The authors would like to thank the many individuals who have worked on and perfected this technique and have been responsible for the generation of many manuscripts both in this lab and others. This work was supported by National Institutes of Health Grant EY12707 (PMS) and an unrestricted grant from Research to Prevent Blindness to Department of Ophthalmology.

REMARQUE: Tous les animaux utilisés dans cette procédure sont traités conformément à l&#39;Association pour la recherche en vision et déclaration d&#39;ophtalmologie pour l&#39;utilisation d&#39;animaux dans ophtalmique et Vision Research ainsi que les lignes directrices établies par le comité de surveillance de l&#39;animal à l&#39;Université de Saint Louis. NOTE: Tous les instruments et solutions chirurgicales sont stérilisés avant la chirurgie pour limiter l&#39;infection microbienne de l&#39;œil. Il convient de noter que, bien que les animaux font l&#39;expérience de la douleur de cette procédure, nous ne comptons pas les analgésiques. La raison en est que tous les analgésiques sont anti-inflammatoires et puisque les réponses de transplantation de la cornée d&#39;une inflammation, l&#39;utilisation de médicaments anti-inflammatoires pourrait compromettre notre capacité à déterminer les facteurs qui sont impliqués dans l&#39;échec de la greffe de cornée. 1. Anesthésie <ol style="margin-left: 40px;"><li> Placez la souris donateurs et bénéficiaires sous anesthésie générale par injections IP de kétamine (86.98 mg / kg) et de xylazine (13,04 mg / kg). </li><li> Gardez la souris receveuse sous anesthésie tout au long de la procédure qui prend généralement 30 minutes à une heure. Par conséquent, surveiller en permanence la souris pour des signes de reprise de conscience. </li><li> Appliquer puralube pommade à l&#39;œil qui ne sera pas subir une intervention chirurgicale après que l&#39;animal est anesthésié pour prévenir la sécheresse. </li></ol> 2. La greffe de cornée <ol style="margin-left: 40px;"><li> Obtention du bouton cornéen du donneur. <ol><li> Une fois que l&#39;animal est anesthésié complètement, réaliser une mydriase adéquate par administration d&#39;un couple de gouttes pour les yeux de 1% de tropicamide et 2,5% de chlorhydrate de phényléphrine. </li><li> Placez la tête de l&#39;animal donneur horizontalement sur une planche placée sur un support mobile robuste. Fixer la tête avec une bande de ruban à travers le goulot de sorte que l&#39;oeil est dans une position horizontale tout au long de la totalité de l&#39;opération. </li><li> L&#39;utilisation d&#39;un trépan de 2 mm de diamètre, dont la pointe a été teint avec du bleu de méthyle, surtapisser le site de la greffe de cornée centrale. </li><li> Avec une lame tranchante, pénétrer la cornée et injecter Healon dans la chambre antérieure de l&#39;approfondir pour réduire le risque de dommages à l&#39;endothélium des bailleurs de fonds et l&#39;objectif sous-jacent. </li><li> Exciser le greffon du donneur avec Vannas ciseaux et placer dans un plat contenant une solution saline équilibrée de Hanks jusqu&#39;à utilisation. </li><li> Après la greffe du donneur a été enlevé, euthanasier la souris donneuse par inhalation de CO 2. </li></ol></li><li> Préparation du lit de greffe. <ol><li> Répétez les mêmes étapes comme décrit dans 2.1.1 à 2.1.2 pour le destinataire. </li><li> L&#39;utilisation d&#39;un diamètre de 1,5 mm trépan, décrire le site de la greffe de destinataire. </li><li> Avec une lame tranchante, pénétrer la cornée et injecter Healon dans la chambre antérieure de l&#39;approfondir pour réduire le risque de dommages à l&#39;objectif sous-jacent. </li><li> Retirez le bouton central de la cornée exposée du destinataire à l&#39;aide Vannas ciseaux et jetez-le. </li></ol></li><li> Suturer lagreffe <ol><li> Placer la cornée du donneur au cours de la greffe lit dans la cornée du receveur. Assurez-vous que Healon adéquate est sous la cornée du donneur à protéger les cellules du donneur endothéliales des dommages causés par un contact