Gli autori desiderano ringraziare FORTE Progetto, finanziato dalla Regione Toscana (POR CReO FESR 2007-2013, Bando Unico R &amp; S 2012), il 7 ° PQ ECHORD UE ++ Experiment LA-ROSES che parzialmente supportate le attività di ricerca, e il 7 ° PQ BiophotonicPlus Progetto &quot;LITE&quot; concesso dalla Regione Toscana.

Lo studio è stato condotto con l&#39;prospetticamente approvazione del comitato etico dell&#39;ospedale; il consenso informato è stato ottenuto. Lo studio è stato in adesione ai principi della Dichiarazione di Helsinki. 1. donatori Endotelio Preparazione <ol><li> Utilizzare una cornea del donatore, come predisposto dalla banca degli occhi locale a temperatura ambiente. </li><li> In sala operatoria, estrarre la cornea del donatore dal suo contenitore consegna e messo a parte la soluzione per la conservazione dei tessuti e la nutrizione che viene utilizzato per il trasporto della cornea. </li><li> Posizionare il donatore corneo-sclerale bordo su una camera anteriore artificiale, in cui un liquido può fluire e creare una pressione variabile; coprire la cornea con la testa di fissaggio del tessuto. </li><li> Collegare camera anteriore artificiale (AAC) attraverso un connettore a 3 vie per una siringa riempita con la conservazione cornea e nutrizione liquido. </li><li> Utilizzare l&#39;anello di compressione per tenere il fermo saldamente in posizione. </li><li> Utilizzare il syringe collegato alla AAC di mantenere la pressione intracameral: riempire la camera anteriore con la soluzione di conservazione del tessuto fino a raggiungere la pressione ottimale (vedi 1.7). Chiudere il connettore. </li><li> Testare la pressione all&#39;interno della camera anteriore con un dito. Modificare la pressione all&#39;interno fino a raggiungere la pressione interna destra (nell&#39;intervallo 60-70 mmHg). Chiudere il connettore a 3 vie. </li><li> Misurare lo spessore della cornea del donatore con Optical Coherence Thomography (OCT). Mantenere la camera anteriore con quella cornea in posizione fissa davanti ottiche dello strumento e poi prendere una acquisizione completa spessore-OCT. </li></ol> 2. laser a femtosecondi Preparazione di donatori Endotelio <ol><li> Eseguire applanazione della cornea del donatore con il dispositivo laser a femtosecondi. Utilizzare il laser femtosec per tagliare il tessuto del donatore con tre successivi tagli: taglio laterale posteriore, pieno lamellare e anteriore taglio laterale. Impostare i seguenti parametri per le tre tagli.<ol><li> Per il taglio completo lamellare (modello raster, iniziare), impostare la profondità di taglio corrispondente al punto più sottile della cornea del donatore (misurata con il OCT come descritto in 1.8), sempre sottraendo 95 micron. Impostare l&#39;energia dell&#39;impulso nell&#39;intervallo 0,8-0,9 μJ (a seconda della profondità di lavoro). Impostare il diametro a 8,7 mm. Impostare la separazione posto tangenziale al 2 micron. Impostare la separazione posto radiale a 2 micron. </li><li> Per il taglio laterale anteriore, impostare la profondità posteriore 30 micron più profonda della precedente completo taglio lamellare. Impostare l&#39;energia dell&#39;impulso a 2.10 μJ; il diametro a 8,6 mm, la separazione posto a 3 micron, mentre la separazione strato a 3 micron. </li><li> Per il taglio laterale posteriore, individuare la profondità anteriore del lato posteriore tagliato 30 micron anteriormente a pieno taglio lamellare. Impostare la profondità posteriore al 900 micron. Impostare l&#39;energia dell&#39;impulso a 2.10 μJ; il diametro a 8,3 mm, la separazione posto a 2 micron; la separazione dello strato di 2 micron. </li></ol></li&gt; </ol> 3. Destinatario Preparazione Eye <ol><li> Preparare il paziente per un intervento chirurgico. Effettuare uno 4,00 millimetri incisione corneale alle ore 12 con un 4,0 millimetri precalibrati lama. Eseguire una paracentesi limbari con una lama pretarata 1,2 mm, posta a 02:00. Fare un altro paracentesi limbari a ore 6 con un coltello pugnalata di 30 °. </li><li> Inserire un mantenitore camera anteriore nella camera anteriore del paziente, attraverso le 2 paracentesi o&#39;clock. </li><li> Effettuare una diametro 8,2 millimetri descemetorexis circolare con il gancio dedicato. </li><li> Striscia la membrana Descemet ed endotelio dallo stroma posteriore e rimuovere i tessuti con un gancio Descemet. </li></ol> 4. Chromophore Preparazione <ol><li> Mettere 1 mg di indocianina Polvere verde in una provetta da 1,5 ml microcentrifuga. </li><li> Aggiungi 9 mg di acqua sterile (la soluzione di acqua ICG è del 10% w / w). </li><li> Mescolare manualmente la polvere ICG e l&#39;acqua con un agitatore di metallo. </li&gt; </ol> 5. La colorazione di donatori Endotelio <ol><li> Mettere l&#39;endotelio donatore sulla parte più larga di una Busin iniettore, con il lato interno a contatto con la superficie dell&#39;iniettore. </li><li> Macchiare il lato interno di endotelio donatore con la soluzione cromoforo, nella sua parte periferica, utilizzando una spatola. Il tessuto tinto corretto ha un colore verdastro omogenea. Attendere 3 minuti prima di iniziare la fase 5.3. </li><li> Tirare l&#39;endotelio dei donatori sulla parte anteriore del Busin iniettore, laminati. </li></ol> 6. Inserimento della Endotelio Donor <ol><li> Afferrare e inserire l&#39;endotelio donatore piegato utilizzando un atraumatica pinza coassiali per endoteliale lenticule. </li><li> Rimuovere il manutentore camera anteriore. </li><li> Suturare l&#39;incisione corneale e la paracentesi (eseguita al passo 3.1) con un unico stich Nylon 10.0. </li><li> Iniettare una bolla d&#39;aria per aprire il tessuto donatore e premerlo contro la cornea ricevente. La bolla d&#39;aria deve completely riempire lo spazio camera anteriore. </li></ol> 7. saldatura laser <ol><li> Posizionare il lenticule donatore nel centro della cornea ricevente, dall&#39;interno di un gancio o movimentazione dell&#39;aria bolla dall&#39;esterno della cornea con una spatola. </li><li> Utilizzare un diodo laser che emette a 810 nm, dotato di un diametro di 300 micron nucleo della fibra ottica sterile, con 0,22 come apertura numerica (NA). </li><li> Tenere la punta della fibra di fuori del bulbo oculare, e fornire la luce laser verso l&#39;endotelio macchiato, attraverso il tessuto corneale trasparente. La punta della fibra è in configurazione senza contatto. </li><li> Utilizzare le seguenti impostazioni per il laser: modalità di emissione di singolo punto, 70-80 durata dell&#39;impulso msec, 35-40 mJ per impulso. He-Ne puntamento del fascio su. </li><li> Invia spot laser singoli alla periferia macchiato dell&#39;endotelio donatore. Fornire i punti in sequenza: l&#39;aspetto finale è un anello di punti in periferia del lenticule donatore. La distanza tra due AdjaCentro spot cent è il doppio di un diametro dello spot. </li><li> Applicare una lente a contatto sull&#39;occhio del paziente, insieme a 0,3% e 0,1% tobramicina desametasone sospensione oftalmica. </li></ol>

