Name: establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment
Uploaded: 2015-06-05T15:00:00.000+00:00
Duration: 7 min 53 s
Description: Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

This work was supported by National Institutes of Health Grant to A.S.M. (EY021784).

Aşağıdaki protokol Thomas Jefferson Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu kurallarına ve Vizyon Araştırma Hayvanların Kullanım ARVO Bildirimi ile uyumludur. Ex Vivo Yara Kültür Lensler 1. Kurulum ve Hazırlık <ol><li> Steril, laminer akış başlığı içinde üç adet 100 mm petri kapları yerleştirin. Oda sıcaklığında, yarım Tris / Dekstroz tamponu ile petri kutularının iki (140 mM NaCI, 5 mM KCI, 0.7 mM Na-2 PO 4, 5 mM D-glikoz, 8.25 mM Tris baz, HCI ile 7.4&#39;e pH td tamponu) doldurun Üçüncü boş bırakarak. Ön sıcak kültür ortamı 37 o C ye (Ortam 199,% 1 L-glutamin ve% 1 penisilin / streptomisin ile takviye edilmiş) Not: in vivo meydana geldiği gibi kültür ortamı iyileşme, standart bir yara, serumsuz olduğu; Bununla birlikte, yara onarımı kültürleri serum ya da diğer faktörleri de tanımlanmış ortam koşullarında başarıyla yetiştirilebilir. </li><li> Bereketli embriyonik d kaldıray inkübatör 15 beyaz leghorn civciv yumurta (hafif hafif sallanarak 37.7 ° C&#39;de tutulan) </li><li> Bir yıkama şişesinden laminar akış çeker ve% 70 etanol ile kabuğun temiz bir dış seçilen yumurta yerleştirin. Steril çözümleri ve araçları kullanarak, laminer akış kaputu aseptik koşullarda aşağıda tüm prosedürleri yürütün. </li><li> Boş 100 mm petri içine yumurta ve yer içeriğini Crack. Standart forseps ve ince makas kullanılarak embriyo başını kesmek. TD tampon içeren bir petri civciv embriyo baş koyun ve düzgün embriyonun geri kalan atmayın. İsteğe bağlı, 15 dakika daha uzun, kısa bir süre için TD tamponda civciv embriyo başlarını tutun. </li><li> Bir petri kapağı üzerinde civciv embriyo başını yerleştirin. Yüksek hassasiyetli forseps kullanarak, aşağıdaki sırayla göz ekli vitreus ile birlikte lensi çıkarın. Gözün arkasında küçük bir açıklık oluşturmak için forseps ile gözün arka sıkıştırın. </li><li> Sonra, w vitreus mizah kavramaki forseps ve yavaşça bir yuvarlanma hareketi ile vitreus römorkör, ekli lens ile vitreus göz yerinden edilecektir. TD tampon içeren kalan petri vitreus yerleştirin objektif /. Lensler artık 30 dakika için TD tamponu içinde kalmasına izin verir. </li><li> Bir mikroskop altında yeni bir petri kapak lensi hareket ettirin. Bu noktada bir mikroskop altında tüm adımları uygulayın. Yüksek hassasiyetli forseps dikkatle mercek dokusuna zarar vermemeye dikkat alarak forseps kenarı kullanılarak lens ile yerinden edilmiş herhangi siliyer cisim (pigmentli hücreler) uzakta fırça ile. Not: siliyer gövde çıkarılması lens endojen olmayan hücre tipleri, yara onarımı kültürü dahil olmasını sağlar. </li><li> Arka lens kapsülü ile dernekten vitreus vücudu kapalı kısma yüksek hassasiyetli forseps ile vitreus mercek ayırın. </li><li> Yüksek hassasiyet kullanarak küçük bir damla içine lensi transferi forsepsbir 35 mm doku kültürü çanağı içinde TD tamponu (yaklaşık 200 ul). </li></ol> 2. Sahne Mock Katarakt Cerrahisi <ol><li> Yukarı bakacak şekilde lensin ön yönü ile 35 mm çanak TD tampon damla lensi yönlendirin. Not: merceğin ön kolay lens epitelyum ön ve ekvator bölgesi arasındaki sınır notlar dokuda yoğun halkasının mevcudiyeti ile tanımlanır. Bunun aksine, mercek elyaf hücrelerinin bağlı olduğu mercek kapsülünün arka işaretlerin yokluğu vardır. </li><li> , Her el bir forseps ile doku tutarak