direct avec l&#39;objectif. </li><li> Utilisation super fine microforceps à pointe, placer la première bouchée de 11-0 suture en nylon sur le côté des bailleurs de fonds, à travers le donateur avec 90% de profondeur de pleine épaisseur au côté du destinataire, puis attacher. </li><li> Une fois que la cornée est ancré en place, effectuer des sutures interrompues midcardinal tels que la cornée a 8 à 10 points de suture au total et la cornée du donneur est bien aligné avec et fixé au destinataire lit de greffe de cornée. </li></ol></li><li> L&#39;approfondissement de la chambre antérieure <ol><li> Approfondir la chambre antérieure par injection HBSS ou bulle d&#39;air dans la chambre antérieure et vérifier doucement l&#39;intégrité de la greffe de cornée pour une fuite avec une éponge de cellulose. NOTE: Si la chambre antérieure ne peut être réformé alors il ya une forte probabilité de cataract qui fera les évaluations futures de la cornée transplantée très difficile et aussi potentiellement conduire à des bailleurs de fonds de la cornée dysfonctionnement endothélial et l&#39;échec ainsi greffe. </li></ol></li><li> L&#39;évaluation finale <ol><li> Observer l&#39;oeil pour déterminer que la pupille est ronde et la profondeur de la chambre antérieure est normal. NOTE: Si l&#39;élève est pas rond, cela indique que pendant la suture de l&#39;iris a été endommagé et donc la greffe est considéré comme un échec technique. </li><li> Appliquer une pommade antibiotique pour les yeux. En option: Fermez la paupière avec une suture de soie 7-0. </li><li> Observez souris jusqu&#39;à ce qu&#39;ils soient complètement éveillé et ensuite individuellement les loger pour un minimum de deux jours après la chirurgie. </li></ol></li></ol> 3. Suture Removal <ol style="margin-left: 40px;"><li> Dans les cas où le couvercle suture est utilisé, anesthésier les souris comme décrit ci-dessus et retirer le couvercle de la suture à 48 h. </li><li> Anesthésier les souris au jour 7 post-opératoire. Retirez le Sutures fixation de la greffe de cornée. Une fois les sutures sont enlevées et l&#39;animal a pleinement éveillé, retourner dans sa cage. </li></ol> 4. l&#39;évaluation clinique <ol style="margin-left: 40px;"><li> Examiner l&#39;œil pour les indications de complications liées aux procédures qui comprennent, la cataracte (opacification du cristallin), hyphéma (sang dans la chambre antérieure), une chambre antérieure qui ne sont pas de la bonne profondeur, ou l&#39;opacité importante de la cornée. Pensez à ceux qui démontrent ces complications comme «compromis» et les euthanasier par inhalation de CO 2. <ol><li> Effectuer tous les examens sur des souris non anesthésiées. Maintenez la souris avec retenue d&#39;une part ainsi de la souris pour que l&#39;autre main peut proptose l&#39;œil afin de permettre une meilleure vue de l&#39;œil. Une fois que les observations sont terminées, revenir à l&#39;animal de sa cage. </li></ol></li><li> Vous avez déjà un observateur familier avec les groupes de traitement évaluer cornées transplantées des signes de greffe de cornée 2 à 3 fois par semaineles épisodes de rejet ou échec de la greffe de la cornée. Utilisez soit le microscope chirurgical ou un biomicroscope lampe à fente horizontale pour ces observations. <ol><li> Évaluer chaque cornée pour l&#39;opacité en utilisant une échelle de 0 à 5. L&#39;échelle est définie comme suit: <ol><li> Attribuez une note de 0 à ces cornées qui ne présentent aucun signe d&#39;opacité. </li><li> Attribuer une note de 1 à ces cornées présentant une opacité superficielle minime. </li><li> Attribuer un score de 2 à cornées qui affichent opacité légère et profonde, mais l&#39;élève sous-jacente et de l&#39;iris sont encore discernable. </li><li> Attribuer un score de 3 à cornée afficher stromale opacité dans laquelle les iris ne peuvent être vus en détail à l&#39;exception des marges d&#39;élèves. </li><li> Attribuer un score de 4 à cornée afficher dense opacité stromale et si aucune des structures sous-jacentes