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

Il seguente protocollo è conforme alle Istituzionale Animal Care and Use Committee linee guida Thomas Jefferson University e con la dichiarazione ARVO per l&#39;uso di animali in Vision Research. 1. Configurazione e preparazione di lenti per la Cultura Ex Vivo Wound <ol><li> Mettere tre 100 piatti mm Petri in una, cappa a flusso laminare sterile. Riempire due dei piatti petri metà con tampone Tris / destrosio (buffer TD; 140 mM NaCl, KCl 5 mM, 0,7 mM Na 2 PO 4, 5 mM D-glucosio, 8,25 mM Tris Base, pH a 7,4 con HCl) a RT , lasciando il terzo vuoto. Terreni di coltura pre-caldo (Media 199 integrato con, 1% di L-glutamina e 1% di penicillina / streptomicina) a 37 ° C. Nota: La ferita norma di guarigione di coltura è senza siero, come avviene in vivo; tuttavia, le culture ferita di riparazione possono essere coltivate con successo in condizioni di media definiti che includono siero o altri fattori. </li><li> Rimuovere fertile d embrionaleay 15 bianco Livorno pulcino uovo da incubatrice (tenutasi a 37,7 ° C, con dolce dondolio) </li><li> Inserire uovo selezionato nella cappa a flusso laminare e pulita all&#39;esterno della scocca con etanolo al 70% da una bottiglia di lavaggio. Condurre tutte le procedure qui di seguito, in condizioni asettiche in cappa a flusso laminare, con soluzioni e strumenti sterili. </li><li> Crack uova e posizionare il contenuto in 100 millimetri Petri vuota. Decapitare embrione con pinze standard e forbici sottili. Mettere la testa pulcino embrione in una capsula di Petri contenente tampone TD e smaltire il resto dell&#39;embrione. Opzionalmente evitare che le testine di embrione di pollo in tampone TD per un breve periodo di tempo, non più di 15 min. </li><li> Mettere la testa embrionali di pollo su un coperchio capsula di Petri. Utilizzando pinze alta precisione, rimuovere l&#39;obiettivo insieme al suo vitreo allegata dall&#39;occhio nella seguente sequenza. Pizzicare la parte posteriore dell&#39;occhio con la pinza per creare una piccola apertura nella parte posteriore dell&#39;occhio. </li><li> Quindi, cogliere l&#39;umore vitreo wesimo pinze e tirare delicatamente il vitreo, con un moto di rollio, il vitreo con l&#39;obiettivo montato saranno sloggiati dall&#39;occhio. Luogo obiettivo / vitreo nella capsula di Petri rimanente contenente tampone TD. Lasciare le lenti di rimanere in tampone TD per non più di 30 minuti. </li><li> Spostare la lente per un nuovo coperchio Petri sotto un microscopio da dissezione. Da questo punto in poi eseguire tutti i passaggi sotto un microscopio da dissezione. Con pinze ad alta precisione lavarsi attentamente tutta corpo ciliare (cellule pigmentate) che sono stati sloggiato con la lente utilizzando il bordo della pinza, facendo attenzione a non danneggiare il tessuto dell&#39;obiettivo. Nota: Rimozione del corpo ciliare assicura che i tipi di cellule che non sono endogeni alla lente non sono inclusi nella cultura riparazione della ferita. </li><li> Separare l&#39;obiettivo dalla vitreo con una pinza ad alta precisione pizzicando fuori dal corpo vitreo dalla sua associazione con la capsula posteriore. </li><li> Usando l&#39;alta precisione Pinza trasferire l&#39;obiettivo in una piccola gocciadi tampone TD (circa 200 ml) in un piatto di coltura di tessuti 35 mm. </li></ol> 2. Esecuzione Mock chirurgia della cataratta <ol><li> Orientare la lente nella goccia di tampone TD nel piatto da 35 mm, con la faccia anteriore della lente rivolta verso l&#39;alto. Nota: Il anteriore della lente è facilmente identificabile dalla presenza di un anello denso nel tessuto che rileva il confine tra anteriore e regione equatoriale dell&#39;epitelio lente. Al contrario, vi è una mancanza di marcature sul posteriore della capsula del cristallino a cui le cellule di fibre lente sono allegati. </li><li> Utilizzando due pinze alta precisione fanno una piccola incisione (circa 850μm) al centro della capsula anteriore della lente, la membrana basale spessa che circonda il tessuto dell&#39;obiettivo, e la sua associata epitelio della lente anteriore, afferrando il tessuto