We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

All the procedures followed in this study accorded with the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of National Taiwan University Hospital.
1. Isolation, Primary Culture Preparation, and Immunostaining of Bovine CECs
<ol>
	<li>Acquire fresh bovine eyes from a local abattoir.</li>
	<li>Disinfect the eyes in a 10% w/v povidone-iodine solution for 3 min. Wash them with pho...

<ol>
	<li>Maurice, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+location+of+the+fluid+pump+in+the+cornea.">The location of the fluid pump in the cornea.</a> J Physiol. 221 (1), 43-54 (1972).</li><li>Joyce, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferative+capacity+of+the+corneal+endothelium.">Proliferative capacity of the corneal endothelium.</a> Progress in Retinal and Eye Research. 22 (3), 359-389 (2003).</li><li>Morishige, N., Sonoda, K. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+and+its+receptor,+basic+fibroblast+growth+factor,+transforming+growth+factor+beta-1,+and+interleukin-1+alpha+messenger+RNA+production+in+human+corneal+endothelial+cells.">Epidermal growth factor and its receptor, basic fibroblast growth factor, transforming growth factor beta-1, and interleukin-1 alpha messenger RNA production in human corneal endothelial cells.</a> Invest Ophthalmol Vis Sci. 32 (10), 2747-2756 (1991).</li><li>Chen, K. H., Harris, D. L., Joyce, N. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+human+corneal+endothelial+cells+in+culture.">Growth of human corneal endothelial cells in culture.</a> Invest Ophthalmol Vis Sci. 30 (2), 248-253 (1989).</li><li>Peh, G. S., Beuerman, R. W., Colman, A., Tan, D. T., Mehta, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+endothelial+cell+expansion+for+corneal+endothelium+transplantation:+an+overview.">Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview.</a> Transplantation. 91 (8), 811-819 (2011).</li><li>Zhu, C., Rawe, I., Joyce, N. 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T., Chen, H. C., Chen, S. Y., Tseng, S. C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+p120+catenin+unlocks+mitotic+block+of+contact-inhibited+human+corneal+endothelial+monolayers+without+disrupting+adherent+junctions.">Nuclear p120 catenin unlocks mitotic block of contact-inhibited human corneal endothelial monolayers without disrupting adherent junctions.</a> J Cell Sci. 125 (15), 3636-3648 (2012).</li><li>Ho, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Matrix+Metalloproteinase+Activity+Reverses+Corneal+Endothelial-Mesenchymal+Transition.">Inhibition of Matrix Metalloproteinase Activity Reverses Corneal Endothelial-Mesenchymal Transition.</a> Am J Pathol. 185 (8), 2158-2167 (2015).</li><li>Kay, E. D., Cheung, C. C., Jester, J. V., Nimni, M. E., Smith, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+collagen+and+fibronectin+synthesis+by+retrocorneal+fibrous+membrane.">Type I collagen and fibronectin synthesis by retrocorneal fibrous membrane.</a> Invest Ophthalmol Vis Sci. 22 (2), 200-212 (1982).</li><li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+Signal+Regulates+Corneal+Endothelial-to-Mesenchymal+Transition.">Notch Signal Regulates Corneal Endothelial-to-Mesenchymal Transition.</a> Am J Pathol. 183 (3), 786-795 (2013).</li><li>Okumura, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ROCK+Inhibitor+Eye+Drop+Accelerates+Corneal+Endothelium+Wound+Healing.">The ROCK Inhibitor Eye Drop Accelerates Corneal Endothelium Wound Healing.</a> Invest Ophthalmol Vis Sci. 54 (4), 2493-2502 (2013).</li><li>Mannermaa, E., Vellonen, K. 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CECs are known for their propensity to undergo EnMT during cell proliferation. To develop strategies for suppressing the EnMT process for therapeutic purposes, a thorough understanding of the EnMT mechanism is necessary. We described 2 models to investigate EnMT, namely the bovine CEC in vitro culture model and rat corneal endothelium cryoinjury model. Our results demonstrated the EnMT process in both models. Furthermore, the EnMT-suppressing effect of marimastat was reproduced in both models, suggesting that th...

