1 Impostazione del campo sterile <ol><li> Don adeguato abbigliamento compreso tappo e la maschera. Indossare occhiali protettivi quando in camera di trasformazione. </li><li> Preparare la zona di trasformazione. Quando si utilizza un locale, mantenere il funzionamento continuo del ventilatore per creare pressione positiva nella camera. </li><li> Raccogliere tutte le forniture. Controllare le date di scadenza, integrità della confezione, e indicatore di sterilizzazione. </li><li> Lavarsi le mani e indossare guanti non sterili. </li><li> Aprite i kit sterili per stabilire un campo sterile. Quando si utilizza un locale per il trattamento, posizionare i kit sul tavolo e asettico aprire le avvolge. Non utilizzare le esterne 1 ½ pollici di involucro come parte del campo sterile in quanto questo segmento è considerato non sterile. </li><li> Usando una tecnica asettica, aperto e rilasciare gli elementi sterili sul campo sterile se non è già parte del pacchetto premade. </li><li> Spostare microscopio in modo che sia al di sopra del campo sterile, attivare la sorgente luminosa del microscopio, e regolare oculari come necessario. </li><li> OBTtessuto corneale ain a essere spogliato (nei mezzi di conservazione) e posto vicino alla zona di lavorazione sterile preparata. Allentare il tappo della camera d&#39;osservazione e lasciare riposare sulla parte superiore della visualizzazione di base della camera senza fili impegnati. Eliminare i guanti non sterili. </li><li> Aprire un paio di guanti sterili e camice sterile con un asciugamano. </li><li> Eseguire lavaggio chirurgico. Indossare camice sterile, quindi guanti sterili. </li><li> Scatole strumento aperto in modo che tutte le forniture sono accessibili. Aprire umidità telo impermeabile per creare l&#39;area di lavoro. </li><li> Collegare l&#39;ago a una siringa da 5 cc. Utilizzando un emostatico, preparare il ago G 30 disponendo una curva 1 mm dalla smussatura, e un&#39;altra curva 1 mm dalla base (per formare una forma a &quot;S&quot;). </li><li> Montare la siringa di vetro per il trypan macchia blu inserendo lo stantuffo nella siringa e il dito stringendo lo stantuffo. Allega cannula. </li><li> Fissare la cannula per la bottiglia di soluzione salina bilanciata (BSS). </li><li> Separare il pugno trapano dal blocco vuotoe mettere da parte. Centra il blocco vuoto all&#39;interno del campo visivo del microscopio. </li><li> Portare il blocco vuoto a fuoco regolando le impostazioni del microscopio. </li></ol> 2 Procedura di donatore di tessuti Preparazione <ol><li> Sollevare il coperchio della camera di visualizzazione utilizzando un foglio quadrato come una barriera. Con la mano opposta e con pinze, afferrare delicatamente cornea da sclera RIM per eliminare cornea. Sostituire il coperchio della camera di osservazione. </li><li> Completamente deprimere la siringa di aspirazione, quindi posizionare cornea lato epiteliale verso il basso sul blocco vuoto e assicurarsi che sia centrato. (Figura 1) </li><li> Posti a sedere leggermente inferiore al blocco vuoto con i posti di guida. Engage vuoto rilasciando lentamente pistone della siringa di aspirazione. Rimuovere l&#39;anello di posti a sedere e mettere da parte. (Figura 2). </li><li> Eseguire una trapanazione parziale di segnare attraverso la membrana di Descemet. </li><li> Regolare microscopio zoom e messa a fuoco in modo che il tessuto sia ben visualizzato. </li><li&gt; Inclinare il blocco vuoto e utilizzare una lancia tampone sterile posto al limbus per rimuovere eccesso di terreno. (Figura 3) </li><li> Posizionare abbastanza macchia al limbus a macchiare il bordo della membrana di Descemet segnato. Lasciare che la macchia di rimanere sull&#39;endotelio per 60-90 sec. (Figura 4) </li><li> Rimuovere macchia: <ol><li> Inclinare il blocco vuoto per versare la macchia della cornea e sul tampone