peuvent être consultés. </li><li> Attribuer un score de 5 à cornée afficher opacité complète et intensive œdème du stroma, avec pupille et l&#39;iris totalement obscurci. </li></ol></li><li><html> Évaluer également tous cornée pour le degré d&#39;infiltration des vaisseaux sanguins (néovascularisation) en utilisant une échelle de notation de 1 à 8. Pour ce faire, voir la cornée en tant que 4 quadrants égaux et déterminer la quantité de vaisseaux sanguins dans chacun de ces quadrants avec une marquer cette gamme de volonté de 0 (pas de navires) à 2 pour vascularisation extensive de ce quadrant. Ajouter les scores individuels de chaque quadrant de calculer le score final de la néovascularisation. </li><li> Classer cornées comme aiguë rejetées si elles ont un score de 3 pour deux observations consécutives de temps les points jusqu&#39;à 5 semaines. </li><li> Classer les souris dont les cornées étaient claires à 5 semaines, mais développer une opacification parfois&gt; 45 jours après la greffe, avec un score de 3 à deux points de temps consécutifs, comme ayant le rejet d&#39;allogreffe de cornée tard terme subi. Utiliser les courbes de survie de Kaplan-Meier pour analyser la survie du greffon. </li></ol></li></ol> 5. La manipulation du modèle en-left: 40px; &quot;&gt; <li> Préparation des cellules individuelles de la rate. <ol><li> Pour préparer des cellules individuelles, premier euthanasier la souris donneuse. Ensuite, retirez la rate. </li><li> Placer la rate dans un tamis cellulaire et perturber avec un piston de seringue d&#39;une seringue de 3 ml. </li><li> Laver les cellules et remettre en suspension dans 10 ml de solution saline équilibrée de Hanks. </li><li> Retirer 10 pl de suspension cellulaire et ajouter 10 ul de 0,4% de bleu trypan et mélanger. Ajoutez à cela hémocytomètre et compter les cellules dans la grille centrale. Le nombre de cellules dans le tube est le nombre de cellules x 10 4 x 2 (facteur de dilution dans le bleu trypan) x 10 (volume dans le tube). </li></ol></li><li> L&#39;injection dans la chambre antérieure. <ol><li> Anesthésier les souris comme décrit précédemment. </li><li> Pratiquer des injections à l&#39;aide d&#39;un microscope à dissection. Pour chaque injection intracamérulaire, utiliser 10 6 cellules de la rate dans un volume de 0,005 ml et 0,25 ml d&#39;un microlitre seringue munie d&#39;une aiguille G 33. NOTE: D&#39;autres manipulationsdu modèle peut être réalisé par le traitement des animaux avec des réactifs qui agissent comme des antagonistes ou agonistes de déterminer le rôle qu&#39;un facteur particulier peut jouer après une chirurgie de la cornée allogreffe orthotopique. </li></ol></li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presurgical+corticosteroid+treatment+improves+corneal+transplant+survival+in+mice.">Presurgical corticosteroid treatment improves corneal transplant survival in mice.</a> Cornea. 32, 1591-1598 (2013).</li><li>Yamazoe, K., Yamazoe, K., Shimazaki-Den, S., Shimazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+factors+for+corneal+graft+recovery+after+severe+corneal+graft+rejection+following+penetrating+keratoplasty.">Prognostic factors for corneal graft recovery after severe corneal graft rejection following penetrating keratoplasty.</a> BMC Ophthalmol. 13, 5 (2013).</li><li>Panda, A., Vanathi, M., Kumar, A., Dash, Y., Priya, 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+graft+rejection.">Corneal graft rejection.</a> Surv. Ophthalmol. 52, 375-396 (2007).</li><li>Patel, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+survival+and+endothelial+outcomes+in+the+new+era+of+endothelial+keratoplasty.">Graft survival and endothelial outcomes in the new era of endothelial keratoplasty.</a> Exp. Eye Res. 95, 40-47 (2012).</li><li>Anshu, A., Price, M. O., Tan, D. T., Price, F. W. <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:+a+revolution+in+evolution.">Endothelial keratoplasty: a revolution in evolution.</a> Surv. Ophthalmol. 57, 236-252 (2013).</li></ol>

