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.

NOTA: Tutti gli animali utilizzati in questa procedura sono trattati in conformità con l&#39;Associazione per la Ricerca e la Visione e la dichiarazione di Oftalmologia per l&#39;uso di animali in Oftalmica e Vision Research, nonché le linee guida stabilite dalla commissione di sorveglianza degli animali a Saint Louis University. NOTA: Tutti gli strumenti chirurgici e le soluzioni sono sterilizzate prima di un intervento chirurgico per limitare l&#39;infezione microbica degli occhi. Va notato che, mentre gli animali fanno vivere il dolore da questa procedura, non impieghiamo analgesici. La ragione di questo è perché tutti gli analgesici sono anti-infiammatori e da risposte trapianto di cornea coinvolgono l&#39;infiammazione, l&#39;uso di farmaci anti-infiammatori potrebbero compromettere la nostra capacità di determinare quali fattori sono coinvolti nel fallimento del trapianto di cornea. 1. Anestesia <ol style="margin-left: 40px;"><li> Mettere i topi donatori e beneficiari in anestesia generale con iniezioni IP di ketamina (86.98 mg / kg) e xilazina (13,04 mg / kg). </li><li> Tenere il mouse recipiente sotto anestesia durante tutta la procedura che richiede in genere 30 minuti a un ora. Di conseguenza, monitorare costantemente il mouse per eventuali segni di riprendere conoscenza. </li><li> Applicare puralube pomata per l&#39;occhio che non sottoporsi ad intervento chirurgico dopo che l&#39;animale è anestetizzato per prevenire la secchezza. </li></ol> 2. innesto corneale <ol style="margin-left: 40px;"><li> Ottenere il bottone corneale del donatore. <ol><li> Una volta che l&#39;animale è completamente anestetizzato, conseguire un&#39;adeguata midriasi mediante somministrazione di un paio di gocce oculari di 1% e 2,5% tropicamide fenilefrina cloridrato. </li><li> Posizionare la testa dell&#39;animale donatore orizzontalmente su una tavola posta su un robusto supporto mobile. Fissare la testa con una striscia di nastro sul collo per assicurare che l&#39;occhio è in posizione orizzontale durante l&#39;intera operazione. </li><li> Utilizzando un trapano 2 mm di diametro, la cui punta è stato tinto con blu metile, fuorilinea il sito del trapianto della cornea centrale. </li><li> Con una lama affilata, penetrare nella cornea e Healon iniettare nella camera anteriore di approfondire per ridurre la possibilità di danni all&#39;endotelio donatore e lente sottostante. </li><li> Accise l&#39;innesto del donatore con Vännäs forbici e riporre in un piatto contenente soluzione salina bilanciata Hanks &#39;fino al momento dell&#39;uso. </li><li> Dopo l&#39;innesto del donatore è stato il rimosso, eutanasia il mouse donatore tramite CO 2 inalazione. </li></ol></li><li> Preparare il letto trapianto. <ol><li> Ripetere la stessa procedura descritta al punto 2.1.1 tramite 2.1.2 per il destinatario. </li><li> L&#39;utilizzo di un trapano di diametro 1,5 mm delineare il sito ricevente del trapianto. </li><li> Con una lama affilata, penetrare nella cornea e Healon iniettare nella camera anteriore di approfondire per ridurre la possibilità di danni alla lente sottostante. </li><li> Rimuovere il pulsante corneale centrale delineato dal destinatario utilizzando Vännäs forbici e scartare. </li></ol></li><li> Sutura deltrapianto <ol><li> Posizionare la cornea del donatore nel trapianto letto nella cornea del