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

Tüm işlemler Oftalmik ve Vision Research Hayvanların Kullanım Vizyon ve Oftalmoloji Tablosunda Araştırma Derneği ile tanınan bu çalışmada izlenen ve Ulusal Tayvan Üniversitesi Hastanesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1. İzolasyon, İlköğretim Kültür Hazırlama ve Bovine CDKÖ immün <ol><li> Yerel bir mezbahaya taze sığır gözleri kazanır. </li><li> 3 dakika boyunca% 10 w / v povidon-iyot çözeltisi gözleri dezenfekte. Fosfat tamponlu tuz (PBS) çözeltisi ile yıkanır. </li><li> steril koşullar altında bir neşter ve makas ile kornea düğmeye hasat. bir mikroskop altında forseps ile Peel Descemet membranı (bu çalışmada unutmayın, sığır gözler yerel bir mezbahaya tarafından iki parçaya ayrıldı, bu nedenle ilk çalışma prosedürü laboratuvarda preenucleated gözlerinin dezenfeksiyon idi). </li><li> try 1 ml Descemet membran inkübe30 dakika boyunca 37 ° C&#39;de psin. 5 dakika boyunca 112 x g&#39;de santrifüj ile sığır CDKÖ toplayın. takviye hormon epitel ortam (SHEM) HEPES-tamponlu Dulbecco tadil edilmiş Eagle ortamı ve% 5 cenin sığır serumu ile takviye edilmiş Ham F12 ortamında,% 0.5 dimetil sülfoksit, insan epidermal 2 ng / ml arasında eşit hacimlerde ihtiva eden 1 ml hücrelerin tekrar büyüme faktörü, 5 mg / ml arasında insülin, 5 mg / transferin ml, 5 ng / selenyum mi, kolera toksin 1 nmol / L, 50 mg / ml gentamisin ve 1.25 mg / amfoterisin B mi </li><li> 6 cm çanak içine hücreleri (gözün başına yaklaşık 1 x 10 5 hücre) Tohum. Sam&#39;ın Kültür onları. Hava içinde% 5 CO2 atmosferinde 37 ° C &#39;de çanak inkübe edin. 3 günde kültür ortamı değiştirin. </li><li> Hücreler kaynaşmaya eriştikten sonra, PBS ile yıkayın ve 5 dakika boyunca 37 ° C&#39;de tripsin 1 ml inkübe. 5 dakika boyunca 112 x g&#39;de santrifüj ile toplayın. Sam&#39;ın 1 ml hücre pelletini yeniden askıya. <ol&gt; <li> Bir hemasitometre hücreleri saymak. Bir 24-yuvalı plaka içinde çukur başına 1 x 10 4 yoğunluğuna ve kültür Sam hücreleri de kapaklı lam üzerinde hücreleri tohumu. marimastat arasında EnMT bastırıcı etkilerini araştırmak için, marimastat uM kültür ortamına ilave 10 Sam hücreleri inkübe ve her 3 günde bir kültür ortamı değiştirin. </li></ol></li><li> Oda sıcaklığında 30 dakika süre ile,% 4 paraformaldehit 250 ul, pH 7.4 ile belirtilen bir zaman noktasında hücreleri saptamak. 5 dakika için% 0.5 Triton X-100, 250 ul geçirgenliği. 30 dakika boyunca% 10 inek serumu albümini ile bloklayın. </li><li> gece boyunca 4 ° C &#39;de, aktif beta-katenin (ABC), salyangoz ve sevk borusu karşı birincil antikor ile inkübe hücreleri. 200: kullanılan birincil antikor seyreltme 1&#39;dir. antikorlar, 10 mM PBS,% 1 w / v sığır serum albümini ve% 0.09 w / v sodyum asid oluşan antikor seyreltici içinde seyreltilir. </li><li> 15 dakika boyunca PBS ile iki kez hücreleri yıkanır ve inkübe1 saat süre ile oda sıcaklığında (antikor seyreltici içinde 1: 100) ve Alexa Fluor-konjuge sekonder antikor ile m. </li><li> 4 &#39;, 6-diamidino-2-fenilindol 2 ug / ml hücreleri kapsayan, hücre çekirdeğine Counterstain, 15 dakika boyunca PBS ile iki kez yıkama hücreleri ve montaj çözümü, anti-solma ile monte edilir. </li><li> 20X objektif büyütme ile bir lazer tarama konfokal mikroskop kullanılarak 405 ve 488 nm uyarma dalga boylarında immunofluorescent görüntüleri elde edebilir. </li></ol> 2. Sıçan Kornea Endotel