<ol>
	<li>Tan, D. T., Dart, J. K., Holland, E. J., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+transplantation.">Corneal transplantation.</a> Lancet. 379 (9827), 1749-1761 (2012).</li><li>El Husseiny, M. A., Manero, F., Gris, O., Elies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+Review+and+Update+of+Surgical+Treatment+for+Corneal+Endothelial+Diseases.">Historical Review and Update of Surgical Treatment for Corneal Endothelial Diseases.</a> Ophthalmol Ther. , (2014).</li><li>Rossi, 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=Laser+tissue+welding+in+ophthalmic+surgery.">Laser tissue welding in ophthalmic surgery.</a> J Biophotonics. 1 (4), 331-342 (2008).</li><li>Pini, R., 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=Combining+femtosecond+laser+ablation+and+diode+laser+welding+in+lamellar+and+endothelial+corneal+transplants.">Combining femtosecond laser ablation and diode laser welding in lamellar and endothelial corneal transplants.</a> Proc. SPIE Ophthalmic Technologies XVIII. 6 (844), 684411-1-684411-7 (2008).</li><li>Rossi, 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=All-laser'+endothelial+corneal+transplant+in+human+patients.">All-laser' endothelial corneal transplant in human patients.</a> Proc. SPIE Ophthalmic Technologies XXII. 8209, 82091O-1-82091O-3 (2012).</li><li>Rossi, F., Pini, R., Menabuoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+model+analysis+on+the+temperature+dynamics+during+diode+laser+welding+of+the+cornea.">Experimental and model analysis on the temperature dynamics during diode laser welding of the cornea.</a> J Biomed Opt. 12 (1), 014031-1-014031-7 (2007).</li><li>Menabuoni, L., 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=Laser-assisted+corneal+welding+in+cataract+surgery:+retrospective+study.">Laser-assisted corneal welding in cataract surgery: retrospective study.</a> J Cataract Refract Surg. 33 (9), 1608-1612 (2007).</li><li>Buzzonetti, L., 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=Laser+Welding+in+Penetrating+Keratoplasty+and+Cataract+Surgery+in+Pediatric+Patients.+Early+Results.">Laser Welding in Penetrating Keratoplasty and Cataract Surgery in Pediatric Patients. Early Results.</a> J Cataract Refract Surg. 39 (12), 1829-1834 (2013).</li><li>Menabuoni, L., 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=The+'anvil'+profile+in+femtosecond+laser-assistedpenetrating+keratoplasty.">The 'anvil' profile in femtosecond laser-assistedpenetrating keratoplasty.</a> Acta Ophthalmol. 91 (6), e494-e495 (2013).</li><li>Canovetti, A., 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=Laser-assisted+penetrating+keratoplasty:+one+year&#8217;s+results+in+patients,+using+a+laser-welded+&#8220;anvil&#8221;-profiled+graft.">Laser-assisted penetrating keratoplasty: one year&#8217;s results in patients, using a laser-welded &#8220;anvil&#8221;-profiled graft.</a> Am J Ophthalmol. 158 (4), 664-670 (2014).</li></ol>