di garza. </li><li> Utilizzare le lance chirurgici per rimuovere l&#39;eccesso macchia dalla zona limbare della cornea, facendo attenzione a non toccare l&#39;endotelio. (Figura 5) </li></ol></li><li> Utilizzare alcune gocce di BSS per sciacquare la cornea di soluzione colorante, e lance tampone sterile per rimuovere l&#39;eccesso BSS. (Figura 6) </li><li> Mettere 1-2 gocce di BSS sul cerchio sclera per impedire tessuto da essiccazione. Ripetere, se necessario, durante tutta la procedura. </li><li> Eseguire dissezione parziale della membrana di Descemet: <ol><li> Cominciando dove collegare il tubos al blocco vuoto, utilizzando pinze aperte, mettere una punta al bordo del segno partitura e la membrana leggermente separati di Descemet e l&#39;endotelio lontano dal stroma. (Figura 7). </li><li> Membrana separata di Descemet e l&#39;endotelio non più di 1 mm dal bordo segnato e continuare la dissezione a 360 ° intorno. (Figura 8). </li><li> Nota la posizione di eventuali micro lacrime mettendo un punto sul blocco vuoto con il marcatore pelle. NOTA: Se il trapano non penetrare completamente attraverso la membrana di Descemet, utilizzare il bordo smussato del preparato 30 ago G per completare il punteggio attraverso la membrana di Descemet, se necessario, seguita da una seconda applicazione di colorazione, se necessario. Se adesività della membrana di Descemet ostacola la dissezione parziale, rimuovere l&#39;endotelio di fuori dell&#39;area trephinated come necessario. </li></ol></li><li> Usare pinze per rimuovere eventuali tag endoteliali che si sovrappongono il marchio punteggio. </li> &lt;li&gt; Si consideri la posizione del micro lacrime precedentemente segnalati e ruotare il blocco vuoto in modo che il più grande è a ore 6. Questo diventerà il cardine del lembo. </li><li> Utilizzando le pinze, afferrare la membrana di Descemet alle ore 12. Separare delicatamente la membrana di Descemet dallo stroma mediante pelatura verso la cerniera. Creare la cerniera bloccando la separazione 2 mm dal marchio punteggio. NOTA: i bordi periferici del lembo inizieranno a scorrere durante la separazione. (Figura 9). </li><li> Adagiare il lembo posteriore in posizione sul stroma. Utilizzare BSS e tampone lance per unscroll l&#39;endotelio e riportarlo alla sua posizione originale. (Figura 10A-C) </li><li> Rimuovere il liquido residuo dal tra la membrana di Descemet e stroma con lance tampone sterile in modo che il lembo rimane in posizione. (Figura 11) </li><li> Utilizzare lance tampone sterile per assorbire l&#39;umidità in eccesso dalla sclera vicino alla cerniera. Utilizza l&#39;indicatore pelle per disegnare unn punta di freccia sul cerchio sclera che punta al centro della cerniera, rimuovere l&#39;inchiostro in eccesso con un tampone sterile lancia. (Figura 12) </li><li> Rilasciare vuoto premendo il pistone della siringa collegata al blocco vuoto. Mentre il vuoto è disinserito, utilizzare pinze per rimuovere cornea dal blocco vuoto. </li><li> Sollevare il coperchio della camera di visualizzazione utilizzando un foglio quadrato come una barriera. Posizionare la cornea nella camera di osservazione, lato endoteliale, e sostituire il coperchio della camera di osservazione. </li><li> Ripetere i passaggi precedenti procedura con compagno di cornea, se applicabile. Usare una garza per pulire il blocco vuoto prima di elaborare il secondo tessuto. </li><li> Serrare il coperchio della camera di osservazione (s) e rimuovere il tessuto dalla zona di lavorazione. </li><li> Abbattere il campo sterile da cessione di articoli secondo Biologicamente Piano di gestione dei rifiuti. Rimuovere i guanti contaminati, e abito. Lavarsi le mani e indossare guanti non sterili. </li><li> Eseguire post-cut valutazione lampada a fessura e la microscopia speculare della cornea (s)e registrare i risultati. </li></ol>

