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 phosphate-buffered saline (PBS) solution.</li>
	<li>Harvest the corneal button with a scalpel and scissors under sterile conditions. Peel the Descemet&#39;s membrane with forceps under a dissecting microscope (note that in this study, the bovine eyes were enucleated by a local abattoir; therefore, the first study procedure was the disinfection of the preenucleated eyes in the laboratory).</li>
	<li>Incubate the Descemet&#39;s membrane in 1 ml of trypsin at 37 &#176;C for 30 min. Collect the bovine CECs by centrifugation at 112 x g for 5 min. Resuspend the cells in 1 ml of supplemented hormonal epithelial medium (SHEM) containing equal volumes of HEPES-buffered Dulbecco&#39;s modified Eagle medium and Ham&#39;s F12 medium, supplemented with 5% fetal bovine serum, 0.5% dimethyl sulfoxide, 2 ng/ml of human epidermal growth factor, 5 mg/ml of insulin, 5 mg/ml of transferrin, 5 ng/ml of selenium, 1 nmol/l of cholera toxin, 50 mg/ml of gentamicin, and 1.25 mg/ml of amphotericin B.</li>
	<li>Seed the cells (approximately 1 x 105 cells per eye) into a 6 cm dish. Culture them in the SHEM. Incubate the dish at 37 &#176;C in an atmosphere of 5% CO2 in air. Change the culture medium every 3 days.</li>
	<li>When the cells reach confluence, wash them in PBS and incubate them in 1 ml of trypsin at 37 &#176;C for 5 min. Collect them by centrifugation at 112 x g for 5 min. Re-suspend the cell pellet in 1 ml of the SHEM.
	<ol>
		<li>Count the cells in a hemocytometer. Seed the cells on cover slides at a density of 1 x 104 per well in a 24-well plate, and culture the cells in the SHEM. For investigating the EnMT-suppressing effect of marimastat, incubate the cells in SHEM with 10 &#181;M of marimastat added into the culture medium, and change the culture medium every 3 days.</li>
	</ol>
	</li>
	<li>Fix the cells at an indicated time point with 250 &#181;l of 4% paraformaldehyde, pH 7.4, for 30 min at room temperature. Permeabilize with 250 &#181;l of 0.5% Triton X-100 for 5 min. Block with 10% bovine serum albumin for 30 min.</li>
	<li>Incubate the cells with primary antibodies against active beta-catenin (ABC), snail, and slug overnight at 4 &#176;C. The dilution of the primary antibodies used is 1:200. The antibodies are diluted in antibody diluent composed of 10 mM PBS, 1% w/v bovine serum albumin, and 0.09% w/v sodium azide.</li>
	<li>Wash the cells twice with PBS for 15 min, and incubate them with the Alexa Fluor-conjugated secondary antibody (1:100 in antibody diluent) at room temperature for 1 hr.</li>
	<li>Counterstain the cell nucleus by covering the cells with 2 &#181;g/ml of 4&#39;,6-diamidino-2-phenylindole, wash the cells twice with PBS for 15 min, and mount them with anti-fading mounting solution.</li>
	<li>Obtain immunofluorescent images at the excitation wavelengths of 405 and 488 nm by using a laser scanning confocal microscope with 20X objective magnification.</li>
</ol>
2. Rat Corneal Endothelium Cryoinjury Model and Intracameral Injection
<ol>
	<li>Anesthetize 12 week-old male Sprague-Dawley rats with intramuscular injections of 2% xylazine (5.6 mg/kg) and tiletamine plus zolazepam (18 mg/kg). Gently pinch the skin of the animals to confirm proper anesthesia in the absence of skin twitching.</li>
