. Block sections with 3% normal goat serum (NGS) for 1 h at RT in humid chamber.</li>
			<li>Incubate tissue with primary antibody (1:100 or as the supplier suggests) in 3% NGS overnight at 4 &#176;C (300 &#181;L per slide).</li>
		</ol>
		</li>
		<li>Day 2
		<ol>
			<li>Rinse slides 3x with PBST (PBS plus 1% Tween 20), 2 min each. Place slides in 3% H2O2 for 10 min to block endogenous peroxidase. Wash 3x with PBST, 2 min each. Incubate sections with HRP secondary antibody (1:250) in 3% NGS for 1 h at RT (300 &#181;L per slide).</li>
			<li>Wash slides 3x PBST, 2 min each. Treat slides with the DAB kit. Add 300 &#181;L/slide for 3 min. Wash slides with dH2O 2x (quick dips).</li>
			<li>Counterstain with Hematoxylin for 20 s. Wash the slide with dH2O 2x (quick dips). Stain with bluing agent for 20 s. Rinse in dH2O for 20 s. Dehydrate tissue by placing slides into increasing concentrations of ethanol (50%, 70%, 100%, 100%, all quick dips).</li>
			<li>Dry slides on a paper towel under the hood for 10-20 min.</li>
			<li>Mount slides using 1 drop of mounting media, cover with coverslip. Label and store at RT.</li>
			<li>Image the slides under a microscope and quantify DAB signal with ImageJ7 (see section 4).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Immunostaining: Fluorescence
	<ol>
		<li>On Day 1 follow steps described in step 2.2.1. Perform the following steps on Day 2.</li>
		<li>Rinse slides 3x in PBST for 2 min each. Incubate sections with fluorophore-tagged secondary antibody in 3% NGS (1:200) for 1 h at RT.</li>
		<li>Wash 3x in PBST, 2 min each. Mount slides using 1 drop of 4&#8242;,6-diamidino-2-phenylindole (DAPI) mounting media and cover with a coverslip. Dry on paper towel under the hood for 30 min. Store in the dark at 4 &#176;C until fluorescence imaging.</li>
	</ol>
	</li>
	<li>Quantification using Image J
	<ol>
		<li>Download the &#34;Fiji&#34; version of ImageJ, which includes the necessary plugins for DAB staining quantification.</li>
		<li>Open an image in Fiji and select Image &#8594;&#160;Color &#8594;&#160;Color Deconvolution.</li>
		<li>Select &#34;H DAB&#34; as the stain and then click OK. Three new images will appear. Select the image that contains only DAB staining.</li>
		<li>To quantify stromal staining only, use the ImageJ eraser function to remove the epithelium from the DAB image.</li>
		<li>Select Analyze &#8594; Measure (or Ctrl + M) and record the value.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