ricevente. Assicurarsi che un&#39;adeguata Healon è sotto la cornea del donatore per proteggere le cellule endoteliali del donatore venga danneggiato da contatto diretto con lente. </li><li> Utilizzando Super Fine microforceps a punta, posizionare il primo morso di 11-0 in nylon sutura nel lato dei donatori, attraverso il donatore con il 90% della profondità del tutto spessore a lato del destinatario, quindi legare. </li><li> Una volta che la cornea è ancorato al suo posto, eseguire punti staccati midcardinal tale che la cornea ha da 8 a 10 punti di sutura totale e la cornea del donatore è rivestito in modo sicuro con e allegato al destinatario letto trapianto di cornea. </li></ol></li><li> Approfondire la camera anteriore <ol><li> Approfondire la camera anteriore iniettando HBSS o bolla d&#39;aria nella camera anteriore e controllare delicatamente l&#39;integrità del trapianto di cornea per perdite con una spugna di cellulosa. NOTA: Se la camera anteriore non può essere riformato allora c&#39;è un&#39;alta probabilità di cataract che renderà le future valutazioni della cornea trapiantata molto difficile e anche potenzialmente portare ad una disfunzione dell&#39;endotelio corneale del donatore e quindi il fallimento del trapianto. </li></ol></li><li> Valutazione finale <ol><li> Osservare l&#39;occhio per determinare che la pupilla è rotonda e la profondità della camera anteriore è normale. NOTA: Se l&#39;alunno non è tutto ciò indica che durante la sutura l&#39;iride è stato danneggiato e, quindi, l&#39;innesto è considerato un guasto tecnico. </li><li> Applicare una pomata antibiotica per gli occhi. Opzionale: Chiudere il coperchio occhio con una sutura di seta 7-0. </li><li> Osservare i topi fino a quando sono completamente sveglio e poi individualmente li ospitano per un minimo di due giorni post-operatorie. </li></ol></li></ol> 3. Sutura di rimozione <ol style="margin-left: 40px;"><li> Nei casi in cui viene utilizzato coperchio sutura, anestetizzare topi come descritto sopra e rimuovere la sutura coperchio a 48 ore. </li><li> Anestetizzare topi il giorno 7 dopo l&#39;intervento. Rimuovere il sutures garantire il trapianto di cornea. Una volta che i punti di sutura vengono rimossi e l&#39;animale si è completamente risvegliato, restituirlo alla sua gabbia. </li></ol> 4. Valutazione clinica <ol style="margin-left: 40px;"><li> Controllare l&#39;occhio per indicazioni di complicazioni procedurali che comprendono, cataratta (opacità del cristallino), ifema (sangue nella camera anteriore), una camera anteriore che non è di profondità adeguata, o significativo l&#39;opacità della cornea. Prendere in considerazione quelli che dimostrano queste complicazioni come &quot;compromesso&quot; e l&#39;eutanasia da CO 2 inalazione. <ol><li> Eseguire tutti gli esami sui topi non anestetizzati. Tenere il mouse con una mano trattenendo così il mouse in modo che l&#39;altra parte può proptose l&#39;occhio per consentire una migliore vista dell&#39;occhio. Una volta che le osservazioni sono stati completati, rispedire l&#39;animale verso la sua gabbia. </li></ol></li><li> Avere un osservatore non hanno familiarità con i gruppi di trattamento valutare cornee trapiantate 2 o 3 volte a settimana per i segni di trapianto di corneaepisodi di rigetto o di fallimento del trapianto di cornea. Utilizzare il microscopio operatorio o un biomicroscopio con lampada a fessura orizzontale