iki yüksek hassasiyetli forseps ön lens kapsülü, objektif dokusunu çevreleyen kalın bazal membran ve ilişkili lens ön epitel merkezinde küçük bir kesi (yaklaşık 850μm) yapmak kullanma ve yavaşça karşıt yönlerde asılarak. </li><li> Lens dokusu, dislod kısmını oluşturan lif hücre kütlesi, KaldırLens epitel eklerinde onu ging ve (Şekil 1A modellenmiş klasik katarakt cerrahisinde kullanılan bir yaklaşım,) hidro-elüsyon lens kapsülü çevreleyen. </li><li> TD tampon 300 ul bir 27.5 g bir iğne ucu ile bir 1 ml şırınga doldurun. Anteryor mercek kapsülünün yapılan kesi içine iğne ucu yerleştirin ve yaklaşık yarım lens içine. </li><li> Yavaşça mercek fiber hücre kitlesinin içine TD tampon enjekte şırınga basınız. 50 ila 200 ul TD, ve asla fazla 300 ul enjekte edilir. Epitel ve lens kapsülünün kendisini gevşeterek lif hücre kütlesi gözlemleyin. </li><li> Yüksek hassasiyetli forseps kullanarak, ön kesi sitesi aracılığıyla objektiften gevşetti lif hücre kütlesi kaldırın. Not: Bu yordam, bu siteye sadece komşu lif hücreleri ekli hücrelerin soyulmuş edilmişti posterior lens bazal membran kapsülü ve bir yaralı lens epitel bırakır. </li></ol> 3. PreparinEx Vivo Kültür g Yaralı Objektif <ol><li> Kapsül çanta ön yüzünde beş kesim yaparak, kültür tabağına yukarıda açıklanan katarakt cerrahisi, hücre tarafı yukarı kaynaklanan objektif kapsül çanta düzleştirin. </li><li> Lensin ekvatora kadar orijinal kesi dik kesin. Aşağı kültür çanak kapsül tarafında takılı epiteli hücre tarafı yukarı ile mercek kapsülünün sonuçtaki beş &quot;flep&quot; dümdüz. Bir yıldızın ya da flowerlike şekli (Şekil 1B bakınız) almalı şimdi ex vivo yaralı lensi unutmayın. </li><li> Yıldızın her noktada usulca aşağı forseps ile, çanak basın kapsülü sabitlemek için. Bu eksplant beş en dış ucunda küçük bir girinti yapmak ve yemek için sürekli eki neden olur. Not: Bu işlem sırasında kapsül zarar mümkündür ve böylece fla uçlarına yakın çanak kapsülü sabitlemek için önemlidirmümkün olduğunca PS yanı sıra mümkün olduğunca emniyet noktalarının en az miktarda yapmak için (genellikle iki, flep başına üç maksimum). </li><li> 35 mm çanak TD tamponunu çıkarın ve önceden ısıtılmış ortam 1,5 mi ile değiştirin. Inkübatör, kapağı ve yer ile 35 mm çanak Kapak (37 ° C,% 5 CO2). </li></ol> Arka Kapsül Yaralı Alanı Yeniden epitelizasyon Kantitatif Analiz Lens Epitel Hücreleri Orjinal Ek Zonu (OAZ), dan, oluşuyor Orta Göç Bölgesi (CMZ), 4. ayrılması. Not: hücreler, hasara tepki olarak, hemen CMZ bölgeye doğru hareket etmeye başlar. Kültürde günden yeterince hücrelerin mikro diseksiyon 23 ile CMZ ve Oaz ayrılması sonrasında, moleküler ve biyokimyasal analiz için CMZ genelinde göç vardır. Bu protokol, kapsülün yaralı bölgeden bir seferde bir kanat (OAZ) kaldırılmasını içerir. <ol><li> Bir Demar gözlemleyinkatyondur, OAZ ve Cmz arasında mikroskop altında açıkça görülebilir (Şekil 2A). İki yüksek hassasiyetli forseps kullanılarak, çizginin her iki tarafında, OAZ / CMZ hattı, başka bir hemen yanında bir kenarına, her iki forseps ile kavramak (Şekil 2A, B, ok). </li><li> Bir elinizi kullanarak / CMZ tarafta bir forseps Öte yandan / forseps ile yavaşça OAZ / CMZ hattı boyunca Oaz çekerken, yaralı kültüre tutunmaya devam ediyor. CMZ kolayca bu hat boyunca Oaz ayrılır. Iki bölge tamamen ayrılır kadar tüm kültür etrafında bu çizgi boyunca devam edin. </li><li> Örneğin Western blot veya ko-immünopresipitasyon 23,25 gibi RNA Dizi 24 veya biyokimyasal analizler gibi moleküler analiz için ayrıldı OAZ ve CMZ fraksiyonlar incelenmesi. </li></ol>