	<li>Instill one drop of 0.5% proparacaine hydrochloride to the right eye of each rat to minimize eye pain and the blink reflex. Instill tetracycline ointment to the left eye to prevent corneal dryness.</li>
	<li>Cool a stainless steel probe (diameter = 3 mm) in liquid nitrogen. Apply the stainless steel probe to the central cornea of the right eye for 30 sec. Frequently instill PBS to the right eye during the procedure to prevent corneal dryness.</li>
	<li>Instill 0.1% atropine and 0.3% gentamicin sulfate immediately after cryoinjury and once daily to relieve ocular pain resulting from ciliary spasm and to prevent infection.
	<ol>
		<li>After the procedure, keep the rats warm using a heat lamp, and observe their recovery every 15 min after anesthesia until they regain motor control. Additionally, apply tetracycline ointment to the right eye to prevent corneal dryness during the recovery period.</li>
	</ol>
	</li>
	<li>Repeat the corneal cryoinjury for 3 consecutive days.</li>
	<li>For delivering marimastat or bFGF into the anterior chamber of the rat eyes, anesthetize the rats as described previously. Instill one drop of 0.5% proparacaine hydrochloride into the right eye to minimize eye pain and the blink reflex.</li>
	<li>Irrigate the ocular surface with sterile PBS. Perform anterior chamber paracentesis under an operating microscope by inserting a 30 G needle attached to a 1 ml syringe at the paralimbal clear cornea in a plane above and parallel to the iris.</li>
	<li>Turn the needle bevel up, and slightly depress the corneal wound to drain some aqueous humor and reduce the intraocular pressure. Inject 0.02 ml of the drug intracamerally. Gently compress the needle tract with a cotton tip during the needle withdrawal.</li>
	<li>Photograph the external eye at an indicated time point under the operating microscope.</li>
</ol>
3. Harvesting the Rat Corneal Button and Immunostaining
<ol>
	<li>To euthanize the rats, place them in a euthanasia chamber and infuse 100% CO2 at a fill rate of 10-30% of the chamber volume per minute. Maintain CO2 infusion for an additional minute after lack of respiration and faded eye color.</li>
	<li>Penetrate the rat eye at the limbus with a sharp blade. Cut the corneas with corneal scissors along the limbus. Flatten the corneas on a slide. Make additional radial incisions if the corneas curl up.</li>
	<li>Fix the corneas with 250 &#181;l of 4% paraformaldehyde, pH 7.4, for 30 min at room temperature. Permeabilize with 250 &#181;l of 0.5% Triton X-100 for 5 min, and block with 10% bovine serum albumin for 30 min.</li>
	<li>Incubate the corneas with primary antibodies against ABC overnight at 4 &#176;C with a dilution of 1:200 in antibody diluent. Wash the corneas twice with PBS for 15 min, and incubate them with the secondary antibody (1:100 in antibody diluent) at room temperature for 1 hr. Counterstain the cell nucleus by covering the cells with 2 &#181;g/ml of 4&#39;,6-diamidino-2-phenylindole, and wash the cells twice with PBS for 15 min.</li>
	<li>Mount the corneas in anti-fading mounting solution. Obtain the immunofluorescent images at excitation wavelengths of 405 and 561 nm with a laser scanning confocal microscope at 20X objective magnification.</li>
</ol>