per queste osservazioni. <ol><li> Valutare ogni cornea opacità usando una scala da 0 a 5. La scala è definito come segue: <ol><li> Assegnare un punteggio da 0 a quelle cornee che non hanno segni di opacità. </li><li> Assegnare un punteggio di 1 a quelle cornee che mostrano il minimo l&#39;opacità superficiale. </li><li> Assegnare un punteggio di 2 a cornee che visualizzano l&#39;opacità mite e più in profondità, ma la pupilla e l&#39;iride sottostante sono ancora distinguibile. </li><li> Assegnare un punteggio di 3 cornea da visualizzare stromale opacità in cui il diaframma non possono essere viste in dettaglio con l&#39;eccezione dei margini pupilla. </li><li> Assegnare un punteggio di 4 a cornea da visualizzare densa opacità stromale e se non strutture sottostanti possono essere visualizzati. </li><li> Assegnare un punteggio di 5 a cornea da visualizzare per l&#39;opacità completa ed intensa edema stromale, con la pupilla e l&#39;iride completamente oscurata. </li></ol></li><li><html> Inoltre valuta ogni cornea per il grado di infiltrazione dei vasi sanguigni (neovascolarizzazione) utilizzando una scala di valutazione da 1 a 8. Per eseguire ciò, visualizzare la cornea come costituito da 4 quadranti uguali e determinare la quantità di vasi sanguigni in ciascuno di questi quadranti con punteggio che spaziano volontà da 0 (nessun vasi) a 2 per esteso vascolarizzazione di quel quadrante. Aggiungere i singoli punteggi di ogni quadrante per calcolare il punteggio finale neovascolarizzazione. </li><li> Classificare cornee come acutamente rifiutati se hanno un punteggio di 3 per due osservazioni consecutive per tempo i punti fino a 5 settimane. </li><li> Classificare i topi le cui cornee erano chiari a 5 settimane, ma sviluppare opacizzazione a volte&gt; 45 giorni dopo l&#39;attecchimento, con un punteggio di 3 per due momenti consecutivi, ad avere subito in ritardo termine rigetto corneale. Utilizzare le curve di Kaplan-Meier di sopravvivenza per analizzare la sopravvivenza del trapianto. </li></ol></li></ol> 5. Manipolazione del Modello in-left: 40px; &quot;&gt; <li> Preparazione delle singole cellule dalla milza. <ol><li> Per preparare singole cellule, prima eutanasia il mouse donatore. Quindi, rimuovere la milza. </li><li> Posizionare la milza in un colino cellulare e interrompere con un pistone della siringa da una siringa da 3 ml. </li><li> Lavare le cellule e risospendere in 10 ml di soluzione salina bilanciata Hanks. </li><li> Rimuovere 10 ml di sospensione cellulare e aggiungere 10 ml di 0,4% trypan blu e mescolare. Aggiungere che emocitometro e contare le cellule nella griglia centrale. Il numero di cellule nel tubo è il numero di celle 10 x 4 x 2 (fattore di diluizione in blu tripan) x 10 (volume nel tubo). </li></ol></li><li> Iniezione nella camera anteriore. <ol><li> Anestetizzare topi come precedentemente descritto. </li><li> Eseguire iniezioni usando un microscopio da dissezione. Per ogni iniezione intracameral, utilizzare 10 6 cellule della milza di un volume di 0,005 ml e una siringa microlitro 0,25 ml con ago 33 G. NOTA: altre manipolazionidel modello può essere effettuata trattando gli animali con reagenti che agiscono sia come antagonisti o agonisti per determinare il ruolo che un determinato fattore può giocare dopo l&#39;intervento chirurgico ortotopico allogenico corneale. </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.

