Les auteurs tiennent à remercier projet FORTE, financé par la Région Toscane (POR Creo FESR 2007-2013, Bando Unico R &amp; S 2012), les EU FP7 ECHORD ++ Expérience LA-ROSES que partiellement pris en charge les activités de recherche, et le 7e PC BiophotonicPlus projet &quot;LITE&quot; accordé par la Région Toscane.

L&#39;étude a été réalisée avec l&#39;approbation du comité d&#39;éthique prospective de l&#39;hôpital; le consentement éclairé a été obtenu. L&#39;étude était dans le respect des principes de la Déclaration d&#39;Helsinki. 1. Préparation donateurs endothélium <ol><li> Utilisez une cornée du donneur tel que préparé par la banque des yeux locale à la température ambiante. </li><li> Dans la salle de chirurgie, tirez la cornée du donneur de son conteneur de livraison et mis à part la solution pour la préservation des tissus et de la nutrition qui est utilisé pour le transport de la cornée. </li><li> Placez le donneur cornéo-sclérale sur une jante chambre antérieure artificielle, dans lequel un liquide peut couler et créer une pression variable; couvrir la cornée avec la tête de retenue de tissu. </li><li> Branchez la chambre antérieure artificielle (AAC) par un connecteur 3-way à une seringue remplie de la préservation de la cornée et du liquide de la nutrition. </li><li> Utilisez l&#39;anneau de compression pour maintenir la retenue solidement en place. </li><li> Utilisez le syringe reliée à l&#39;AAC pour maintenir la pression intracamérulaire: remplir la chambre antérieure avec la solution de conservation de tissu jusqu&#39;à atteindre la pression optimale (voir 1.7). Fermez le connecteur. </li><li> Test de la pression à l&#39;intérieur de la chambre antérieure avec un doigt. Changez la pression à l&#39;intérieur jusqu&#39;à atteindre la pression interne droite (dans la gamme 60-70 mmHg). Fermez le connecteur 3-way. </li><li> Mesurer l&#39;épaisseur de la cornée du donneur avec cohérence optique Thomography (OCT). Maintenir la chambre antérieure avec cette cornée dans une position fixe à l&#39;avant de l&#39;optique de l&#39;instrument et puis prendre une acquisition épaisseur-OCT plein. </li></ol> 2. Préparation Laser femtoseconde de l&#39;endothélium des donateurs <ol><li> Effectuer aplanissement de la cornée du donneur dans le cadre du dispositif de laser femtoseconde. Utilisez le laser femtosec pour couper le tissu du donneur avec trois coupes ultérieures: coupe latérale postérieure, plein lamellaire et la coupe de côté antérieur. Définissez les paramètres suivants pour les trois coupes.<ol><li> Pour la coupe pleine lamellaire (de configuration de trame, commencer), régler la profondeur de coupe correspondant au point de la cornée du donneur (mesurée avec les PTOM comme décrit dans 1.8) plus mince, toujours soustrayant 95 um. Réglez l&#39;énergie d&#39;impulsion dans la gamme 0,8-0,9 pJ (en fonction de la profondeur de travail). Réglez le diamètre à 8,7 mm. Réglez la séparation spot tangentielle à 2 pm. Réglez la séparation spot radiale à 2 pm. </li><li> Pour la coupe de côté antérieur, régler la profondeur de 30 um plus profond que la coupe lamellaire complète précédente postérieure. Réglez l&#39;énergie d&#39;impulsion à 2.10 pJ; le diamètre de 8,6 mm; la séparation spot à 3 pm, tandis que la séparation de la couche à 3 pm. </li><li> Pour la coupe de côté postérieur, localiser la profondeur antérieure de la face postérieure coupé 30 um avant qu&#39;en coupe lamellaire complet. Réglez la profondeur postérieure à 900 um. Réglez l&#39;énergie d&#39;impulsion à 2.10 pJ; le diamètre de 8,3 mm; la séparation de place à 2 pm; la séparation de