Cryoinjury Modeli ve İntrakameral Enjeksiyon <ol><li> % 2 ksilazin (5.6 mg / kg) ve tiletamin artı zolazepam (/ kg 18 mg) kas içine enjeksiyonu ile 12 haftalık erkek Sprague-Davvley sıçanları anestezisi. Yavaşça cilt seğirmesi yokluğunda uygun anestezi onaylamak için hayvanların derisini çimdik. </li><li> Göz ağrısı ve göz kırpma refleksi en aza indirmek için her sıçanın sağ gözüne% 0.5 proparacaine hidroklorür bir damla aşılamak. Aşılamaksol göze tetrasiklin merhem kornea kuruluğunu önlemek için. </li><li> sıvı nitrojen içinde paslanmaz çelikten bir prob (çap = 3 mm) soğutun. 30 sn sağ gözünün kornea santralinde paslanmaz çelik prob uygulayın. Sıkça kornea kuruluğunu önlemek için işlem sırasında sağ gözüne PBS aşılamak. </li><li> hemen cryoinjury sonra% 0.1 atropin ve% 0.3 gentamisin sülfat aşılamak ve günde bir kez siliyer spazm kaynaklanan göz ağrısı rahatlatmak için ve enfeksiyonu önlemek için. <ol><li> İşlemden sonra, sıçanlar bir ısı lambası kullanarak sıcak tutmak ve onlar motor kontrolünü yeniden kadar anestezi sonrası kendi iyileşme her 15 dakika gözlemleyin. Ayrıca, iyileşme döneminde kornea kuruluğunu önlemek için sağ gözüne tetrasiklin merhem uygulayın. </li></ol></li><li> 3 gün üst üste kornea cryoinjury tekrarlayın. </li><li> Daha önce açıklandığı gibi sıçan gözlerin ön odasına marimastat veya bFGF verilmesi için, fareler uyutmak. bir damla aşılamaksağ göz içine% 0.5 proparacaine hidroklorür göz ağrısı ve göz kırpma refleksi en aza indirmek için. </li><li> steril PBS ile göz yüzeyi yıkayın. iris yukarıda ve paralel bir düzlemde paralimbal açık bir korneadaki bir 1 ml şırınga bağlanmış bir 30 G iğne sokarak bir ameliyat mikroskobu altında Ön bölme parasentezi gerçekleştirmek. </li><li> İğne yukarı konik çevirin ve hafif bazı sulu mizah drenaj ve göz içi basıncını düşürmek için kornea yara bastırın. intrakameral ilacın 0.02 ml enjekte edilir. Yavaşça iğne çekilmesi sırasında pamuk uçlu iğne yolu sıkıştırmak. </li><li> ameliyat mikroskobu altında belirtilen zaman noktasında dış göz fotoğraf. </li></ol> 3. Hasat Sıçan Kornea Düğme ve immün <ol><li> Fareleri euthanize için, ötenazi odasına koyun ve dakikada odası hacminin% 10-30 bir dolgu hızında% 100 CO 2 demlenmeye. Bir CO 2 infüzyon korumaksolunum yetersizliği ve soluk göz rengi sonra ek dakika. </li><li> keskin bir bıçak ile limbusta sıçan göz nüfuz eder. limbusta boyunca kornea makasla korneaları kesin. bir slayt korneaları dümdüz. korneaları kıvrılıp, ek radyal kesiler olun. </li><li> Oda sıcaklığında 30 dakika süre ile,% 4 paraformaldehit 250 ul, pH 7.4 ile kornealar düzeltildi. 5 dakika için% 0.5 Triton X-100, 250 ul geçirgenliği, ve 30 dakika boyunca,% 10 büyükbaş hayvan serum albümini ile bloke eder. </li><li> 1 oranında sulandırılmış bir gece boyunca 4 ° C &#39;de ABC karşı birincil antikor ile kornealar inkübe: 200 antikoru seyreltici içinde. 15 dakika boyunca PBS ile iki kez kornealar yıkayın ve ikincil antikor ile inkübe: 1 saat süre ile oda sıcaklığında (1 antikoru seyreltici içinde, 100). Hücre 2 ug 4 / ml &#39;, 6-diamidino-2-fenilindol ile hücrelerin kaplayarak nükleus ve 15 dakika boyunca PBS ile iki kez yıkama hücreleri Counterstain. </li><li> montaj çözümü, anti-solmaya korneaları monte edin. ob20X objektif büyütme bir lazer tarama konfokal mikroskop ile immunofluorescent 405 uyarma dalga boylarındaki görüntüleri ve 561 nm tain. </li></ol>