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 organ culture model system is particularly useful for testing putative healing agents since the wound is localized and thus there is a clear margin between wounded and non-wounded tissue in the same cornea (Figure 4). In addition, a mechanical trephine wound allows direct access to the stroma, which is excellent for administration of siRNAs into the wound (Figure 5). Although it is not shown here, viral transduction into corneal tissue in organ culture has also been demonstrated32,33,34. Another permutation of this assay would be to infect corneas with a reporter construct of interest and image gene expression in real time after wounding. In terms of translation to in vivo studies, this same procedure can be accomplished in rabbits35 and thus organ culture on human, pig, or rabbit corneas can be compared to in vivo results. In our experience, the data that we obtained using the organ culture model with siRNA treatment have translated into similar findings in vivo (unpublished data). Since the organ culture corneas lack a functional limbal vasculature, tears, and aqueous humor, each investigator must assess if this will be a useful model for their studies. Resident activation of immune cells has been demonstrated, but the exact parallel to in vivo studies is not yet clear1.
A key step in the protocol is not to penetrate the cornea by wounding too deeply. This will be obvious as the anterior chamber fluid will leak in this case. If this occurs, the globe should be discarded. To produce an even mechanical wound, grip the lip of the demarcated tissue within the trephined area with a forcep and then move the surgical blade parallel to the corneal surface to cut the tissue away within the boundaries of the trephine wound. The corneal surface should not dry out thus we recommend that after making the wound and cutting out the cornea from the globe, place the cornea face down in PBS until the agar mixture is at the correct temperature. Making sure that the agar is not too hot will avoid endothelial damage.
A limitation of this model is that the use of a trephine to produce a wound is uneven and cannot be reproduced identically from cornea to cornea compared to laser-induced wounds36. However, naturally occurring wounds are not all equivalent in depth and a large body of data suggest that any breach in the basement membrane generates myofibroblast development and haze in the stroma, whereas regeneration of the basement membrane leads to diminished scarring37,38,39. This pig corneal organ culture model employs a severe wound in which the basement membrane is removed within the area of the trephine. Development of myofibroblasts and fibrotic markers in the corneal stroma have been consistently and reproducibility achieved using this model system. Some epithelial staining is usually evident that darkens in the wounded and regrown epithelium. This has been demonstrated in other corneal organ culture reports40 but is absent in most corneas from in vivo mouse and rabbit studies41,42. However, a study performed in an in vivo canine model demonstrated strong epithelial &#945;-SMA staining in the epithelium after wounding43. The siRNA that promoted regenerative healing in our studies also significantly reduced this epithelial immunostaining, suggesting that the epithelium may be undergoing EMT (fibrotic scarring) when it regrows in organ culture. In addition, omission of a primary antibody in the staining protocol of a wounded cornea resulted in the total absence of staining7 suggesting that the immunostaining is specific. Frozen sections (not shown) were similar to paraffin sections in this regard. However, because the slight but variable background staining in the epithelium, our lab has only quantified the stromal staining, which appears to have no background histological issues7.
If wounding human corneas, obtaining corneas not used for transplant instead of full globes may be more cost effective40. In this case, the cornea will be wounded without the aid of the pressure afforded by the globe. Additional wounding strategies may include corneal burns, which have been extensively utilized in vivo to produce a scar42.
In summary, the advantages of this system for a 3D tissue wound healing assay is its reproducibility and cost savings with only standard equipment needs, making it an excellent resource for observing and quantifying the effects of agents on tissue healing.