The authors have no competing financial interests.

Le modèle murin de greffe de cornée décrite ici permet à l&#39;enquêteur d&#39;étudier le rejet d&#39;allogreffe de cornée humaine dans un modèle qui permet de prédire quels sont les facteurs associés à la fois meilleur rejet 15,17,18,20, 26-30 et 21-25 acceptation de la cornée allogreffes. Contrairement à la transplantation de la cornée humaine, dans laquelle les patients reçoivent soit un traitement de stéroïde topique ou systémique pour traiter ou prévenir une ou l&#39;autre d...

La transplantation de la cornée est un des types les plus communs et succès de la transplantation réalisée chez l&#39;homme. Les raisons pour lesquelles cette intervention chirurgicale est pratiquée sont le résultat de blessures, de maladies infectieuses 1, ou d&#39;autres formes de la maladie de la cornée non-infectieuse 2. Les chiffres de l&#39;Association of America Banque d&#39;yeux indiquent que plus de 46 000 ont été réalisées en 2011 (voir le site Web à l&#39;adresse: restoresight.org/eye_banks/eye_banks.html). Une indication de son succès est que les taux de défaillance d&#39;un an pour greffes de cornée allogéniques vont de 10 à 15% et à 5 ans, le succès est au-delà de 70% 3-8. Comme de nombreuses études ont démontré le succès des allogreffes de la cornée est directement lié au fait que l&#39;oeil est un site immunologiquement privilégié. Les facteurs responsables de l&#39;état des cornées comme un site de privilège immunitaire comprennent l&#39;absence de ces deux vaisseaux sanguins et lymphatiques dans la cornée, une absence relative de cellules présentatrices d&#39;antigènes, facteurs produite par la cornée qui Suppress funtions immunitaires effectrices 9-15, faible expression de antigènes du CMH 16, et l&#39;expression de FasL 17-20. Cependant, en dépit de ces facteurs prédisposants ces greffes de succès, ils ne subissent rejet 3-7. Par conséquent, la compréhension de ces mécanismes qui interviennent dans ce rejet et de tester différentes thérapies pour prévenir le rejet est d&#39;une importance critique. À cette fin, nous décrivons ici un modèle murin de greffe de la cornée qui a été en usage depuis plus de 20 ans pour étudier la greffe de cornée dans un environnement expérimental contrôlé. Comme les réponses de transplantation impliquent de nombreux facteurs différents qui travaillent de concert, qui sera ultime de déterminer si le tissu transplanté échoue ou réussisse, il est impossible de comprendre l&#39;importance de ces facteurs dans un modèle in vitro. Par conséquent, les études sur des animaux intacts sont nécessaires pour déterminer quels facteurs sont importants à succès ou failure du tissu transplanté. Alors que d&#39;autres espèces d&#39;animaux ont été utilisés pour étudier la greffe de cornée, le modèle murin a plusieurs avantages par rapport à l&#39;utilisation d&#39;autres espèces. Le premier est l&#39;existence de nombreuses souches de souris qui expriment certains transgènes ou ont été gène ciblées à l&#39;absence d&#39;expression de facteurs immunologiques spécifiques dont la fonction dans la transplantation peut être mieux étudié. En outre, il ya beaucoup de réactifs (deux facteurs recombinantes et d&#39;anticorps qui neutralisent facteurs) qui sont spécifiques pour les souris et qui ne existent pour de nombreuses autres espèces d&#39;animaux. En raison de l&#39;existence de ces facteurs, ce modèle a été largement utilisée pour identifier les facteurs impliqués dans la cornée allogreffe réponses aiguës 15, 17,18,20 -29. En outre, un grand nombre de facteurs impliqués dans la transplantation de la cornée sont également connus pour être fonctionnels dans la transplantation d&#39;autres tissus.