la couche à 2 pm. </li></ol></li&gt; </ol> 3. Préparation bénéficiaire Eye <ol><li> Préparer le patient pour la chirurgie. Faire une incision de la cornée 4,00 mm à 12 heures avec une lame de 4,0 mm précalibrés. Faire une paracentèse limbique avec une lame préétalonné 1,2 mm, placé à 02 heures. Faire un autre ponction limbique à 6 heures avec un coup de couteau 30 °. </li><li> Insérez une chambre mainteneur antérieure dans la chambre antérieure du patient, à travers les 2 paracentèse o&#39;clock. </li><li> Effectuer un diamètre de 8,2 mm descemetorexis circulaire avec le crochet dédié. </li><li> Dépouiller la membrane Descemet et de l&#39;endothélium du stroma postérieur et enlever les tissus avec un crochet de Descemet. </li></ol> 4. Préparation Chromophore <ol><li> Mettez 1 mg de poudre Indocyanine vert dans un tube de 1,5 ml. </li><li> Ajouter 9 mg de l&#39;eau stérile (la solution aqueuse est ICG 10% en poids / poids). </li><li> Manuellement mélanger la poudre ICG et l&#39;eau avec un agitateur métallique. </li&gt; </ol> 5. Coloration de l&#39;endothélium des donateurs <ol><li> Mettre l&#39;endothélium donneur sur la partie la plus large d&#39;un Busin-injecteur, avec la face intérieure en contact avec la surface de l&#39;injecteur. </li><li> Colorer le côté intérieur de l&#39;endothélium du donneur avec la solution de chromophore, dans sa partie périphérique, à l&#39;aide d&#39;une spatule. Le tissu correctement colorés a une couleur verdâtre homogène. Attendre 3 min avant l&#39;étape 5.3 de départ. </li><li> Tirez l&#39;endothélium des bailleurs de fonds sur la partie antérieure de la Busin-injecteur, laminés. </li></ol> 6. Mise en place de l&#39;endothélium des donateurs <ol><li> Saisir et insérez l&#39;endothélium des donateurs plié en utilisant une pince coaxiaux atraumatiques pour endothéliale lenticulaire. </li><li> Retirer la chambre antérieure mainteneur. </li><li> Suturer l&#39;incision de la cornée et de la paracentèse (effectuée à l&#39;étape 3.1) avec un Nylon 10,0 seule Stich. </li><li> Injecter une bulle d&#39;air de se dérouler le tissu du donneur et pour le presser contre la cornée bénéficiaire. La bulle d&#39;air doit completely remplir l&#39;espace de la chambre antérieure. </li></ol> 7. Soudage laser <ol><li> Placer la lenticule donneur dans le centre de la cornée de destinataire, à partir de l&#39;intérieur d&#39;un crochet ou le déplacement de la bulle d&#39;air de l&#39;extérieur de la cornée avec une spatule. </li><li> Utilisation d&#39;un laser à diode émettant à 810 nm, équipé d&#39;un diamètre de 300 um de noyau de fibre optique stérile, à 0,22 comme une ouverture numérique (NA). </li><li> Gardez la pointe de la fibre à l&#39;extérieur du globe oculaire, et de fournir la lumière laser vers l&#39;endothélium taché, à travers le tissu de la cornée transparente. La pointe de la fibre est dans la configuration non-contact. </li><li> Utilisez les paramètres suivants pour le laser: mode Spot d&#39;émission unique, 70-80 durée d&#39;impulsion ms, 35-40 mJ par impulsion. He-Ne visant faisceau sur. </li><li> Livrer spots laser unique à la périphérie teinté de l&#39;endothélium des bailleurs de fonds. Livrer les taches séquentiellement: l&#39;aspect final est un anneau de taches dans la périphérie de la lenticule des bailleurs de fonds. La distance entre deux adjacentre de taches de cent est le double d&#39;un diamètre de spot. </li><li> Appliquer une lentille de contact sur l&#39;oeil du patient, ainsi que 0,3% de tobramycine et 0,1% de dexaméthasone suspension ophtalmique. </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.