The authors declare that they have no competing financial interests.

Il &quot;tutto laser&quot; trapianto endoteliale è un approccio originale per il trapianto di cornea mini-invasiva. Tutte le procedure descritte nel protocollo sono state eseguite in sala operatoria, osservando la procedura igienica e sterilizzazione che sono pratiche comuni durante interventi chirurgici, come l&#39;uso di sterilizzato guanti, camice, maschera e cappello. La soluzione è stata preparata ICG in sala operatoria, appena prima della sua applicazione nella colorazione endotelio ...

In questo lavoro presentiamo un approccio originale alla cheratoplastica endoteliale, basato sull&#39;uso di un laser a femtosecondi per preparare tessuto donatore e un quasi diodo laser infrarosso per saldare sul letto destinatario. Misurazione intraoperatoria della cornea del donatore è necessario progettare correttamente le dimensioni del tessuto donatore. Cheratoplastica endoteliale è stato proposto negli ultimi anni per sostituire cheratoplastica perforante nel trattamento endoteliale 1,2 malattia. Il vantaggio principale di questa tecnica è un veloce recupero visivo, rispetto al cheratoplastica perforante, anestesia ridotta durante l&#39;intervento chirurgico, un ridotto rischio di rigetto del trapianto e la preservazione dell&#39;integrità dell&#39;occhio. Il fattore di rischio principale è postoperatorio lenticule donatore lussazione. La tecnica standard viene eseguita inserendo l&#39;endotelio donatore nella sua posizione finale in cui viene mantenuta mediante iniezione di una bolla d&#39;aria: suture vengono utilizzati a causa del Chara meccanica, biofisica e dimensionalicteristics dell&#39;endotelio. Inoltre, il recupero acuità visiva può essere limitato soprattutto a causa di una mancata corrispondenza tra tessuti donatore e ricevente a causa di un tessuto spesso trapiantato. Qui vi presentiamo una procedura nello svolgimento cheratoplastica endoteliale in grado di superare questi problemi principali. L&#39;endotelio donatore può essere assicurata nella sua posizione finale mediante l&#39;uso della tecnica di saldatura laser. Questo è un processo fototermico controllata e localizzata: può essere indotta all&#39;interfaccia donatore / ricevente. E &#39;stata studiata negli ultimi dieci anni e proposto in cheratoplastica perforante e nel trapianto di endotelio 3-5. Il vicino luce infrarossa (lunghezza d&#39;onda: 810 nm) emessa da un laser a bassa potenza diodo è consegnato verso il tessuto biologico al sito della ferita. La cornea è naturalmente trasparente a questa lunghezza d&#39;onda: per rendere questo tessuto di assorbire la luce laser, è necessario macchiare con un cromoforo. Il colorante è proposto un saturate sterilesoluzione acquosa d di indocianina verde (ICG). Abbiamo dimostrato che quando il tessuto corneale è correttamente macchiata di questa preparazione ICG, mostra un picco di assorbimento a 810 nm 6. Inoltre, ICG è ampiamente usato in diagnostica clinica e la sua sicurezza è stata già dimostrata in soggetti umani. La cornea colorato assorbe l&#39;energia luminosa laser a diodi e l&#39;effetto risultante principale è un aumento di temperatura controllata al sito di saldatura. Nessun effetti termici sono indotte nei tessuti non marcate. Il miglioramento della temperatura induce denaturazione termica reversibile nel collagene stromale, con una closuring immediata delle pareti della ferita sul raffreddamento. Questo effetto di saldatura laser è stato in primo luogo dimostrato nella chirurgia della cataratta 7,8 e cheratoplastica penetrante 9,10. Un approccio ottimizzato che presentiamo in questo articolo è stato