<ol>
	<li>Fernandez, M., Afshari, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Keratoplasty:+From+DLEK+to+DMEK.">Endothelial Keratoplasty: From DLEK to DMEK.</a> Middle East African Journal of Ophthalmology. 17 (1), 5-8 (2010).</li><li>Kruse, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Stepwise+Approach+to+Donor+Preparation+and+Insertion+Increases+Safety+and+Outcome+of+Descemet+Membrane+Endothelial+Keratoplasty.">A Stepwise Approach to Donor Preparation and Insertion Increases Safety and Outcome of Descemet Membrane Endothelial Keratoplasty.</a> Cornea. 30 (5), 580-587 (2011).</li><li>Dapena, I., Moutsouris, K., Droutsas, K., Ham, L., van Dijk, K., Melles, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+'No-Touch'+Technique+for+Descemet+Membrane+Endothelial+Keratoplasty.">Standardized 'No-Touch' Technique for Descemet Membrane Endothelial Keratoplasty.</a> Archives of Ophthalmology. 129, 88-94 (2011).</li><li>Tourtas, T., Laaser, K., Bachmann, B., Cursiefen, C., Kruse, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet+Membrane+Endothelial+Keratoplasty+versus+Descemet+Stripping+Automated+Endothelial+Keratoplasty.">Descemet Membrane Endothelial Keratoplasty versus Descemet Stripping Automated Endothelial Keratoplasty.</a> American Journal of Ophthalmology. 153 (6), 1082-1090 (2012).</li><li>Guerra, F., Anshu, A., Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Keratoplasty:+fellow+eyes+comparison+of+Descemet+stripping+automated+endothelial+keratoplasty+and+Descemet+membrane+endothelial+keratoplasty.">Endothelial Keratoplasty: fellow eyes comparison of Descemet stripping automated endothelial keratoplasty and Descemet membrane endothelial keratoplasty.</a> Cornea. 30 (12), 1382-1386 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2012 Eye Banking Statistical Report: Surgical Use and Indications for Corneal Transplant Statistical Report Analysis. , (2012).</li><li>Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+stripping+endothelial+keratoplasty.">Descemet's stripping endothelial keratoplasty.</a> Current Opinion Ophthalmology. 18 (4), 290-294 (2007).</li><li>Woodward, M., Titus, M., Mavin, K., Shtein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Donor+Tissue+Preparation+for+Endothelial+Keratoplasty.">Corneal Donor Tissue Preparation for Endothelial Keratoplasty.</a> Journal of Visualized Experiments. (64), e3847 (2012).</li><li>Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+membrane+endothelial+keratoplasty+surgery:+update+on+the+evidence+and+hurdles+to+acceptance.">Descemet's membrane endothelial keratoplasty surgery: update on the evidence and hurdles to acceptance.</a> Current Opinion Ophthalmology. 24 (4), 329-335 (2013).</li><li>Venzano, D., Pagani, P., Randazzo, N., Cabiddu, F., Traverrso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet+Membrane+air-bubble+separation+in+donor+cornea.">Descemet Membrane air-bubble separation in donor cornea.</a> Journal of Cataract and Refractive Surgery. 36 (12), 2022-2027 (2010).</li><li>Price, F., Price, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DSEK: What you need to know about endothelial keratoplasty. , (2009).</li><li>Studeny, P., Sivekova, D., Lieheova, K., Vokrojova, M., Kuchynka, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+technique+of+lamellar+keratoplasty+(DMEK-S).">Hybrid technique of lamellar keratoplasty (DMEK-S).</a> Journal of Ophthalmology. 2013, 2013.254383 (2013).</li><li>Muraine, M., Gueudry, J., He, Z., Piselli, S., Lefevre, S., Toubeau, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+technique+for+the+preparation+of+corneal+grafts+for+Descemet+membrane+endothelial+keratoplasty.">Novel technique for the preparation of corneal grafts for Descemet membrane endothelial keratoplasty.</a> American Journal of Ophthalmology. 156 (5), 851-859 (2013).</li><li>Lie, J. T., Birbal, R., Ham, L., van der Wees, J., Melles, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+tissue+preparation+for+Descemet+membrane+endothelial+keratoplasty.">Donor tissue preparation for Descemet membrane endothelial keratoplasty.</a> Journal of Cataract Refractive Surgery. 34 (9), 1578-1583 (2008).</li><li>Kruse, F. E., Laaser, K., Cursiefen, C., Heindl, L. M., Schlotzer-Schrehardt, U., Riss, S., Bachmann, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+approach+to+donor+preparation+and+insertion+increases+safety+and+outcome+of+Descemet+membrane+endothelial+keratoplasty.">A stepwise approach to donor preparation and insertion increases safety and outcome of Descemet membrane endothelial keratoplasty.</a> Cornea. 30 (5), 580-587 (2011).</li><li>Rudolph, M., Kaaser, L., Bachmann, B., Cursiefen, C., Epstein, D., Kruse, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+higher-order+aberrations+after+Descemet&#8217;s+membrane+endothelial+keratoplasty.">Corneal higher-order aberrations after Descemet&#8217;s membrane endothelial keratoplasty.</a> Ophthalmology. 119 (3), 528-535 (2012).</li><li>Price, F., Price, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+endothelial+keratoplasty.">Evolution of endothelial keratoplasty.</a> Cornea. 32, 28-32 (2013).</li><li>Dapena, I., Dapena, L., Dirisamer, M., Ham, L., Melles, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+acuity+and+endothelial+cell+density+following+Descemet+membrane+endothelial+keratoplasty+(DMEK).">Visual acuity and endothelial cell density following Descemet membrane endothelial keratoplasty (DMEK).</a> Arch Soc Esp Oftalmol. 86 (12), 395-401 (2011).</li></ol>