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4.
The limited proliferative potential of human CECs has been attributed to several factors, including reduced expression of the epidermal growth factor and its receptor in aging cells5, antiproliferative TGF&#946;2 in the aqueous humor6, and contact inhibition2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited8,9. Furthermore, CECs may undergo a phenotypic change during ex vivo expansion, resembling epithelial-mesenchymal transition (EMT)10-13. Endothelial-mesenchymal transition (EnMT) is characterized by cell junction destabilization, apical-basal polarity loss, cytoskeletal rearrangement, alpha smooth muscle actin expression, and type I collagen secretion14. All of these characteristics may abrogate the normal function of CECs, hampering the use of ex vivo cultured CECs in tissue engineering. Moreover, EnMT has been associated with the pathogenesis of several corneal endothelial diseases, including Fuchs endothelial corneal dystrophy and retrocorneal membrane formation15,16. Therefore, understanding the mechanism of EnMT may aid in manipulating the EnMT process and facilitate the regeneration of CECs to enable competent function. 
In this study, we described a method for isolating bovine CECs from the corneal button. In the primary culture in vitro, the EnMT process, including a phenotypic change, the nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, was observed. We further described a method for demonstrating EnMT in vivo by using a rat corneal endothelium cryoinjury model. Using these 2 models, we demonstrated that marimastat, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, can suppress the EnMT process. The described protocols facilitate the detailed analysis of the EnMT mechanism and the development of strategies for manipulating the EnMT process for further clinical application.

<table border="0" cellpadding="0" cellspacing="0" width="1447">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">trypsin</td>
			<td style="width:269px;">ThermoFisher Scientific</td>
			<td style="width:143px;">12604-013</td>
			<td style="width:340px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle medium and Ham&#39;s F12 medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human epidermal growth factor</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">insulin, transferrin, selenium&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cholera toxin</td>
			<td>Sigma</td>
			<td>C8052-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentamicin</td>
			<td>ThermoFisher Scientific</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">amphotericin B</td>
			<td>ThermoFisher Scientific</td>
			<td>15290-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>111219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">marimastat</td>
			<td>Sigma</td>
			<td>M2699-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-active beta-catenin antibody</td>
			<td>Millpore</td>
			<td>05-665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-snail antibody</td>
			<td>Santa cruz</td>
			<td>sc28199</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-slug antibody</td>
			<td>Santa cruz</td>
			<td>sc15391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11001</td>
			<td>for staining of ABC of bovine CECs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11003</td>
			<td>for staining of ABC of rat corneal endothelium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-rabbit IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11008</td>
			<td>for staining of snail and slug of bovine CECs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">antibody diluent</td>
			<td>Genemed Biotechnologies</td>
			<td>10-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">laser scanning confocal microscope</td>
			<td>ZEISS</td>
			<td>LSM510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">xylazine&#160;</td>
			<td>Bayer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tiletamine plus zolazepam</td>
			<td>Virbac</td>
			<td>N/A</td>
			<td>veterinary drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">proparacaine hydrochloride ophthalmic solution</td>
			<td>Alcon</td>
			<td>N/A</td>
			<td>veterinary drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1% atropine</td>
			<td>Wu-Fu Laboratories Co., Ltd</td>
			<td>N/A</td>
			<td>clinical drug&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.3% gentamicin sulfate</td>
			<td>Sinphar Group</td>
			<td>N/A</td>
			<td>clinical drug&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">basic fibroblast growth factor</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0024</td>
			<td>clinical drug&#160;</td>
		</tr>
	</tbody>
</table>

in vitro and in vivo models to study corneal endothelial-mesenchymal transition

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

After the isolation of bovine CECs, the cells were cultured in vitro. Figure 1 presents the phase contrast images of the bovine CECs. The hexagonal shape of the cells at confluence indicated that the cells were not contaminated by corneal stromal fibroblast during cell isolation. Figure 2 depicts the immunostaining that was performed using antibodies against ABC, snail, and slug at an indicated time point. Apart from phenotypic changes in the <em...

Watch this Scientific Journal Video about In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition at JoVE.com