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The authors declare that they have no competing financial interests.

Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni...

klinik olarak anlamlı, sahte katarakt cerrahisi, burada açıklanan ex vivo epitel yara iyileşmesi modeli bir yaralanma tepki olarak epitel dokuların onarımını düzenleyen mekanizmaları araştırmak için bir araç sağlamak için geliştirilmiştir. Bu modeli oluştururken için amaçlanmıştır Temel özellikler, yakından onarma işlemini bir kültür ayarı, 2), tamir düzenleyici unsurları modüle kolaylığı yaralama in vivo cevabı çoğaltılmış ve görüntü 3) yeteneği 1) sağlayan koşullar dahil gerçek zamanlı olarak bütünüyle içinde. meydan, bu nedenle, hücrelerin doğal mikroçevresinin, epitel yara onarımı incelemek mümkün olduğu bir kültür modeli oluşturmak ve işlemek için oldu. Bu yara onarım modelinin mevcudiyeti onarım sürecini düzenleyen matriks proteinleri, sitokinler ve kemokinlerin endojen sinyal ipuçları belirlenmesi için yeni olanaklar açar. Buna ek olarak, bir model incelenmesi için idealdir nasıln epitel yara alan 2,3, reepitelizasyonu için kolektif bir levha olarak ve yaralı epiteli 4 kolektif göç yönetmenlik işlev yara kenarında mezenkimal lideri hücrelerinin soy belirlemek için hareket edebilmektedir. Bu model de etkili yara iyileşmesini teşvik etmek ve anormal yara onarım 5 engelleyebilir terapötikleri belirlemek için bir platform sağlar. Mevcut yara tamir modelleri bir dizi kültürü ve günümüzde yara onarım süreci hakkında bilinenlerin çoğu sağladı vivo, hem de zaten vardır. Böyle kornea 6-12 ve cilt 13-17 gibi hayvan modellerinde yaralanma, olarak, katkıları da dahil olmak üzere süreçte yer olabilir tüm tamir arabulucuların bağlamında yaralama doku yanıtını incelemek için fırsat var damarlar ve sinir sistemi. Ancak, bu deneyimlerden manipüle sınırlamalar vardırin vivo olarak zihinsel koşullar ve zaman içinde sürekli olarak, in vivo olarak onarım yanıtı görüntüleme çalışmaları yürütmek üzere henüz mümkün değildir. Bunun aksine, örneğin çizilmeye yara çok in vitro yara onarımı kültürü modelleri, kolayca manipüle edilebilir ve zaman içinde takip ancak in vivo dokusunda yara iyileşmesinin okuyan çevresel bağlamı yoksundur. Ex vivo modeller sürecinde herhangi bir zaman noktasında onarım moleküler regülatörleri modüle yeteneği ile birleştiğinde hücrelerin mikroçevresinin bağlamında zamanla sürekli yaralanma onarım işlemini okuyan avantajı sunarken, bu uygun birkaç model var parametreleri. İşte fizyolojik yaralama bir epitel dokusunun yanıtını yeniden kültürleri şifa yüksek tekrarlanabilir ex vivo epitel yara oluşturmak için bir prosedür açıklanmaktadır. Bir doku kaynağı, bir ex vivo moc olarak civciv embriyo lens kullanarakk katarakt cerrahisi yapılmaktadır. Lens avasküler, innerve ve eşlik eden stroma 18,19 serbest değil, o kendi kendine yeten bir kalın bazal membran kapsül içinde olduğundan bu çalışmalar için kullanılacak ideal bir dokudur. İnsan hastalıkta, katarakt cerrahisi nedeniyle lensin opasifikasyon için görme kaybı giderir ve lens toplu içermektedir lens fiber hücre kitlesinin, kaldırılmasını içerir. Aşağıdaki katarakt cerrahisi görme yapay göz içi lensi yerleştirilmesi yoluyla geri yüklenir. katarakt ameliyatı prosedürü, lif hücrelerinin çıkarılması, Fiber hücreleri tarafından işgal edilmiş olan mercek kapsülünün arka bölgenin yeniden epitelizasyon ile yanıt verir bitişik lens epitelyum, bir yaralanma tepkisini indükler. Katarakt cerrahisi, çoğu yara tamir yanıtları gibi, bazen lens Arka Capsu olarak bilinen miyofibroblastlar ortaya çıkması ile ilişkili yara iyileşmesi yanıt bir anormal fibrotik sonucunu ortaya çıkmaktadırle Opasitesi 20-22. Katarakt ameliyatı yara iyileşmesi modeli oluşturmak için, katarakt cerrahisi prosedürü fizyolojik bir yaralanma üretmek için civciv embriyo göz kaldırılır lenslerde taklit edilir. Mercek epitelyal hücreleri tarafından çevrelenmiş bir çok tutarlı dairesel bir yara bölgesinde lens elyaf hücrelerinin sonuçları Mikrocerrahi çıkarılması. Bu hücre popülasyonu sıkıca mercek bazal membran kapsüle bağlı kalır ve cerrahi prosedür ile yaralandı. epitel hücreleri lider hücreleri 1 olarak onarım sürecinde bilinen vimentin zengin mezenkimal hücre popülasyonu tarafından yönetilen yara, iyileşmek için endojen bazal membran arındırılır alan üzerine göç ederler. Bu model ile yaralanma bir epitel yanıtı kolaylıkla görülebilir ve hücrelerin mikroçevresinin bağlamında zamanla izledi. Hücreler ifadesi ya da yara iyileşmesi bir rol oynaması beklenmektedir moleküllerin aktivasyonu modifikasyonlara kolaylıkla temin edilebilir. Th bir güçlü özelliğiModel izole etmek ve yara iyileşmesi çerçevesinde göç özgü değişiklikleri incelemek için yeteneğidir olduğunu. çalışmalar için yaş uyumlu ex vivo olarak yara iyileşme kültürler çok sayıda hazırlanması için yeteneği, bu modelin bir diğer avantajdır. Böylece, bu model sistemi yara iyileşme süreci üzerindeki etkisi yara onarım mekanizmalarını ve test terapötik ayrı kızdırmak için eşsiz bir fırsat sunuyor. ex vivo sahte katarakt cerrahisi modeli yaralanma tamir mekanizmaları çalışmak için kritik bir kaynak sağlayarak, geniş uygulama olması bekleniyor.

