Os autores agradecem a FORTE projeto, financiado pela Região Toscana (POR Creo FESR 2007-2013, Bando Unico R &amp; S 2012), os FP7 ECHORD UE ++ Experiência LA-ROSES que apoiaram parcialmente as actividades de investigação, eo 7. ° PQ BiophotonicPlus Project &quot;LITE&quot; concedido pela Região Toscana.

O estudo foi realizado com a aprovação do Comitê de Ética prospectivamente do hospital; foi obtido consentimento informado. O estudo foi em adesão aos princípios da Declaração de Helsinki. 1. Preparação de Doadores endotélio <ol><li> Use uma córnea doadora elaborado pelo banco de olhos local à temperatura ambiente. </li><li> Na sala de cirurgia, puxe a córnea do doador de seu contêiner de entrega e separou a solução para preservação de tecido e nutrição que é usado para o transporte de córnea. </li><li> Coloque o doador córneo-escleral aro mais de uma câmara anterior artificial, na qual um líquido possa fluir e criar uma pressão variável; cobrir a córnea com a cabeça de retenção de tecido. </li><li> Ligue a câmara anterior artificial (AAC) através de um conector de 3 vias para uma seringa cheia com a preservação da córnea e nutrição líquida. </li><li> Use o anel de compressão para manter o retentor firmemente no lugar. </li><li> Use a syrInge ligado à AAC para manter a pressão intracameral: encher a câmara anterior com a solução de preservação do tecido até atingir a pressão óptima (ver 1.7). Feche o conector. </li><li> Testar a pressão dentro da câmara anterior com um dedo. Alterar a pressão no interior, até atingir a pressão interna direita (na gama de 60-70 mmHg). Feche o conector de 3 vias. </li><li> Meça a espessura da córnea do doador com Coerência Óptica Thomography (OCT). Manter a câmara anterior com que córnea em uma posição fixa na frente das óptica do aparelho e, em seguida, tomar uma aquisição espessura-OCT completo. </li></ol> 2. Preparação de laser femtosegundo endotélio Donor <ol><li> Execute aplanação da córnea do doador sob o dispositivo a laser de femtossegundos. Use o laser femtosec para cortar o tecido do doador com três cortes subsequentes: corte lateral posterior, lamelar completo e corte lateral anterior. Defina os seguintes parâmetros para os três cortes.<ol><li> Para o corte total lamelar (padrão de quadro, a começar), definida a profundidade de corte que corresponde ao ponto mais fino da córnea do doador (medido com a OCT como descrito em 1.8), sempre subtraindo 95 uM. Definir a energia de impulsos na gama de 0,8-0,9 μJ (dependendo da profundidade de trabalho). Definir o diâmetro de 8,7 milímetros. Defina a separação ponto tangencial a 2 m. Defina a separação ponto radial até 2 m. </li><li> Para o corte lateral anterior, defina a profundidade posterior 30 mm mais profundo do que o corte lamelar anterior completo. Defina a energia de pulso para 2,10 μJ; o diâmetro de 8,6 mm; a separação local a 3 um, enquanto a camada de separação a 3 um. </li><li> Para o corte do lado posterior, localize a profundidade anterior da face posterior 30 um corte anterior de corte lamelar completo. Defina a profundidade posterior a 900 mm. Defina a energia de pulso para 2,10 μJ; o diâmetro e 8,3 mm; a separação local a 2 uM; a separação da