Il modello murino di trapianto di cornea qui descritta consente al ricercatore di studiare rigetto corneale umano in un modello che è predittivo di quali fattori sono più associati sia con il rifiuto 15,17,18,20, 26-30 e 21-25 accettazione di cornea trapianti. A differenza di trapianto di cornea umana, in cui i pazienti sono indicati sia per il trattamento steroideo topico o sistemico sia per trattare o prevenire il rigetto 31, questo modello è in genere utilizzato per determinare que...

Il trapianto corneale è uno dei tipi più comuni di successo e trapianti eseguiti negli esseri umani. Le ragioni per cui si esegue questa chirurgia sono il risultato di lesioni, malattie infettive 1, o di altre forme di malattie della cornea non infettive 2. I dati della Eye Bank Association of America indicano che più di 46.000 sono stati eseguiti nel 2011 (si veda il sito Web all&#39;indirizzo: restoresight.org/eye_banks/eye_banks.html). L&#39;indicazione del suo successo è che i tassi di fallimento di un anno per gli innesti corneali allogenici vanno dal 10 al 15% a 5 anni e il successo è superiore al 70% 3-8. Come molti studi hanno dimostrato, il successo di alloinnesti corneali è direttamente legato al fatto che l&#39;occhio è un sito immunologicamente privilegiato. I fattori responsabili dello stato cornee come sito privilegio immunitario comprendono la mancanza di entrambi i vasi sanguigni e linfatici nella cornea, una relativa assenza di cellule presentanti l&#39;antigene, fattori prodotta dalla cornea che Abbateres funtions effettrici del sistema immunitario 9-15, bassa espressione di MHC antigeni 16, e l&#39;espressione di FasL 17-20. Tuttavia, nonostante questi fattori predisponenti questi innesti di successo, essi vanno incontro a rigetto 3-7. Di conseguenza, la comprensione quei meccanismi che mediano questo rifiuto e testare varie terapie per prevenire il rigetto è di importanza critica. A tal fine, si descrive qui un modello murino di trapianto di cornea, che è stato in uso per oltre 20 anni per studiare il trapianto di cornea in un ambiente controllato sperimentale. Poiché risposte trapianto coinvolgono diversi fattori che lavorano in concerto che sarà definitiva determinare se il tessuto trapiantato riesce o riesce, non è possibile comprendere l&#39;importanza di tali elementi in qualsiasi modello in vitro. Di conseguenza, gli studi su animali intatti sono necessari per determinare quali sono i fattori importanti per il successo o il failure del tessuto trapiantato. Mentre altre specie di animali sono stati utilizzati per studiare trapianto di cornea, il modello murino ha diversi vantaggi rispetto all&#39;utilizzo di altre specie. Il primo è l&#39;esistenza di molti ceppi di topi che esprimono alcuni transgeni o sono stati dei geni-bersaglio a non avere l&#39;espressione di specifici fattori immunologici la cui funzione nel trapianto può essere meglio studiata. In aggiunta, ci sono molti reagenti (entrambi i fattori ricombinanti e anticorpi che neutralizzano i fattori) che sono specifici per i topi e che non esistono per molte altre specie di animali. A causa dell&#39;esistenza di questi fattori, questo modello è stato ampiamente utilizzato per identificare i fattori rilevanti che partecipano acute risposte trapianto allogenico corneali 15, 17,18,20 -29. Inoltre, molti dei fattori coinvolti nel trapianto di cornea sono anche noti per essere funzionale nel trapianto di altri tessuti.

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			<td>305122</td>
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			<td height="41" style="height:41px;width:216px;">2.5% Phenylephrine HCl</td>
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			<td height="41" style="height:41px;width:216px;">1% Tropicamide</td>
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			<td style="width:119px;">NDC-24208-585-59</td>
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			<td style="width:71px;">Hamilton</td>
			<td style="width:119px;">7654-01</td>
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			<td height="21" style="height:21px;width:216px;">33 gauge needle</td>
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			<td height="41" style="height:41px;width:216px;">Cell Strainer (100 &#956;m nylon)</td>
			<td style="width:71px;">BD Falcon</td>
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			<td height="41" style="height:41px;width:216px;">Hemocytometer</td>
			<td style="width:71px;">Cardinal Health</td>
			<td style="width:119px;">B3175</td>
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			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T8154</td>
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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.

Il modello murino di trapianto di cornea è stato utilizzato da oltre 20 anni con successo per caratterizzare i meccanismi di entrambi corneale rigetto del trapianto corneale 19-23 e accettazione 13, 15,16,18, 24-27. Questo modello è stato utilizzato per stabilire l&#39;importanza di FasL nella cornea del trapianto di accettazione, in quanto gli animali che non hanno FasL non erano in grado di accettare trapianti corneali 15. E &#39;stato anche utilizzato per dimostrare che la crescita ...

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.

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

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

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9.6K Views.  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.

Video: 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.

Trapianto ortotopico aortica: un modello di ratto per studiare lo sviluppo di vasculopatia cronica

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

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Medicina

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.

Un metodo per isolamento Islet murini e trapianto renale subcapsulare

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.

Murino cervicale Cuore trapianto Modello utilizzando una tecnica Cuff Modificato

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.

Il trapianto di valvola polmonare Utilizzando un modello murino di trapianto cardiaco eterotopico

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.

Cateterizzazione cardiaca nei topi per misurare il rapporto pressione del volume: Indagare l&#39;effetto 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 modello di cultura dell'organo endoteliale porcina utilizzando i pulsanti corneali divisi

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).

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

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

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.

Complicazioni ridotte dopo la riconnessione arteriosa in un modello di ratto di trapianto di fegato ortotopico

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-trapianto di rene ortotopico in un modello suino utilizzando la conservazione degli organi 24 ore su 24 e la telemetria continua

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.

Trapianto intramiocardico di idrogel iniettabili a carico MSC dopo infarto del miocardio in un modello murino

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