Hassan Tausif e il dottor Navasuja Chandrasekaran hanno nulla da rivelare. Drs. Mian, Shtein, e Woodward sono dirigenti medici per la Banca degli Occhi del Michigan. Mr. Mavin, il signor Tito, e la signora Johnson sono dipendenti di Midwest Banche degli Occhi.

Lo strato di cellule endoteliali corneali gioca un ruolo fondamentale nel mantenimento della chiarezza della cornea attraverso la disidratazione. Malattie delle cellule endoteliali possono provocare significativa compromissione visiva. DMEK mira a sostituire le cellule endoteliali malate con un innesto donatore costituito da membrana e strato di cellule endoteliali di Descemet. Donatori preparazione dei tessuti con un DM intatta e adeguata conta delle cellule endoteliali sono fondamentali per la sopravvivenza del tessut...

Cheratoplastica endoteliale (EK) si è rapidamente sostituito cheratoplastica perforante (PKP), come la procedura di scelta per il trattamento di malattie endoteliali rappresentano oltre il 50% di tutti i trapianti eseguiti negli Stati Uniti nel 2012 6. PKP è limitata dalla riabilitazione visiva prolungato con alta astigmatismo e l&#39;instabilità di rifrazione, la perdita di integrità globo e le complicanze legate sutura. Procedure primi EK generazione, tra cui Profondo lamellare endoteliale Cheratoplastica (DLEK) coinvolto dissezione manuale di stroma corneale con trapianto di tessuto dello spessore del donatore parziale. Queste procedure sono riusciti a trapianto endoteliale tessuto del donatore, pur mantenendo una superficie corneale integro ma erano tecnicamente impegnativo e portato a limitata acuità visiva meglio corretta a causa di un irregolare della cornea lamellare 1 interfaccia. La procedura EK attuale di scelta è di Descemet Stripping Endothelial Keratoplasty (DSEK) che offre clar cornealelità con recupero più rapido della visione, minimizza postoperatorio errore di rifrazione e astigmatismo, e diminuisce il rischio di deiscenza della ferita traumatica, pur mantenendo la superficie corneale 7. Questa procedura comporta la rimozione della membrana di Descemet e l&#39;endotelio malata e la sostituzione con parziale donatore spessore del tessuto corneale comprendente un nuovo strato di cellule endoteliali. La cornea del donatore è sezionato con un microcheratomo per preparare il tessuto posteriore, essendo trapiantato 8. Preparazione del tessuto donatore dall&#39;occhio-banche è stato determinante per la rapida crescita e, probabilmente, il successo di questa procedura. Il prossimo aumento di 16 volte nelle procedure DSEK negli Stati Uniti tra il 2005 e il 2012 corrisponde a quasi la metà del tessuto in corso di preparazione da parte delle banche degli occhi-6. Anche se DSEK può migliorare l&#39;acuità visiva a 20/40 o meglio, la maggior parte dei pazienti non raggiungono 20/20 a causa di interfaccia stromale foschia e maggiore lucedisperdere l&#39;aggiunta di donatore del tessuto stromale 7. La tecnica chirurgica EK è progredita al di Descemet Membrane Endothelial Keratoplasty (DMEK) che prevede il trapianto di membrana unico donatore di Descemet (DM) ed endotelio. Questa procedura ha una maggiore percentuale di pazienti che raggiungono 20/20, più veloce recupero visivo, e un minor rischio di rigetto endoteliale 9. Tuttavia, la preparazione del tessuto del donatore è più difficile a causa delle dimensioni più sottili innesto.