<table><tbody><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>S271-3</td><td>Use at 140 mM in TD Buffer</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>P217-500</td><td>Use at 5 mM in TD Buffer</td></tr><tr><td>Sodium Phosphate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>S0876</td><td>Use at 0.7 mM in TD Buffer</td></tr><tr><td>D-glucose (Dextrose)</td><td>Fisher Scientific</td><td>D16-500</td><td>Use at 0.5 mM in TD Buffer</td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-1</td><td>Use at 8.25 mM in TD Buffer</td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>A144-500</td><td>Use to pH TD buffer to 7.4</td></tr><tr><td>Media 199</td><td>GIBCO</td><td>11150-059</td><td></td></tr><tr><td>L-glutamine</td><td>Corning/CellGro</td><td>25-005-CI</td><td>Use at 1% in Media199</td></tr><tr><td>Penicillin/streptomycin</td><td>Corning/CellGro</td><td>30-002-CI</td><td>Use at 1% in Media199</td></tr><tr><td>100 mm petri dishes</td><td>Fisher Scientific</td><td>FB0875711Z</td><td></td></tr><tr><td>Stericup Filter Unit</td><td>Millipore</td><td>SCGPU01RE</td><td>Use to filter sterilize Media</td></tr><tr><td>Dumont #5 forceps (need 2)</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>35 mm Cell Culture Dish</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>27 G 1 ml SlipTip with precision glide needle</td><td>BD</td><td>309623</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>Standard Forceps</td><td>Fine Science Tools</td><td>91100-12</td><td></td></tr><tr><td>Other Items Needed: General dissection instruments,&amp;nbsp; fertile white leghorn chicken eggs, check egg incubator (humidified, 37.7&amp;deg;C), laminar flow hood, binocular stereovision dissecting microscope</td><td></td><td></td><td></td></tr></tbody></table>

establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Ex Vivo Model hücrelerin doğal mikroçevresinin yara iyileşme sürecini incelemek için oluşturulan Hücrelerin doğal mikroçevresinin içinde epitel yara iyileşmesini düzenleyen katılan mekanizmalarını araştırmak için, klinik olarak anlamlı ex vivo sahte katarakt cerrahisi modeli oluşturuldu. Bu model nedeniyle yapısal özellikleri konusunda pek çok avantajlar sunmaktadır mercek dokularından oluşturulur: 1) objektif mercek kapsülü olarak adlandırıla...

Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

Bir Klinik İlgili kurulması<em&gt; Ex Vivo</emYerli mikroçevresinin Epitelyal Yara Tamir incelenmesi için&gt; Mock Katarakt Cerrahisi Modeli

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

6.5K Görüntüleme.  Thomas Jefferson University. Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment. 

Video: Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Cilt Onarımını Doğru Değerlendirmek için İnsan Ex vivo Yara Modeli ve Tam Montajlı Boyama Yaklaşımı

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Gelişim Biyolojisi

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;

Embriyonik Yaralı Civciv Kornealarında İzsiz Doku Rejenerasyonunun İncelenmesi

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

Kornea epitel aşınma oküler Burr ile bir Model olarak kornea için yara iyileşme

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

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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 ...

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 ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

Kornea Yara İyileşmesini İncelemek İçin Epitelyal Aşınma Modeli

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

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, ...

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched in epitheliotropic growth factors that are essential in corneal wound healing. Unlike AS, PRP is based on a differential centrifugation system, yielding more platelet-derived growth factors. Autologous conditioned serum (ACS) not only preserves the preparation of AS and PRP, but also focuses on immune-modulating properties, which are important in inflammatory diseases.
The lack of standardized protocols and high preparation costs are limitations for the clinical application of ACS. This video experiment demonstrates a standard operating procedure for preparing modified autologous conditioned serum (mACS) eye drops. First, glycerol was added into heparin syringes as the blood cell stabilizer during hypoxic incubation. To activate the blood cells, a 4 h incubation at 37 &#176;C was initiated. Then, the blood samples were centrifuged at 3,500 &#215; g for 10 min at room temperature. After filtration of the supernatant through a 0.22 &#181;m filter, the mACS eye drops were fully prepared.
A tentative try-out of the therapeutic effect of mACS showed that it may have competitive advantages over conventional AS in the corneal wound healing in ex vivo mouse eyes. The AS used in this study was prepared according to published studies and the clinical practice in our hospital. Therefore, the efficacy of mACS on ocular surface diseases could be evaluated in future research through in vivo animal studies and clinical trials.

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

This article describes a protocol to simplify the process and render the preparation of autologous conditioned serum (ACS) less expensive. No special syringes or surface-coated glass beads are needed. Moreover, the modified ACS (mACS) has competitive advantages over conventional autologous serum in the corneal wound healing of murine eyes ex vivo.

Modifiye Otolog Şartlandırılmış Serum Üretimi ve Murin Kornea Epitelinde İyileşme Potansiyelinin Ex Vivo Değerlendirilmesi

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched ...

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover&#160;was&#160;used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3rd day after modeling, the rats&#39; corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.

Kornea Sütürleri ile Kombine Kornea Epitel Küretajına Dayalı Sıçanlarda Şiddetli Kornea İnflamasyonu Modelinin Oluşturulması

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. ...

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.

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