camada de 2 um. </li></ol></li&gt; </ol> 3. Destinatário Preparação Eye <ol><li> Preparar o paciente para a cirurgia. Faça uma incisão na córnea 4,00 milímetros em 12 horas com 4,0 milímetros de lâmina pré-calibrado. Faça uma paracentese de limbo com um 1,2 milímetros de lâmina pré-calibrado, colocado a duas horas. Faça outro paracentesis limbo às 6 horas com um 30 ° faca facada. </li><li> Insira um mantenedor da câmara anterior na câmara anterior do paciente, através das 2 paracentesis o&#39;clock. </li><li> Execute uma diâmetro 8,2 milímetros descemetorexis circular com o gancho dedicado. </li><li> Tira a membrana Descemet e endotélio do estroma posterior e remover os tecidos com um gancho de Descemet. </li></ol> 4. Cromóforo Preparação <ol><li> Coloque 1 mg de Indocianina pó verde em um tubo de microcentrífuga de 1,5 ml. </li><li> Adicionar 9 mg de água estéril (a solução ICG água é de 10% w / w). </li><li> Misturar o pó manualmente ICG e a água com um agitador de metal. </li&gt; </ol> 5. Coloração de endotélio Donor <ol><li> Coloque o endotélio dador para a parte mais larga de um neg-injector, com o lado de dentro em contacto com a superfície do injector. </li><li> Manchar o lado interior do endotélio do dador com a solução cromóforo, na sua parte periférica, usando uma espátula. O tecido corretamente manchado tem uma cor esverdeada homogénea. Esperar 3 min antes de iniciar o passo 5.3. </li><li> Puxe o endotélio doador na parte anterior da Busin-injector, rolou. </li></ol> 6. Inserir o endotélio Donor <ol><li> Segure e insira o endotélio doador dobrado usando uma pinça atraumática coaxiais para lenticule endotelial. </li><li> Remover o mantenedor da câmara anterior. </li><li> Suturar a incisão na córnea ea paracentese (realizada no passo 3.1) com um único stich Nylon 10.0. </li><li> Injectar uma bolha de ar para desdobrar o tecido do doador e para pressioná-lo contra a córnea destinatário. A bolha de ar deve completely preencher o espaço na câmara anterior. </li></ol> 7. solda a laser <ol><li> Coloque a lentícula doador no centro da córnea do receptor, a partir do interior de um gancho ou de mover o ar do lado de fora da bolha da córnea com uma espátula. </li><li> Usar um laser de diodo emitindo a 810 nm, equipado com um diâmetro de 300 ^ M de núcleo de fibra óptica estéril, com 0,22 como uma abertura numérica (NA). </li><li> Mantenha a ponta da fibra de fora do globo ocular, e entregar a luz laser para o endotélio coradas, através do tecido da córnea transparente. A ponta da fibra está em configuração sem contato. </li><li> Use as seguintes configurações para o laser: modo de emissão local único, 70-80 duração do impulso ms, 35-40 mJ por impulso. He-Ne visando feixe diante. </li><li> Entregar os pontos de laser único na periferia manchado do endotélio doador. Entregar os pontos sequencialmente: o aspecto final é um anel de manchas na periferia da lentícula dador. A distância entre dois adjamanchas centavo centro é o dobro de um diâmetro do ponto. </li><li> Aplicar uma lente de contato no olho do paciente, juntamente com 0,3% de tobramicina e 0,1% dexametasona suspensão oftálmica. </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.