con una pinza in ogni mano e tirando delicatamente in direzioni opposte. </li><li> Rimuovere la massa cellulare fibra, che costituisce la maggior parte del tessuto dell&#39;obiettivo, dislodging dai suoi allegati al epitelio lente e circostante capsula del cristallino da idro-eluizione (un approccio utilizzato nella chirurgia della cataratta classico, modellato in Figura 1A). </li><li> Riempire una siringa da 1 ml con una punta dell&#39;ago 27,5 G con 300 ml di tampone TD. Inserire la punta dell&#39;ago nella incisione fatta nella capsula anteriore del cristallino, e circa a metà strada nella lente. </li><li> Deprimere delicatamente la siringa per iniettare il buffer di TD nella massa cellulare fibra lente. Iniettare fra 50 e 200 microlitri TD, e mai più di 300 microlitri. Osservare la massa cellulare fibra stessa allentando dalla capsula epitelio e la lente. </li><li> Utilizzando pinze ad alta precisione, rimuovere la massa cellulare fibra allentato dalla lente attraverso il sito di incisione anteriore. Nota: Questa procedura lascia il posteriore della lente membrana basale capsula per cui le cellule della fibra erano stati allegati denudati delle cellule, e di un epitelio lente infortunato proprio adiacente a questo sito. </li></ol> 3. Preparing dell&#39;obiettivo Ferito per Ex Cultura Vivo <ol><li> Appiattire il sacco capsulare obiettivo che risulta dalla chirurgia della cataratta sopra descritto sul piatto di coltura, lato della cella su, facendo cinque tagli nella faccia anteriore del sacco capsulare. </li><li> Tagliare perpendicolare al sito di incisione originale attraverso all&#39;equatore della lente. Appiattire le risultanti cinque &quot;sportelli&quot; di capsula del cristallino con epitelio attaccato sul lato cultura piatto capsula giù, cellula verso l&#39;alto. Nota la lente ex vivo ferito ora dovrebbe assumere una stella o una forma a fiori (vedi Figura 1B). </li><li> Per fissare la capsula al piatto, premere dolcemente con le pinze in ogni punto della stella. Questo farà una piccola rientranza a cinque punte più esterne del espianto e il risultato in attacco sostenuto al piatto. Nota: È possibile danneggiare la capsula durante questa procedura e quindi è importante garantire la capsula al piatto più vicino alle punte del flaps possibile, così da rendere il minor numero di punti di fissaggio come possibile (in genere due, massimo di tre per falda). </li><li> Rimuovere il buffer TD dal piatto 35mm e sostituirlo con 1,5 ml di mezzi di pre-riscaldato. Coprire il piatto 35 millimetri con il suo coperchio e posto in incubatrice (37 ° C, 5% di CO 2). </li></ol> 4. Separazione della centrale migrazione Zone (CMZ), dove riepitelizzazione dell&#39;Area ferito della capsula posteriore si verifica, dal Attachment Originale della Zona (OAZ) delle cellule epiteliali lente, per l&#39;analisi quantitativa. Nota: le cellule iniziano a muoversi nella regione CMZ immediatamente in risposta al danno. Di giorno una cultura abbastanza cellule migrano attraverso la CMZ per l&#39;analisi molecolare e biochimica, dopo la separazione della CMZ e la OAZ da micro-dissezione 23. Questo protocollo comporta la rimozione di un lembo (OAZ) alla volta dalla zona ferita della capsula. <ol><li> Osservare una demarcatione, chiaramente visibili al microscopio da dissezione, tra OAZ e CMZ (vedi Figura 2A). Utilizzando due pinze alta precisione, afferrare con entrambe le pinze a bordo della linea OAZ / CMZ, quella appena adiacente all&#39;altra, su entrambi i lati della linea (vedere la Figura 2A, B, freccia). </li><li> Usando una mano / una pinza sul lato CMZ continuano a tenere su la cultura feriti, mentre con l&#39;altra mano / pinza, tirare delicatamente il OAZ lungo la linea OAZ / CMZ. La CMZ separa facilmente dalla OAZ lungo questa linea. Continuare lungo questa linea intorno all&#39;intera cultura finché le due regioni sono completamente separati. </li><li> Studiare i separati OAZ e CMZ frazioni per l&#39;analisi molecolare, come 24 o biochimica analisi di RNA-Seq quali Western Blot o co-immunoprecipitazione 23,25. </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...