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The authors have no competing financial interests to declare.

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 these 2 models share the same mechanism.
The bovine CEC in vitro culture model offers ease of manipulation. In addition, compared with the human or primate CEC in vitro culture models employed in previous studies17,18, bovine eyes are larger and easier to acquire, and thus have more CECs available. In our study, we acquired the enucleated bovine eyes from a local abattoir. Because of the fresh nature of the bovine eyes, the exact number of bovine CECs harvested is subject to variation, at approximately 1 x 105 cells per eye. By contrast, the number of human CECs in a normal cornea is approximately 3 x 105 per eye. This number is lower in study-grade corneas and is even lower in residual corneal rims after transplantation. Manipulations during the harvesting procedure will further reduce the number of cells obtained. Furthermore, bovine CECs can proliferate and undergo EnMT spontaneously; therefore, the bovine CEC in vitro culture model is useful in investigating the EnMT mechanism and screening EnMT-suppressing chemicals, such as marimastat. Although there remains concern regarding species-related differences, such as the proliferative capacity of CECs, our results demonstrated that the EnMT signaling pathway of bovine CECs is similar to that of human CECs, including the activation of Wnt/&#946;-catenin18,19.
During the isolation of bovine CECs, peeling the Descemet&#39;s membrane is critical. In some bovine eyes, the Descemet&#39;s membrane may have tight adhesion with the corneal stroma, leading to excessive stromal tissue on the peeled Descemet&#39;s membrane. Further trypsinization may lead to the release of stromal keratocytes that transform into corneal fibroblasts, which affect cell purity and therefore the experimental results. In our study, we peeled the Descemet&#39;s membrane carefully under a microscope. To further ensure cell purity, we first cultured the harvested cells in a 6 cm dish until cell confluence. Only when the cells were hexagonal in shape were they used in further experiments.
To further substantiate the findings of the bovine CEC in vitro culture model, we used the rat corneal endothelium cryoinjury model adopted in previous studies20,21. After cryoinjury, the rat CECs underwent EnMT during regeneration, manifested by the nuclear translocation of active &#946;-catenin, indicating that activation of Wnt/&#946;-catenin signaling is conserved among species during CEC regeneration. In addition, this model is useful in investigating the function of CECs because low CEC function leads to obvious corneal edema. Combined with other equipments, such as ultrasound biomicroscope, anterior segment optical coherence tomography, and confocal microscopy, corneal thickness can be quantitatively evaluated and thus reflect CEC function.
To stimulate the regeneration of CECs and suppress EnMT after wound healing, we administered intracameral injections of bFGF followed by marimastat injection. The treatment significantly reduced the extent of corneal edema. Previous studies have used the topical application of CEC growth-stimulating agents, such as ROCK inhibitor, and EnMT-suppressing agents, such as Notch inhibitor21,22. However, the penetration of drugs into the CEC layer is limited by the corneal epithelium and stroma, which may influence the treatment efficacy23. In contrast to topical application, intracameral injection enables direct access to the corneal endothelium by introducing drugs directly into the anterior chamber. Therefore, the growth-stimulating and EnMT-suppressing effect of chemicals can be assessed unbiasedly following cryoinjury. In our study, we performed anterior chamber paracentesis to lower the intraocular pressure before intracameral injection. Without paracentesis, an abrupt increase in intraocular pressure causes corneal edema or even prolapse of the iris through the needle tract. Gently compressing the tract during needle withdrawal aids in sealing the wound and preventing intraocular infection.