Le «tout laser» endothéliale greffe est une approche originale de la greffe de cornée minimalement invasive. Toutes les procédures décrites dans le protocole ont été effectués dans la salle de chirurgie, en observant la procédure d&#39;hygiène et de stérilisation qui sont des pratiques courantes lors de chirurgies, telles que l&#39;utilisation de stérilisée gants, blouse, masque et chapeau. On a préparé la solution ICG dans la salle d&#39;opération, peu de temps avant son ap...

Dans ce travail, nous présentons une approche originale de la kératoplastie endothéliale, basé sur l&#39;utilisation d&#39;un laser femtoseconde pour préparer tissu du donneur et une diode laser proche infrarouge à souder sur le lit receveur. Mesure peropératoire de la cornée du donneur est nécessaire de concevoir correctement les dimensions de tissu du donneur. Kératoplastie endothéliale a été proposée dans les dernières années pour remplacer kératoplastie dans le traitement de la maladie 1,2 endothéliale. Le principal avantage de cette technique est une récupération visuelle plus rapide, par rapport à la kératoplastie, l&#39;anesthésie réduite pendant la chirurgie, une diminution du risque de rejet du greffon et de la préservation de l&#39;intégrité de l&#39;oeil. Le principal facteur de risque est postopératoire donateurs lenticulaire dislocation. La technique standard est effectuée en insérant l&#39;endothélium des bailleurs de fonds dans sa position finale où il est maintenu par l&#39;injection d&#39;une bulle d&#39;air: pas de sutures sont utilisés en raison de la chara mécanique, biophysique et dimensionscteristics de l&#39;endothélium. En outre, la récupération de l&#39;acuité visuelle peut être limitée en raison principalement d&#39;une inadéquation entre les tissus donateurs et bénéficiaires en raison d&#39;un épais tissu transplanté. Ici, nous présentons une procédure dans l&#39;exécution de kératoplastie endothéliale qui peut surmonter ces problèmes principaux. L&#39;endothélium du donneur peut être assuré dans sa position finale par l&#39;utilisation de la technique de soudage au laser. Ceci est un processus photothermique contrôlée et localisée: elle peut être induite à l&#39;interface donneur / receveur. Il a été étudié au cours des dix dernières années et proposé dans kératoplastie et la greffe de l&#39;endothélium 3-5. La lumière du proche infrarouge (longueur d&#39;onde: 810 nm) émis par une diode laser de faible puissance est délivré vers le tissu biologique au niveau du site de la plaie. La cornée est naturellement transparent à cette longueur d&#39;onde: afin de rendre ce tissu à absorber la lumière laser, il est nécessaire de teindre avec un chromophore. Le colorant est proposé un saturer stérilesolution d&#39;eau d de vert d&#39;indocyanine (ICG). Nous avons démontré que lorsque le tissu cornéen est correctement colorée avec cette préparation d&#39;ICG, elle montre un pic d&#39;absorption à 810 nm 6. En outre, l&#39;ICG est largement utilisé dans le diagnostic clinique et sa sécurité a déjà été démontrée chez des sujets humains. La cornée teinté absorbe l&#39;énergie de lumière laser à diode et le principal effet résultant est une élévation de la température contrôlée au niveau du site de soudage. Pas d&#39;effets thermiques sont induites dans les tissus non colorées. La mise en valeur de la température provoque une dénaturation thermique réversible dans le collagène du stroma, avec un closuring immédiate des parois de la plaie lors du refroidissement. Cet effet de soudage au laser a d&#39;abord été mise en évidence dans la chirurgie de la cataracte et 7,8 kératoplastie 9,10. Une approche optimisée que nous présentons dans le présent document a été étudié pour l&#39;application dans la kératoplastie endothéliale. Dans l&#39;opération proposée, spots laser simples (des dizaines durables de msec) are délivrée au tissu, résultant en un effet photothermique localisée dans la dimension du spot (quelques centaines de um de diamètre): l&#39;effet induit est un soudage laser dur, constitué d&#39;une photocoagulation du collagène confinée à l&#39;interface donneur / hôte. Le résultat de la dénaturation du collagène au niveau du site est soudée une forte adhérence entre les tissus de donneur et l&#39;hôte, fournissant ainsi un effet de suture qui est impossible à obtenir avec la technique standard (points). Le tissu retrouve son apparence naturelle dans un court suivi (1 mois) et l&#39;adhérence entre les tissus donneur / accueil est améliorée par le soudage prévu dans le stade très précoce de la phase de guérison. Pour éviter l&#39;autre risque principal de la kératoplastie endothéliale, à savoir la transplantation d&#39;un tissu de donneur d&#39;épaisseur, intraoperatorie tomographie par cohérence optique (OCT) est utilisé: un dispositif commercial de mesurer l&#39;épaisseur du donneur cornée, de telle sorte qu&#39;un bon profil de coupe peut être conçu avec lafemtosec laser. Le projet de «tout laser» endothéliale greffe semble donc d&#39;améliorer les résultats cliniques de cette chirurgie mini-invasive.

<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.

Le «tout laser&quot; procédure chirurgicale est proposé d&#39;effectuer la greffe de cornée minimalement invasive. La procédure est facile à réaliser (voir la figure 1): par rapport à une greffe endothéliale norme uniquement les étapes de mesure de l&#39;épaisseur de la cornée, la coloration du tissu du donneur et la prestation de la lumière laser sont ajoutés. Les avantages obtenus compensent largement un temps opératoire augmenté de quelques minutes. L&#39;utilisation de peropératoire...

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 «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 vues.  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.

Vidéo: 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.

Utilisation quantitative PCR en temps réel pour déterminer prise de greffe de donneurs de cellules dans un modèle murin concurrentiel greffe de moelle osseuse

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

Recherche

Médecine

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.

Murin de transplantation cardiaque hétérotopique 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 ...

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.

Technique de porc approvisionnement du foie et la transplantation orthotopique utilisant un 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 ...

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.

Modèle murin de rejet vasculaire allo-induite et Transplant artériosclérose

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.

Rein murin 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 ...

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.

Procédé amélioré pour la création d’un In Vitro barrière hémato - encéphalique modèle basé sur les cellules endothéliales du cerveau porcin

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.

Réaction optimisée de Griess pour UV-Vis et détermination à l'œil nu de la primaquine antipaludique

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.

Lésion d’ischémie-reperfusion d’organe en simulant des changements hémodynamiques dans le modèle de greffe de foie de rat

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 - une base de données électronique intégrée sur les dossiers de santé et la recherche pour les receveurs de greffe de rein

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.

Essai de transcytose cellulaire endothéliale comme modèle in vitro pour évaluer la perméabilité interne de la barrière hémato-rétinienne

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