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, 4F). This tissue was immunostained for alpha-smooth muscle actin (&#945;-SMA), the expression of which characterizes myofibroblasts. There is a dramatic increase in &#945;-SMA immunostaining in the stroma as detected by colorimetric DAB substrate. There was also an increase in epithelial reactivity that may suggest EMT transition7 (see the Discussion). Disorganization in the epithelium and stroma was evident. A wounding experiment at lower magnification and with fluorescent immunostaining instead of DAB is shown (Figure 4G, 4H). The wound margin is visible as well as a gradient of active myofibroblasts from the anterior to posterior stroma.
Although fibrotic markers are expressed by one week (Figure 4), to obtain consistent and reliable development of fibrotic markers, a two-week time point was chosen. In Figure 5 an assay using a one-time application of control or gene-targeting siRNA to be tested for promoting regenerative healing is demonstrated. In this case, the targeting siRNA was for USP10, a deubiquitinase. Pathological myofibroblasts demonstrated increased adhesion through the accumulation of &#945;v-integrins in focal adhesions21. Our previous studies showed that &#945;v&#946;1 and &#945;v&#946;5 are important fibrotic integrins in corneal stromal healing7. Integrins bind ECM outside the cell and together they are internalized. The internalized integrin is ubiquitinated and sent for degradation in the lysosome or the ubiquitin tag is removed by a deubiquitinase (DUB) and the integrin is recycled to the cell surface. We discovered that an increase in the gene expression of the DUB (USP10) increased the rate of ubiquitin removal from the integrin subunits &#946;1 and &#946;5 leading to a resultant accumulation of &#945;v/&#946;1/&#946;5, on the cell surface, with subsequent TGF&#946; activation and induction of fibrotic markers7. Knockdown of USP10 in corneal organ culture prevented the appearance of fibrotic markers7.&#160;An example of these results is shown in Figure 5. As above, &#945;-SMA is utilized as a marker for myofibroblasts. Another indicator of scarring is Fibronectin-EDA (FN-EDA), a splice variant of FN that contains an RGD, &#945;v integrin binding domain22,23,24. It is also termed cellular FN (c-FN). It serves as a key fibrotic marker since FN-EDA is not in circulating plasma but instead is only expressed and secreted by cells under fibrotic conditions25. In Figure 5A-5C immunostaining for &#945;-SMA is shown. Compared to unwounded (Figure 5A), wounding plus control siRNA (Figure 5B) showed a dramatic increase in &#945;-SMA protein expression, whereas addition of USP10 siRNA7 dramatically reduced expression in the stroma and epithelium. Similarly, compared to unwounded (Figure 5D), wounding plus control siRNA (Figure 5E) demonstrated a dramatic increase in fibronectin-EDA protein expression compared to treatment with USP10 siRNA (Figure 5F). Immunohistology for the target protein (in this case, USP10) was used to demonstrate successful knockdown7. In addition, performing qRT-PCR can assure gene knockdown in the tissue or also to assay for other fibrotic markers7. ImageJ can be used to quantify signal in the stroma only or total signal (Figure 5G). At least 3 corneas for each condition being tested should be used to quantify immunostaining to generate statistical significance as we have shown here and previously published7. Other proteins that have been routinely utilized for fibrotic markers are collagen III expression and an increase in integrin expression1,26.
In Figure 6, the use of ex vivo cornea culture is demonstrated as a toxicology assay. In this experiment, corneas were either left unwounded (Figure 6A), wounded (Figure 6B), or wounded and treated with 10 &#181;M Spautin-127, which was added to the cell culture media for increasing periods of time before wash out (Figure 6C-6F). Spautin-1 is a drug that non-specifically targets USP1027. Because of our success with USP10 siRNA, Spautin treatment was tested for effectiveness in preventing scarring. Unlike the siRNA, Spautin at this concentration was toxic to the tissue. Increasing time with Spautin-1 in culture prevented re-epithelialization and resulted in qualitative cell death, disorganized matrix and stromal vacuoles suggesting that Spautin-1 does not promote healing at the concentration assayed. Standard histological assays can be employed to quantify cell proliferation or apoptosis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58562/58562fig1.jpg" /> 
Figure 1: Cross-section of a human eye with an expanded view of the cornea. In primates and chickens, histologically there are five distinct layers: epithelium, Bowman&#39;s membrane, stroma, Decement&#39;s membrane, and endothelium28,29. In all other mammals, Bowman&#39;s membrane is not visible histologically. At the transmission electron microscopic level, a basement membrane is observed separating the corneal epithelium and stroma in all corneas including those with a Bowman&#39;s membrane. An intact Bowman&#39;s membrane or basement membrane separating the epithelium from the stroma is a necessary to prevent scarring in all mammals. Image reprinted with permission from AllAboutVision.com (http://www.allaboutvision.com/resources/cornea.htm). <a href="https://www.jove.com/files/ftp_upload/58562/58562fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58562/58562fig2v2.jpg" /> 