O transplante endotelial &quot;todo o laser&quot; é uma abordagem original para o transplante de córnea minimamente invasiva. Todos os procedimentos descritos no âmbito do protocolo foram realizados na sala de cirurgia, observando-se o procedimento de higiene e esterilização que são práticas comuns durante cirurgias, tais como o uso de luvas esterilizadas, vestido, máscara e boné. A solução ICG foi preparado na sala de cirurgia, logo antes de sua aplicação na coloração do endo...

Neste trabalho apresentamos uma abordagem original à ceratoplastia endotelial, baseado no uso de um laser femtosegundo para preparar tecido do doador e um laser de diodo infravermelho próximo para soldá-lo para a cama destinatário. Medição intra-operatório da córnea doadora é necessário para projetar corretamente as dimensões do tecido doador. Ceratoplastia endotelial tem sido propostos nos últimos anos para substituir ceratoplastia penetrante no tratamento de doença endotelial 1,2. A principal vantagem desta técnica é uma recuperação visual mais rápida, com relação a ceratoplastia penetrante, anestesia reduzida durante a cirurgia, uma diminuição do risco de rejeição do enxerto e a preservação da integridade do olho. O principal fator de risco é pós-operatória luxação doador lenticule. A técnica padrão é realizado através da inserção do endotélio doador na sua posição final, em que é mantida através da injecção de uma bolha de ar: sem suturas são utilizadas por causa da chara mecânica, biofísico e dimensionalcteristics do endotélio. Além disso, a recuperação da acuidade visual pode ser limitada, principalmente devido a uma incompatibilidade entre os tecidos do doador e do receptor, devido a um tecido grosso transplantado. Aqui nós apresentamos um procedimento na execução de ceratoplastia endotelial que pode superar esses problemas principais. O endotélio dador pode ser assegurada na sua posição final pela utilização da técnica de soldadura a laser. Este é um processo fototérmico controlada e localizada: ele pode ser induzida na interface dador / receptor. Tem sido estudada nos últimos dez anos e proposto em ceratoplastia penetrante e no transplante de endotélio 3-5. A luz de infravermelho próximo (comprimentos de onda: 810 nm) emitida por um diodo de laser de baixa potência é fornecida para o tecido biológico no local da ferida. A córnea é naturalmente transparente para este comprimento de onda: a fim de fazer este tecido para absorver a luz de laser, é necessário pintar com um cromóforo. O corante proposto é uma saturar estérilsolução de água d de indocianina verde (ICG). Demonstrou-se que quando o tecido da córnea é adequadamente coradas com esta preparação ICG, que mostra um pico de absorção a 810 nm 6. Além disso, o ICG é amplamente utilizado no diagnóstico clínico e a sua segurança foi já demonstrada em seres humanos. A córnea absorve a luz coradas energia laser de diodo e o principal efeito resultante é a subida de temperatura controlada no local de soldadura. Sem efeitos térmicos são induzidas nos tecidos não coradas. O aumento da temperatura provoca desnaturação térmica reversível no colagénio do estroma, com uma closuring imediata das paredes da ferida após arrefecimento. Este efeito de soldagem a laser foi primeiramente demonstrada em cirurgia de catarata e ceratoplastia penetrante 7,8 9,10. Uma abordagem otimizada que estamos apresentando neste trabalho foi estudado para aplicação em ceratoplastia endotelial. Na cirurgia proposta, pontos de laser de solteiro (com duração de dezenas de milissegundos) arE fornecida ao tecido, resultando num efeito fototérmico localizada dentro da dimensão local (algumas centenas de m de diâmetro): o efeito é induzida uma soldadura a laser de disco, composto por um fotocoagulação do colagénio confinado na interface doador / hospedeiro. O resultado da desnaturação do colagénio no local da soldadura é uma forte aderência entre os tecidos doadores e hospedeiros, proporcionando assim um efeito de sutura que é impossível de obter com a técnica padrão (pontos). O tecido recupera sua aparência natural em um curto seguimento (1 mês) e a aderência entre tecidos doador / hospedeiro é melhorada pela solda fornecido na fase muito precoce da fase de cura. Para evitar o outro risco principal do ceratoplastia endotelial, ou seja, o transplante de um tecido dador de espessura, intracirúrgico tomografia de coerência óptica (OCT) é utilizado: um dispositivo comercial mede a espessura da córnea do doador, de modo que um perfil de corte correcto pode ser concebido com aa laser femtosec. O transplante endotelial proposta de &quot;tudo a laser&quot; parece, assim, para melhorar os resultados clínicos desta cirurgia minimamente invasiva.

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

an &#8220;all-laser&#8221; endothelial transplant

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

O procedimento cirúrgico &quot;todo o laser&quot; é proposta para realizar transplantes de córnea minimamente invasiva. O procedimento é fácil de realizar (ver Figura 1): no que diz respeito a um transplante endotelial padrão somente as etapas de medir a espessura da córnea, coloração do tecido do doador e fornecer a luz de laser são adicionados. As vantagens conseguidas pela maior parte compensar um aumento do tempo de cirurgia de alguns minutos. A utilização intra-operatória de OCT para m...

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.

Um &quot;All-a laser&quot; Endotelial 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 visualizações.  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.

Vídeo: 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.

Usando Quantitative PCR em tempo real para determinar enxerto célula dadora num modelo murino de osso Competitiva Marrow Transplantation

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

Medicina

Murine Heterotopic Heart Transplant Technique

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

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

Técnica de transplante heterotópico Murino Coração

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.

Técnica para as aquisições de fígado de porco e Transplante ortotópico usando um 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.

Modelo rato de rejeição vascular induzida por Aloimune e Transplante Arteriosclerose

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.

Rim murino técnica de transplante

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.

Melhor método para o estabelecimento de uma In Vitro barreira hemato - encefálica modelo baseado em células endoteliais do cérebro de suínos

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.

Reação aperfeiçoada de Griess para a determinação UV-VIS e nu-olho do Primaquine anti-malarial

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.

Lesão por Isquemia-Reperfusão de Órgãos simulando alterações hemodinâmicas no modelo de transplante de fígado de rato

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 - um Banco de Dados Integrado de Pesquisa e Registro Eletrônico de Saúde para Receptores de Transplante Renal

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.

Ensaio de Transcitose de Células Endoteliais como Modelo In Vitro para Avaliar a Permeabilidade da Barreira Interna Sangue-Retina

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