La clinicamente rilevante, la chirurgia della cataratta finto, ex vivo epiteliale modello guarigione delle ferite qui descritto è stato sviluppato per fornire uno strumento per indagare i meccanismi che regolano la riparazione dei tessuti epiteliali in risposta ad un infortunio. Le caratteristiche principali che sono state finalizzate per nella creazione di questo modello inclusi 1) creare condizioni che strettamente replicato la risposta in vivo a ferire in un ambiente culturale, 2) la facilità di modulare gli elementi regolatori di riparazione, e 3) capacità di immagine il processo di riparazione, nella sua interezza, in tempo reale. La sfida, quindi, è stato quello di creare un modello di cultura in cui è stato possibile studiare e manipolare, riparazione della ferita epiteliale in microambiente nativo delle cellule. La disponibilità di questo modello ferita riparazione apre nuove possibilità per individuare gli spunti di segnalazione endogeni di matrice proteine, citochine e chemochine che regolano il processo di riparazione. Inoltre, il modello è ideale per l&#39;esame come unn epitelio è in grado di muoversi come un foglio collettivo per ri-epithelialize della ferita 2,3, e per la determinazione della stirpe di cellule mesenchimali capo sul bordo della ferita che funzionano nel dirigere la migrazione collettiva dell&#39;epitelio feriti 4. Questo modello offre anche una piattaforma con cui identificare terapie che potrebbero promuovere efficace la guarigione delle ferite e prevenire ferita aberrante riparazione 5. Ci sono già un certo numero di modelli disponibili ferita riparazione, sia nella cultura e in vivo, che hanno fornito la maggior parte di ciò che si conosce il processo di riparazione delle ferite oggi. In modelli animali di lesioni, come cornea 6-12 e 13-17 della pelle, vi è la possibilità di studiare la risposta del tessuto al ferimento nel contesto di tutti i mediatori riparazione che potrebbero essere coinvolti nel processo, compresi i contributi dal vascolarizzazione e del sistema nervoso. Tuttavia, vi sono limitazioni per manipolare il expericondizioni mentali in vivo, e non è ancora possibile condurre studi di imaging della risposta di riparazione in vivo, continuamente nel tempo. Al contrario, la maggior parte dei modelli di coltura in vitro ferita riparazione, come la ferita graffiatura, possono essere facilmente manipolati e seguiti nel tempo ma mancano contesto ambientale di studiare guarigione delle ferite nel tessuto in vivo. Mentre ex vivo modelli offrono il vantaggio di studiare il processo di riparazione pregiudizio continua nel tempo nel contesto del microambiente delle cellule associata alla capacità di modulare i regolatori molecolari di riparazione in qualsiasi punto di tempo nel processo, ci sono alcuni modelli che si adattano questi parametri. Qui è descritto una procedura per generare altamente riproducibile ex vivo epiteliali guarigione della ferita culture che riproducono la risposta di un tessuto epiteliale di un ferimento fisiologico. Utilizzando la lente pulcino embrione come fonte di tessuto, un ex vivo mocviene eseguita la chirurgia della cataratta k. L&#39;obiettivo è un tessuto ideale da utilizzare per questi studi dal momento che è indipendente all&#39;interno di una spessa capsula membrana basale, avascolare, non è innervato, e priva di qualsiasi stroma associato 18,19. Nella malattia umana, chirurgia della cataratta Indirizzi perdita della vista a causa opacizzazione della lente, e comporta la rimozione della massa cellulare fibre lente, che comprende la maggior parte della lente. Dopo visione chirurgia della cataratta viene ripristinata mediante l&#39;inserimento di una lente intraoculare artificiale. La procedura di chirurgia della cataratta, attraverso la rimozione di cellule di fibre, induce una risposta lesioni nell&#39;epitelio lente adiacente, che risponde riepitelizzazione dell&#39;area posteriore della capsula del cristallino che era stato occupato dalle cellule della fibra. Nella chirurgia della cataratta, come nella maggior parte delle risposte di riparazione della ferita, si verifica talvolta un esito fibrotico aberrante risposta guarigione della ferita, associata alla comparsa di miofibroblasti, che nella lente è noto come posteriore capsule opacizzazione 20-22. Per generare il modello guarigione delle ferite chirurgia della cataratta, una procedura di chirurgia della cataratta è imitato in lenti rimosso dall&#39;occhio pulcino embrione per produrre una lesione fisiologica. Rimozione microchirurgica delle fibre lente risultati cellule in una ferita circolare molto consistente circondata dalle cellule epiteliali lente. Questa popolazione di cellule rimane fermamente attaccata alla membrana capsula del cristallino seminterrato ed è ferito dalla procedura chirurgica. Le cellule epiteliali migrano sulla zona dissodata della membrana basale endogena per guarire la ferita, guidata da una popolazione di cellule mesenchimali ricche di vimentina noti nel processo di riparazione come cellule capo 1. Con questo modello la risposta di un epitelio di lesione può essere facilmente visualizzata e seguita con il tempo nel contesto del microambiente delle cellule. Le cellule sono facilmente accessibili per le modifiche dell&#39;espressione o l&#39;attivazione di molecole che dovrebbero svolgere un ruolo nella riparazione delle ferite. Una potente funzione di thè il modello è la capacità di isolare e studiare i cambiamenti specifici di migrazione nel quadro di guarigione della ferita. La capacità di preparare un gran numero di ex vivo cicatrizzanti culture abbinati invecchiato per studi è un altro vantaggio di questo modello. Così, questo modello di sistema offre l&#39;opportunità unica di prendere in giro a parte i meccanismi di riparazione delle ferite e terapie di prova per il loro effetto sul processo di guarigione delle ferite. Il finto modello ex vivo chirurgia della cataratta dovrebbe avere ampia applicabilità, fornendo una risorsa fondamentale per lo studio dei meccanismi di riparazione del pregiudizio.