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			<td height="21" style="height:21px;width:216px;">Zeiss Surgical Microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Rebuilt</td>
			<td></td>
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			<td height="21" style="height:21px;">1 mL Syringe</td>
			<td>BD</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 mL Syringe</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">10 mL Syringe</td>
			<td>BD</td>
			<td>309602</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vannus Scissors</td>
			<td style="width:71px;">Stortz</td>
			<td style="width:119px;">E-3387</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">11-0 Sutures</td>
			<td style="width:71px;">Alcon</td>
			<td style="width:119px;">717939M</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trephine 2.0mm</td>
			<td style="width:71px;">Katena</td>
			<td style="width:119px;">K 2-7520</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Trephine 1.5 mm</td>
			<td style="width:71px;">Katena</td>
			<td style="width:119px;">K 2-7510</td>
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			<td height="41" style="height:41px;width:216px;">Tricaine Hydrochloride 0.5%</td>
			<td style="width:71px;">Alcon</td>
			<td style="width:119px;">NDC 0065-0741-12</td>
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			<td height="21" style="height:21px;width:216px;">Healon</td>
			<td style="width:71px;">Abbott</td>
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			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">11251-20</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">7-0 Sutures</td>
			<td style="width:71px;">Alcon</td>
			<td align="right" style="width:119px;">8065</td>
			<td></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:216px;">2.5% Phenylephrine HCl</td>
			<td style="width:71px;">Alcon</td>
			<td style="width:119px;">NDC 61314-342-02</td>
			<td></td>
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			<td height="41" style="height:41px;width:216px;">1% Tropicamide</td>
			<td style="width:71px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">NDC-24208-585-59</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hamilton Syringe</td>
			<td style="width:71px;">Hamilton</td>
			<td style="width:119px;">7654-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">33 gauge needle</td>
			<td style="width:71px;">Hamilton</td>
			<td align="right" style="width:119px;">90033</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Cell Strainer (100 &#956;m nylon)</td>
			<td style="width:71px;">BD Falcon</td>
			<td align="right" style="width:119px;">352360</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Hemocytometer</td>
			<td style="width:71px;">Cardinal Health</td>
			<td style="width:119px;">B3175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T8154</td>
			<td></td>
		</tr>
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</table>

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.

Le modèle murin de greffe de la cornée a été utilisé pour plus de 20 ans à caractériser avec succès des mécanismes à la fois de l&#39;allogreffe de cornée rejet 19-23 et la cornée allogreffe acceptation 13, 15,16,18, 24-27. Ce modèle a été utilisé pour établir l&#39;importance de l&#39;expression de FasL dans la cornée allogreffe acceptation, par le fait que les animaux qui manquent de FasL sont pas en mesure d&#39;accepter les allogreffes de cornée 15. Il a également...

Watch this Scientific Journal Video about Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation at JoVE.com

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

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.

Murin de transplantation de la cornée: Un modèle pour étudier la forme la plus courante de la transplantation d&#39;organes solides

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

murine-corneal-transplantation-model-to-study-most-common-form-solid

Research

JoVE Journal

Medicine

9.6K vues.  Saint Louis University. 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.

Vidéo: Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Orthotopique aortique transplantation: un modèle de rat pour étudier le développement de la vasculopathie chronique

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Recherche

Médecine

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Une méthode pour isoler l&#39;îlot murin et transplantation rénale sous-capsulaires

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Murin col de l&#39;utérus Cœur, la transplantation en utilisant une technique de modification de manchette

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Transplantation de valve pulmonaire en utilisant un modèle de souris de transplantation cardiaque hétérotopique

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Cathétérisme cardiaque chez la souris pour mesurer le volume de pression Relation: Étudier les effets Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Un modèle de culture d'orgue endothéliale de porc cornée utilisant des boutons cornéens fendus

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

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).

Développement d’un modèle expérimental non invasif assisté au laser de la perte de cellules endothéliales cornéennes

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

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

Complications réduites après reconnexion artérielle dans un modèle de rat de transplantation orthotopique de foie

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

Auto-transplantation orthotopique de rein dans un modèle porcin utilisant la conservation d’organe de 24 heures et la télémétrie continue

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Transplantation intramyocardique d’hydrogels injectables à chargement MSC après l’infarctus du myocarde dans un modèle murin

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...