<table><tbody><tr><td>VisionBlue</td><td>D.O.R.C., Zuiland, The Netherlands</td><td>NA</td><td></td></tr><tr><td>Barron vacuum punch</td><td>Katena, Denville, New Jersey</td><td>K20-2112</td><td></td></tr></tbody></table>

corneal donor tissue preparation for descemet's membrane endothelial keratoplasty

Di Descemet Membrane Endothelial Keratoplasty (DMEK) è una forma di trapianto di cornea in cui solo uno strato di singola cellula, l&#39;endotelio corneale, insieme alla sua membrana basale (membrana di Descemet) viene introdotto sul stroma posteriore del destinatario 3. A differenza di Descemet Stripping Automated Endothelial Keratoplasty (DSAEK), dove viene introdotto stroma donatore aggiuntivo, viene creata alcuna interfaccia innaturale stroma-to-stroma. Come risultato, la naturale anatomia della cornea è conservato il più possibile consentendo un migliore tempo di recupero e acuità visiva 4. Cheratoplastica endoteliale (EK) è la procedura di scelta per il trattamento della disfunzione endoteliale. I vantaggi di EK includono un rapido recupero della visione, la conservazione dell&#39;integrità oculare e il minimo cambiamento di rifrazione a causa di uso di una piccola incisione periferica 1. DSAEK utilizza tessuto del donatore preparato con parziale stroma spessore e endotelio. La rapida success e l&#39;utilizzo di questa procedura può essere attribuito alla disponibilità di eye-banca preparati tessuti pretagliati. I vantaggi di preparazione eye-banca del tessuto del donatore includono l&#39;eliminazione del bisogno di attrezzature specializzate in sala operatoria e la disponibilità di back up tessuto del donatore in caso di perforazione del tessuto durante la preparazione. Inoltre, la preparazione ad alto volume di tessuto del donatore da parte di tecnici eye-Banca può fornire una migliore qualità del tessuto del donatore. DSAEK può aver limitato migliore acuità visiva corretta a causa della creazione di una interfaccia stromale tra il donatore e il ricevente della cornea. L&#39;eliminazione di questa interfaccia con il trapianto di membrana unico donatore di Descemet e l&#39;endotelio nella DMEK possono migliorare i risultati visivi e ridurre le complicanze dopo EK 5. Simile a DSAEK, successo a lungo termine e l&#39;accettazione di DMEK dipende dalla facilità di disponibilità di pretagliati, eye-banca preparato tessuto del donatore. Qui vi presentiamo un approccio graduale alla preparazione dei tessuti del donatore che possono ridurre alcuni barriERS oculari banche affrontano nella fornitura di innesti DMEK.