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The authors have no competing financial interests to declare.

CESler hücre proliferasyonu sırasında EnMT geçmesi için kendi eğilimi bilinmektedir. Tedavi amaçlı EnMT sürecini bastırmak için stratejiler geliştirmek, EnMT mekanizmasının tam bir anlayış gereklidir. Biz EnMT, in vitro kültür modeli ve sıçan kornea endotel cryoinjury modelinde yani sığır CEC araştırmak için 2 modeller nitelendirdi. Bizim sonuçları Her iki modelde de EnMT süreci göstermiştir. Ayrıca, marimastat ve EnMT baskılayıcı etkisi bu 2 model aynı mekanizmayı ...

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.

Sığır CECS izole edildikten sonra, hücreler, in vitro olarak kültürlendi. 1 sığır CDKÖ faz kontrast görüntüler sunar Şekil. Izdiham hücrelerin altıgen şekil hücreleri hücre izolasyonu sırasında kornea stromal fibroblast tarafından kontamine olmadığını belirtti. 2 belirtilen bir zaman noktasında ABC, salyangoz ve sümüklü böcek karşı antikorlar kullanılarak gerçekleştirilmiştir immün tasvir <s...

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

sığır endotel hücrelerinin ana bir kültürü kornea endotelial-mezenkimal geçiş mekanizmasını araştırmak için kullanıldı. Ayrıca, bir fare endotel cryoinjury modeli in vivo olarak endotel-mezenkimal geçiş göstermek için kullanılmıştır.

Vitro ve in vivo Modelleri Kornea Endotel-mezenkimal Geçiş Çalışması için

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

Research

JoVE Journal

Developmental Biology

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

7.9K Görüntüleme.  Far Eastern Memorial Hospital.  sığır endotel hücrelerinin ana bir kültürü kornea endotelial-mezenkimal geçiş mekanizmasını araştırmak için kullanıldı. Ayrıca, bir fare endotel cryoinjury modeli in vivo olarak endotel-mezenkimal geçiş göstermek için kullanılmıştır. 

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.

Kornea doku Mühendisliği: İnsan kornea sinir Stromal etkileşimlerin bir Vitro modeli

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

Gelişim Biyolojisi

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.

Sıçangiller Kornea Nakli: Katı Organ Nakli En Yaygın Formu Eğitim için Bir Model

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

Tıp

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.

Kornea mikrocep Testi: Fare Eye Angiogenezis Bir Model

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

Nörobilim

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.

Biyomalzeme Membranlar Üzerinde İnsan ve Koyun Korneal Endotel Hücre Tabakalarının Büyümesi

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

Biyomühendislik

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.

Ex vivo kornea Organ kültür modeli için yaranın çalışmaları

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.

Split Korneal Düğmeleri Kullanarak Bir Domuz Korneal Endotel Organ Kültür Modeli

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).

Kornea Endotel Hücre Kaybının Noninvaziv, Lazer Destekli Deneysel Modelinin Geliştirilmesi

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.

Kornea Epitelinin Mekanik ve Kimyasal Yaralanmaları için Ex Vivo ve In Vivo Hayvan Modelleri

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.

Mezenkimal endotel geçiş büyüme faktörü β sinyal dönüşüm tarafından indüklenen moleküler Analizi

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

Biyoloji

Endotelyalden mezenkimal geçişi incelemek için in vivo kalp nakli

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.

Yazar Spotlight: Endotelyal-mezenkimal geçiş çalışması için yenidoğan heterotopik sıçan kalp nakli modeli  

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.

Endotelyal-mezenkimal geçiş çalışması için yenidoğan heterotopik sıçan kalp nakli modeli

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