Figure 2: Diagram of the cellular events that lead to corneal scarring. This diagram depicts the basic events that unfold in the anterior cornea after wounding. (A) Depiction of the epithelium, basement membrane, stroma, and the quiescent cells embedded in the stroma, i.e, keratocytes.(B) The red triangle depicts a wound, which can be mechanical, an ulcer, virus, or persistent infection. (C) After wounding, in which the Bowman&#39;s or basement membrane is breached, the cells around the wound apoptose. (D and E) An influx of cells repopulate the wound from resident keratocytes or bone marrow-derived fibroblasts and transition into activated fibroblasts or directly into myofibroblasts. (F) These adherent pathological myofibroblasts create an autocrine loop of TGF&#946;&#160;activation and secretion of disorganized fibrotic matrix that promotes corneal haze and scar formation. In a regeneratively healed wound, myofibroblasts appear but have apoptosed in the healed tissue6,29,30. <a href="https://www.jove.com/files/ftp_upload/58562/58562fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58562/58562fig3.jpg" /> 
Figure 3: Receiving and processing corneas for organ culture. (A) Pig eyes are received with lids to protect the cornea during shipping. (B) Image of the globe after tissue is removed. (C) Image of a 6 mm trephine. (D) Wounding of the central cornea with a trephine. (E) Image of the mounted cornea after removal from the globe. <a href="https://www.jove.com/files/ftp_upload/58562/58562fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58562/58562fig4.jpg" /> 
Figure 4: Corneal tissue after wounding. Immunohistological analysis of unwounded (control) or wounded corneas. Pig corneas were either left unwounded (control) or wounded. Tissue sections were immunostained with antibody to alpha-smooth muscle actin (&#945;-SMA) to identify myofibroblasts. (A and B) Control, unwounded. (C and D) Wounded and fixed at 6 h post-wounding. Epithelium is removed (arrow). (E and F) Wounded and fixed at 6 days post-wounding. Stroma has filled-in and the epithelium has regrown (arrow head). Representative activated myofibroblasts are denoted with asterisks (*). (G, H) Low magnification images of &#945;-SMA immunostaining 2 weeks after wounding: &#945;-SMA (red), DAPI (blue). Images were captured using an upright fluorescence/brightfield microscope with a CCD camera. Scale bar = 100 &#181;m (A, C, E); 50 &#181;m (B, D, F);&#160;200 &#181;m (G, H). <a href="https://www.jove.com/files/ftp_upload/58562/58562fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58562/58562fig5.jpg" /> 
Figure 5. Testing regenerative healing agents. Pig corneas were either (A and D) unwounded (control), (B and E) wounded and treated with control siRNA, or (C and F) wounded and treated with USP10 siRNA. Immunostaining for (A-C) &#945;-SMA or (D-F) Fibronectin-EDA. After treatment with USP10 siRNA, &#945;-SMA was reduced by 2.2 &#177; 0.6 fold ***p &#60; 0.001 and FN-EDA 3.3 &#177; 1.2 fold **p &#60; 0.01. Images were captured using an upright fluorescence/brightfield microscope with a CCD camera. Scale bar = 50 &#181;m. (G) Corneal stromal staining as quantified by ImageJ. Statistical significance was calculated by one-way ANOVA with Bonferroni&#39;s test. Figure has been adapted with permission from Gillespie et al.7. <a href="https://www.jove.com/files/ftp_upload/58562/58562fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58562/58562fig6.jpg" /> 
Figure 6: Testing agents for effects on reepithelialization - toxicology studies. Immunostaining for &#945;-SMA. Pig corneas were (A) unwounded, and (B) wounded. (C-F) Cornea were wounded and incubated with 10 &#181;M Spautin-1. The inhibitor was washed out and replaced by media after (C) 2 days, (D) 4 days, (E) 6 days, (F) 14 days. All media changes during the incubation period included Spautin as indicated. All corneas were fixed and embedded in paraffin after 2 weeks in culture. Images were captured using an upright fluorescence/brightfield microscope with a CCD camera. Scale bar = 50 &#181;m.&#160;<a href="https://www.jove.com/files/ftp_upload/58562/58562fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

11.5K Views.  SUNY Upstate Medical University. 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.

Video: Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

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 ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

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 ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

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, ...