La corretta separazione dei DM e endotelio da stroma è essenziale nella preparazione di tessuti pretagliati per DMEK. Cinquanta cornee dei donatori con una età media di 65 ± 6 anni (range 55-75), la morte di tempo di elaborazione di 5,9 ± 2,3 giorni (range 1-10) e la densità delle cellule endoteliali (ECD) di 2.616 ± 321 cellule / mm 2 ( gamma 2,049-3,247) sono stati utilizzati per valutare la fattibilità e la riproducibilità della tecnica di preparazione chirurgica da due tecnici della banca degli oc...

Watch this Scientific Journal Video about Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty at JoVE.com

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Cheratoplastica endoteliale è una tecnica chirurgica in continua evoluzione come risultato progressi nel trapianto di innesti sottili con ogni successione 1. Qui vi presentiamo una tecnica per la dissezione dei tessuti manuale controllato per consentirne la sicura separazione e ripetibile di endotelio e la membrana di Descemet da donatori pulsanti corneosclerale per il trapianto 2.

Corneale Preparazione donatore di tessuti per di Descemet Membrane Endothelial Keratoplasty

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

corneal-donor-tissue-preparation-for-descemet-s-membrane-endothelial

Research

JoVE Journal

Medicine

15.2K Views.  University of Michigan.  Cheratoplastica endoteliale è una tecnica chirurgica in continua evoluzione come risultato progressi nel trapianto di innesti sottili con ogni successione 1. Qui vi presentiamo una tecnica per la dissezione dei tessuti manuale controllato per consentirne la sicura separazione e ripetibile di endotelio e la membrana di Descemet da donatori pulsanti corneosclerale per il trapianto 2. 

Video: Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Adenovirale Terapia genica per diabetici cheratopatia: Effetti sulla guarigione delle ferite e Stem Cell Marker Expression in Human Organ cornee-coltura e le cellule epiteliali Limbal

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Ricerca

Biologia dello sviluppo

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

Una tecnica semplificata per<em&gt; In situ</emEscissione&gt; della cornea e l&#39;eviscerazione del tessuto retinico dal globo oculare umano

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

Medicina

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

Preparazione del tessuto corneale dei donatori per Keratoplasty Endothelial

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

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.

Ingegneria tissutale corneale: Un In Vitro modello delle interazioni Stromal-nervo della Cornea umana

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

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

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.

Murini trapianto di cornea: Un modello per studiare la forma più comune di trapianto di organi solidi

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

An &#8220;All-laser&#8221; Endothelial Transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

Un &quot;All-laser&quot; Endothelial Transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

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.

Crescita degli strati cellulari endoteliali umani e ovini sulle membrane biomateriali

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

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

Microscopia a coerenza ottica a campo intero per l'analisi istologica delle caratteristiche stromali negli innesti corneali

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

Immunologia e infezioni

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).

Sviluppo di un modello sperimentale non invasivo e assistito dal laser della perdita di cellule endoteliali corneale

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

Corneale Preparazione donatore di tessuti per di Descemet Membrane Endothelial Keratoplasty

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