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><tbody><tr><td>trypsin</td><td>ThermoFisher Scientific</td><td>12604-013</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium and Ham&#039;s F12 medium</td><td>ThermoFisher Scientific</td><td>11330</td><td></td></tr><tr><td>fetal bovine serum</td><td>ThermoFisher Scientific</td><td>26140-079</td><td></td></tr><tr><td>dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>human epidermal growth factor</td><td>ThermoFisher Scientific</td><td>PHG0311</td><td></td></tr><tr><td>insulin, transferrin, selenium&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>41400-045</td><td></td></tr><tr><td>cholera toxin</td><td>Sigma</td><td>C8052-1MG</td><td></td></tr><tr><td>gentamicin</td><td>ThermoFisher Scientific</td><td>15750-060</td><td></td></tr><tr><td>amphotericin B</td><td>ThermoFisher Scientific</td><td>15290-026</td><td></td></tr><tr><td>paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>111219</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787&amp;nbsp;</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>marimastat</td><td>Sigma</td><td>M2699-25MG</td><td></td></tr><tr><td>anti-active beta-catenin antibody</td><td>Millpore</td><td>05-665</td><td></td></tr><tr><td>anti-snail antibody</td><td>Santa cruz</td><td>sc28199</td><td></td></tr><tr><td>anti-slug antibody</td><td>Santa cruz</td><td>sc15391</td><td></td></tr><tr><td>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><td>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><td>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><td>antibody diluent</td><td>Genemed Biotechnologies</td><td>10-0001</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole</td><td>ThermoFisher Scientific</td><td>D1306</td><td></td></tr><tr><td>mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>laser scanning confocal microscope</td><td>ZEISS</td><td>LSM510</td><td></td></tr><tr><td>xylazine&amp;nbsp;</td><td>Bayer</td><td>N/A</td><td></td></tr><tr><td>tiletamine plus zolazepam</td><td>Virbac</td><td>N/A</td><td>veterinary drug</td></tr><tr><td>proparacaine hydrochloride ophthalmic solution</td><td>Alcon</td><td>N/A</td><td>veterinary drug</td></tr><tr><td>0.1% atropine</td><td>Wu-Fu Laboratories Co., Ltd</td><td>N/A</td><td>clinical drug&amp;nbsp;</td></tr><tr><td>0.3% gentamicin sulfate</td><td>Sinphar Group</td><td>N/A</td><td>clinical drug&amp;nbsp;</td></tr><tr><td>basic fibroblast growth factor</td><td>ThermoFisher Scientific</td><td>PHG0024</td><td>clinical drug&amp;nbsp;</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 in vitro culture, a corresponding nuclear translocation of ABC and EMT regulators was observed. Figure 3 illustrates the effect of marimastat, a broad-spectrum MMP inhibitor, on the EnMT process of the in vitro cultured bovine CECs. Figure 4 comprises the external eye photographs of rats after cryoinjury followed by intracameral injection. Figure 5 shows the immunostaining of the rat corneal button that was performed using antibodies against ABC after cryoinjury followed by intracameral injection. The nuclear translocation of ABC was observed in the PBS group and was significantly increased in the bFGF group, indicating activation of the Wnt/&#946;-catenin signaling and EnMT process. After intracameral injection of bFGF followed by marimastat injection, the nuclear staining of ABC was diminished, suggesting the EnMT-inhibiting effect of marimastat.
<img alt="Figure 1" src="/files/ftp_upload/54329/54329fig1.jpg" /> 
Figure 1: Phase contrast images of in vitro cultured bovine CECs. After being seeded on the culture plate, the bovine CECs initially appeared fibroblast-like on days 3 and 6. They became more hexagonal upon reaching complete confluence on day 9. Scale bar = 50 &#181;m. Representative images of 3 replicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54329/54329fig2.jpg" /> 
Figure 2: Immunostaining of in vitro cultured bovine CECs. During the in vitro culture of the bovine CECs, the nuclear translocation of ABC, snail, and slug was detected through day 14. ABC: active &#946;-catenin. Scale bar = 100 &#181;m. Representative images of 3 replicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54329/54329fig3.jpg" /> 
Figure 3: Immunostaining of in vitro cultured bovine CECs with or without marimastat at different cell confluence levels. Immunostaining demonstrated that ABC, snail, and slug were evident in the nucleus of the bovine CECs with or without 10 &#181;M of marimastat on day 3. However, marimastat significantly reduced the nuclear staining of ABC, snail, and slug on day 9 when the bovine CECs became fully confluent. Scale bar = 100 &#181;m. Representative images of 3 replicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54329/54329fig4.jpg" /> 
Figure 4: External eye photographs of rats at indicated time points after cryoinjury. Following cryoinjury for 3 consecutive days, rats were subjected to intracameral injection of 0.02 ml of PBS or 50 ng/ml bFGF on day 3. On day 6, 0.02 ml of 10 &#181;M marimastat was injected intracamerally in the bFGF/Mari group, whereas PBS was injected in the other 2 groups (n = 9 in each group). External eye photographs revealed reduced corneal edema after bFGF injection, whereas marimastat further reduced corneal edema compared with bFGF alone. n = 9 in each group. Scale bar = 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54329/54329fig5.jpg" /> 
Figure 5: Immunostaining of rat corneal buttons. Immunostaining of the rat corneal buttons that were harvested on day 9 revealed little nuclear staining of ABC in the PBS group. In the bFGF group, there was extensive nuclear staining of ABC, which was significantly reduced in the bFGF/Mari group. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.9K Views.  Far Eastern Memorial Hospital.  Een primaire kweek van runder corneale endotheelcellen werd gebruikt om het mechanisme van corneale endotheel-mesenchymale overgang te onderzoeken. Verder werd een rat hoornvliesendotheel cryoinjury model gebruikt om corneale endotheel-mesenchymale overgang in vivo te demonstreren. 

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

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Medicine

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Neuroscience

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Bioengineering

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

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

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

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

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 ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

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, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

Biology

In Vivo Heart Transplantation to Study Endothelial-to-Mesenchymal Transition

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

Author Spotlight: A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...

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

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