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

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

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

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

The overall goal of this procedure is to investigate the mechanism of Corneal Endothelial-mesenchymal Transition In Vitro and In Vivo. This method can help answer questions about Corneal Endothelial-mesenchymal Transition in the field of Corneal Endothelial regeneration. The advantage of this technique is that it can be applied to both In Vitro and the In Vivo models.And is easy to manipulate. The implications of this technique is tend towards therapy of disease related to Corneal Endothelial-mesenchymal Transition. Such as formation, because the models can be used to select to us that inhibits the process of Corneal Endothelial-mesenchymal Transition.The the procedure will be miss Jung-Shen Chang, a technician from a laboratory. To begin this procedure, disinfect fresh bovine eyes in a 10%weight per volume Povidone-iodine solution for three minutes. Then, wash them with PBS.Harvest the corneal button with a scalpel and scissors and wash it with PBS Under a dissecting microscope, peel the Descemet's Membrane with forceps. After that, incubate the Descemet's Membrane in one milliliter of Trypsin at 37 degrees Celsius for 30 minutes. Next, collect the bovine CECs by centrifugation at 112 times g for five minutes.After centrifugation, re-suspend the cells in one milliliter of SHEM. Seed the cells in a six centimeter dish and culture them in SHEM. Subsequently, incubate the cells at 37 degrees Celsius in 5%CO2 and change the culture medium every three days.When the cells reach confluence, wash them in PBS. Then, incubate them in one millimeter of Trypsin at 37 degrees Celsius for five minutes. Collect them by centrifugation at 112 times g for five minutes.After five minutes, re-suspend the cell pellet in one millimeter of SHEM. Next, count the cells in a Hemacytometer. Following that, seed the cells on cover slides in a 24-well plate and culture the cells in SHEM.For investigating the ENMT-suppressing effect of Maramastat, incubate the cells in SHEM with 10 micromolar Maramastat in the culture medium and change the culture medium every three days. Then, fix the cells at an indicated time point with 250 microliters of 4%Paraformaldehyde for 30 minutes at room temperature. Afterward, permeablize them with 250 microliters of 0.5%Triton X-100 for five minutes.And block them with 10%BSA for 30 minutes. Subsequently, incubate the cells with primary antibodies against ABC, snail, and slug overnight at four degrees Celsius. The next day, wash the cells twice with PBS for 15 minutes each time.And incubate them with the Alexa Fluor conjugated secondary antibody at room temperature for one hour. After that, counterstain the cell nucleus by covering the cells with DAPI at two micrograms per milliliter. Wash the cells twice with PBS for 15 minutes each time.And mount them with anti-fading mounting solution before obtaining the immunoflourescent images. After anesthetizing a 12-week old male SD rat, gently pinch the skin of the animal to confirm proper anesthesia in the absence of skin twitching. Then, apply a drop of 0.5%Proparacaine Hydrochloride to the rat's right eye to minimize pain and the blink reflex.Subsequently, apply Tetracycline ointment to the left eye to prevent corneal dryness. Next, cool a stainless steel probe in liquid Nitrogen. Apply the stainless steel probe to the central cornea of the right eye for 30 seconds.Frequently instill PBS to the right eye during the procedure to prevent corneal dryness and apply 0.1%Atropine and 0.3%Gentamicin Sulfate immediately after cryoinjury once daily to relieve ocular pain resulting from Ciliary spasm and to prevent infection. After the procedure, keep the rat warm using a heat lamp and observe its recovery until it regains motor control. Apply Tetracycline ointment to the right eye to prevent corneal dryness during the recovery period and repeat the corneal cryoinjury procedure for three consecutive days.For delivering Maramastat or basic FGF into the interior chamber of the rat eyes, apply a drop of 0.5%Proparacaine Hydrochloride to the right eye of the anesthetized rat to minimize pain and the blink reflex. Next, irrigate the ocular surface with sterile PBS. Under an operating microscope, perform anterior chamber paracentesis by inserting a 30 gauge needle attached to a one millimeter syringe at the paralimbal clear cornea in a plain, above, and parallel to the iris Then, turn the needle bevel up and slightly depress the corneal wound to drain some aqueous humor and reduce the interocular pressure.Inject 0.02 milliliters of the drug intracamerally and gently compress the needle tract with a cotton tip during the needle withdraw. This figure presents the phase contrast images of the bovine CECs and cultures. The hexagonal shape of the cells at confluence indicates that the cells are not contaminated by corneal stromal fibroblasts during cell isolation.During the In Vitro culture of bovine CECs, the nuclear translocation of ABC, snail, and slug was detected through day 14. This figure illustrates the effect of Maramastat, a broad spectrum MMP inhibitor on the ENMT process of the In Vitro cultured bovine CECs. Here, are the external eye photographs of rats after cryoinjury, followed by intracameral injection.The photographs show reduced corneal edema after basic FGF injection. Whereas Maramastat further reduced corneal edema compared with basic FGF alone. And this figure shows immunostaining of the rat corneal button using antibody against ABC after cryoinjury followed by intracameral injection.The nuclear translocation of ABC is observed in the PBS group and is significantly increased in the basic FGF group, indicating activation of Wnt Beta-catenin signalling and the ENMT process. After intracameral injection, a basic FGF followed by Maramastat, the nuclear staining of ABC is diminished, suggesting the ENMT-inhibiting effect of Maramastat. After its development, this technique pave the way for researchers in the field of Corneal Endothelial-mesenchymal Transition, In Vitro and the In Vivo.After watching this video, you should have a good understanding of how to isolate bovine corneal endothelial cells and to perform corneal cryoinjury as well as intracameral injection

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Research

JoVE Journal

Developmental Biology

7.8K 次观看.  Far Eastern Memorial Hospital. The overall goal of this procedure is to investigate the mechanism of Corneal Endothelial-mesenchymal Transition In Vitro and In Vivo. This method can help answer questions about Corneal Endothelial-mesenchymal Transition in the field of Corneal Endothelial regeneration. The advantage of this technique is that it can be applied to both In Vitro and the In Vivo models.And is easy to manipulate. The implications of this technique is tend towards therapy of disease related to Corneal Endo...