<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 Modello creato per studiare il processo di guarigione delle ferite nei microambiente nativo le cellule &#39; Per studiare i meccanismi coinvolti nella regolazione guarigione delle ferite di un epitelio all&#39;interno microambiente nativo delle cellule, un clinicamente rilevante ex vivo finto modello chirurgia della cataratta è stato creato. Questo modello è stato creato da tessuto lente che offre molti vantaggi per le sue proprietà intrinseche: 1) l&#39;obiettiv...

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.

Istituzione di un clinicamente rilevanti<em&gt; Ex Vivo</em&gt; Mock chirurgia della cataratta modello per indagare epiteliale Wound Repair in un microambiente Native

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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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 Views.  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

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.

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 e in modelli in vivo per studiare transizione corneale endoteliale-mesenchimale

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

Ricerca

Biologia dello sviluppo

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Combinazione di Microstereolithography e Electrospinning per la Produzione di Membrane Equipaggiato con nicchie per la rigenerazione della cornea

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Bioingegneria

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation results in calcium mobilization events that propagate from cell to cell, which are essential for initiating cellular motility into the wound bed, promoting efficient wound healing. The Trinkaus-Randall lab has developed a methodology for imaging the corneal wound healing response in ex vivo murine globes in real time. This approach involves enucleating an intact globe from a mouse that has been euthanized per established protocols and immediately incubating the globe with a calcium indicator dye. A counterstain that stains other features of the cell can be applied at this stage to assist with imaging and show cellular landmarks. The protocol worked well with several different live cell dyes used for counterstaining, including SiR actin to stain actin and deep red plasma membrane stain to stain the cell membrane. To examine the response to a wound, the corneal epithelium is injured using a 25 G needle, and the globes are placed in a 3D printed holder. The dimensions of the 3D printed holder are calibrated to ensure immobilization of the globe throughout the duration of the experiment and can be modified to accommodate eyes of different sizes. Live cell imaging of the wound response is performed continuously at various depths throughout the tissue over time using confocal microscopy. This protocol allows us to generate high-resolution, publication-quality images using a 20x air objective on a confocal microscope. Other objectives can also be used for this protocol. It represents a significant improvement in the quality of live cell imaging in ex vivo murine globes and permits the identification of nerves and epithelium.

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

The present work describes a novel experimental protocol that utilizes a 3D printed holder to enable high-resolution live cell imaging of enucleated globes. Through this protocol, the cellular calcium signaling activity in wounded corneal epithelium from ex vivo globes can be observed in real time.

Imaging di cellule vive di globi ex vivo intatti utilizzando un nuovo supporto stampato in 3D

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation ...

Biochimica

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;

Studiare la rigenerazione dei tessuti senza cicatrici nelle cornee embrionali dei pulcini feriti

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.

Guarigione delle ferite di abrasione epiteliale corneale con oculare Burr come un modello per Cornea

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 ...

Medicina

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 modello di cultura di organo corneale per la guarigione delle ferite studi

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 ...

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.

Un modello di abrasione epiteliale per lo studio della guarigione delle ferite corneali

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.

Modelli animali ex vivo e in vivo per lesioni meccaniche e chimiche dell'epitelio corneale

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.

Produzione di siero autologo condizionato modificato e valutazione ex vivo del suo potenziale cicatrizzante nell'epitelio corneale murino

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

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.

Istituzione di un clinicamente rilevanti

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