studiato per l&#39;applicazione in cheratoplastica endoteliale. Nella chirurgia proposto, spot laser singoli (decine durata di msec) are consegnato al tessuto, con conseguente effetto fototermico localizzata nella dimensione spot (alcune centinaia di micron di diametro): l&#39;effetto indotto è una saldatura laser disco, costituito da una fotocoagulazione del collagene confinato all&#39;interfaccia donatore / host. Il risultato della denaturazione collagene nel sito saldato è una forte adesione tra i tessuti donatore e ospite, fornendo così un effetto di sutura che è impossibile ottenere con tecniche standard (punti). Il tessuto riacquista il suo aspetto naturale in un breve follow-up (1 mese) e l&#39;adesione tra i tessuti del donatore / ospite è migliorata dalla saldatura previsto nella fase iniziale della fase di guarigione. Per evitare l&#39;altro rischio principale della cheratoplastica endoteliale, cioè il trapianto di un tessuto donatore spessa, intrachirurgica coerenza ottica tomografia (OCT) è utilizzato: un dispositivo commerciale misura lo spessore della cornea del donatore, in modo che un profilo corretto del taglio può essere progettato con illaser femtosec. La proposta di &quot;tutto laser&quot; trapianto endoteliale sembra quindi migliorare i risultati clinici di questo tipo di chirurgia mini-invasiva.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Indocyanine Green</td>
			<td>Pulsion Medical Systems, Germany</td>
			<td>ICG-PULSION (http://www.pulsion.com/international-english/perfusion/icg-pulsion/)</td>
			<td>Alternative product: IC-GREEN, Akorn&#160; Inc., Lake Forest, Illinois- US (http://www.icginjection.com/)</td>
		</tr>
		<tr>
			<td>Femtosecond Laser&#160;</td>
			<td>Abbott Medical Optics,&#160; Abbott Laboratories Inc. Abbott Park, Illinois, USA</td>
			<td>iFS150 (http://www.abbottmedicaloptics.com/products/refractive/ilasik/ifs-advanced-femtosecond-laser)</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Coherence Tomography (OCT)</td>
			<td>Carl-Zeiss Meditec, Dublin, California- US (http://www.zeiss.com/meditec/en_de/home.html)</td>
			<td>Visante</td>
			<td></td>
		</tr>
		<tr>
			<td>Diode Laser</td>
			<td>E.l.En. Group s.pa., Calenzano-FI, Italy (http://www.elengroup.com/en/divisions/medical)</td>
			<td>Mod. WELD 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial Anterior Chamber</td>
			<td>CORONET, corneal graft products. Network Medical Products Ltd. Coronet House, Kearsley Road, Ripon, North Yorkshire, HG4 2SG, UK</td>
			<td>&#160;Artificial Anterior Chamber (A.A.C.) with large and small tissue-retaining heads. Code 51-935 (http://www.networkmedical.co.uk/ophthalmic_artificial_anterior_chamber.html)</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution for tissue preservation and nutrition&#160;&#160;</td>
			<td>&#160;AL.CHI.MI.A. Srl, Viale Austria 14, 35020 - Ponte S. Nicol&#242; - PD 
			&#160;ITALY</td>
			<td>Carry-C media for corneal deturgescence and transport at room temperature - 12 x 50 ml (http://www.alchimiasrl.com/en/organ-culture-at-31&#176;-c-eb/carry-c-eb)</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Il &quot;tutto laser&quot; intervento chirurgico viene proposto di effettuare il trapianto di cornea mini-invasiva. La procedura è facile da eseguire (vedi Figura 1): rispetto ad un trapianto endoteliale normale solo le fasi di misurare lo spessore corneale, la colorazione del tessuto donatore e fornire la luce laser vengono aggiunti. I vantaggi ottenuti compensano ampiamente un aumentato tempo chirurgico di pochi minuti. L&#39;uso di intraoperatoria Office per misurare lo spessore cornea del donatore ...

Watch this Scientific Journal Video about An â€œAll-laserâ€ Endothelial Transplant at JoVE.com

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

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 ...

Research

JoVE Journal

Medicine

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

9.3K Views.  Italian National Research Council. 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.

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

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Utilizzando quantitativa real-time PCR per la determinazione cellulare attecchimento dei donatori in un modello murino competitivo trapianto di midollo osseo

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Ricerca

Medicina

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

Murini eterotopica cuore Tecnica Trapianto

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

Tecnica di Suini appalti Fegato e trapianto ortotopico con un attivo Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Modello murino della alloimmune indotta Rifiuto vascolare e trapianto Arteriosclerosi

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

Rene Murine Tecnica trapianto

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Migliorato il metodo per la creazione di un In Vitro barriera emato - encefalica modello basato sulle cellule endoteliali Porcine cervello

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead&#160;to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

Reazione Griess ottimizzata per UV-Vis e determinazione a occhio nudo di Primaquine antimalarico

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Lesione da ischemia-riperfusione dell'organo simulando cambiamenti emodinamici nel modello di trapianto di fegato di ratto

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

TBase - an Integrated Electronic Health Record and Research Database for Kidney Transplant Recipients

TBase is an electronic health record (EHR) for kidney transplant recipients (KTR) combining automated data entry of key clinical data (e.g., laboratory values, medical reports, radiology and pathology data) via standardized interfaces with manual data entry during routine treatment (e.g., clinical notes, medication list, and transplantation data). By this means, a comprehensive database for KTR is created with benefits for routine clinical care and research. It enables both easy everyday clinical use and quick access for research questions with highest data quality. This is achieved by the concept of data validation in clinical routine in which clinical users and patients have to rely on correct data for treatment and medication plans and thereby validate and correct the clinical data in their daily practice. This EHR is tailored for the needs of transplant outpatient care and proved its clinical utility for more than 20 years at Charit&#233; - Universit&#228;tsmedizin Berlin. It facilitates efficient routine work with well-structured, comprehensive long-term data and allows their easy use for clinical research. To this point, its functionality covers automated transmission of routine data via standardized interfaces from different hospital information systems, availability of transplant-specific data, a medication list with an integrated check for drug-drug interactions, and semi-automated generation of medical reports among others. Key elements of the latest reengineering are a robust privacy-by-design concept, modularity, and hence portability into other clinical contexts as well as usability and platform independence enabled by HTML5 (Hypertext Markup Language) based responsive web design. This allows fast and easy scalability into other disease areas and other university hospitals. The comprehensive long-term datasets are the basis for the investigation of Machine Learning algorithms, and the modular structure allows to rapidly implement these into clinical care. Patient reported data and telemedicine services are integrated into TBase in order to meet future needs of the patients. These novel features aim to improve clinical care as well as to create new research options and therapeutic interventions.

TBase combines an electronic health record with an innovative research database for kidney transplant recipients. TBase is built upon an in-memory database platform, connected to different hospital systems, and used for regular outpatient care. It automatically integrates all relevant clinical data including transplantation-specific data creating a unique research database.

TBase - una cartella clinica elettronica integrata e un database di ricerca per i riceventi di trapianto di rene

TBase is an electronic health record (EHR) for kidney transplant recipients (KTR) combining automated data entry of key clinical data (e.g., laboratory ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

Saggio di transcitosi delle cellule endoteliali come modello in vitro per valutare la permeabilità della barriera emato-retinica interna

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...