Nós gostaríamos de reconhecer Gundula Hertl o seu apoio.

Todos os procedimentos neste protocolo foram aprovados por diretrizes e legislação de bem-estar Animal alemão. Considere o uso de 10 a 12 semanas de idade NMRI nus ratos e camundongos C57Bl/6 (doador: fêmea velha; destinatário: masculino) durante os experimentos. Use Camundongos NMRI para isolamento ilhéu. Manter os ratos sob condições definidas de acordo com regulamentos locais instalações animais.
1. indução do Diabetes por estreptozotocina
<ol><li>Dia -4, medir o peso corporal de 10 a 12 semanas de idade NMRI nude os ratos e camundongos C57Bl/6, usando uma máquina de pesagem.</li>
	<li>De acordo com o peso do corpo medido, prepare uma dose de 180 mg/kg de estreptozotocina em 0,1 mL de soro fisiológico por rato. Nota: Estreptozotocina deve ser recentemente preparada antes do uso e coberta com papel de alumínio, como é sensível à luz.</li>
	<li>Os ratos syngeneic e alogênico injete cada um com uma única dose de 0,1 mL de estreptozotocina (180 mg/kg) intraperitonealmente usando uma agulha de 26-G.</li>
	<li>Dia -3, -2 e -1 coletam 2-4 µ l de sangue por punção das veias de cauda de ratos não jejuou.</li>
	<li>Use 2-4 µ l de amostra de sangue por tira teste para medir a glicose no sangue em camundongos NMRI e camundongos C57Bl/6 Nota: Neste momento, 80% dos ratos devem se tornar diabético (20 mmol/L).<ol>
<li>Use rato para transplante de ilhotas quando seus níveis de glicose atingem 20 mmol/L.</li>
	</ol>
</li>
</ol>2. isolamento de ilhotas pancreáticas de ratos
<ol><li>Anestesia os ratos com cetamina (100 mg/kg de peso) e xilazina (20 mg/kg de peso corporal) por injeção intraperitoneal com uma agulha de 26-G. Nota: Os volumes de ketamina e xilazina foram determinados de acordo com o peso corporal dos animais (0,10 mL/10g).</li>
	<li>Manter os ratos em posição supina e desinfectar com solução de iodo-povidona. Confirme a eutanásia adequada, verificando o reflexo de pedal. Nota: Em caso de C57Bl/6 raspe a região abdominal.</li>
	<li>Segure a pele com uma pinça fina e fazer uma incisão com uma tesoura para expor na cavidade abdominal. Coloque uma compressa de gaze estéril, o situs intestino para a esquerda e fora da cavidade abdominal.</li>
	<li>Ajustar o estômago em outra direção para localizar o pâncreas e outros associados tecidos.</li>
	<li>Atingir a aorta e fazer um pequeno corte para drenar o sangue. Use gaze esterilizada para absorção de sangue.</li>
	<li>Remova cuidadosamente o intestino. Separe o pâncreas do estômago e baço.</li>
	<li>Excisar o pâncreas e coloque-o em um prato de Petri fresco. Remova todos os materiais indesejados, tais como gordura, usando fórceps.</li>
	<li>Encher uma seringa de 5 mL com 4 mL de colagenase e injetar no pâncreas, com uma agulha de 26-G (solução: 30 mg de colagenase em 12 mL de solução de Hank).</li>
	<li>Picar o pâncreas com uma tesoura para aumentar a área de superfície para uma melhor digestão e transferir para um tubo de tampa vermelha 12 mL. Nota: Hank está equilibrada solução salina: HBSS 10 x (100 mL), penicilina-estreptomicina (10 mL), gentamicina (1 mL), ciprofloxacina (10 mL), HEPES (35 mL), água destilada, H2O (900 mL)</li>
	<li>Execute uma digestão por 10 min a 37 ° C em um banho de água a agitação. Vórtice a amostra três vezes em intervalos regulares por 15 s.</li>
	<li>Misture o pâncreas digerido vigorosamente à mão por 3 min e coloque-o no gelo por 10 s.</li>
	<li>Adicione 6 mL de solução de Hank fria para parar a reação de digestão e centrifugar 560 x g por 3 min à temperatura ambiente.</li>
	<li>Remover o sobrenadante e dissolva a pelota em 10 mL de meio de soro (P/FCS) bezerro Parker/fetal à temperatura ambiente. Nota: Média P/FCS: TCM-199 10 x (100 mL), FCS (50 mL), penicilina-estreptomicina (10 mL), HEPES (20 mL), água destilada, H2O (900 mL)</li>
	<li>Identifica as ilhotas com base na sua morfologia (esferoide, cor marrom-dourado) sob um microscópio de luz. Evite ilhotas com centros de escuros. Verifique se há pureza e viabilidade através da realização de ditizona coloração sob um microscópio de luz. Nota: Ditizona vincula-se ao íon zinco e fornece a mancha de cor vermelha para os ilhéus.</li>
	<li>Use uma pipeta de 1 mL para contar e escolher Ilhéus sob um microscópio de luz e transferi-los para um prato de Petri fresco (60 x 15 mm), contendo 4 mL do meio P/FCS. Nota: O volume de P /meio de FCS numa caixa de Petri não deve exceder 4 mL para garantir a disponibilidade de oxigênio.</li>
	<li>Manter as ilhotas isoladas em 4 mL de meio de P/FCS durante a noite a 37 ° C numa incubadora.</li>
</ol>3. preparação dos animais para transplante de ilhotas
<ol><li>Remover as ilhotas isoladas da incubadora a 37 ° C e colocá-los em um banco do fluxo. Centro de Ilhéus com um movimento circular oscilante da caixa de Petri.</li>
	<li>Encha uma seringa de 1 mL (agulha 23-G azul) com 0,1 mL do meio P/FCS. Em seguida, adicione 350 Ilhéus em 0,3 mL de solução de Hank. Manter a seringa na posição vertical para permitir que os ilhéus resolver.</li>
	<li>Diminuir o volume de 0,05 mL e substituir a agulha 23-G azul com uma agulha de 26-G marrom. A mesma seringa, adicione 0,15 mL de Ficoll (médio gradiente de densidade) na parte superior para alcançar um volume final de 0,2 mL na seringa. Evite bolhas de ar.</li>
	<li>Mantenha a seringa na posição vertical e girá-lo ligeiramente para evitar a aglutinação de ilhotas. Os ilhéus vão resolver no cone da seringa dentro de 2-3 min.</li>
	<li>Medir os níveis corpo peso e sangue glicose dos ratos destinatários; os níveis de glicose do sangue devem ser em torno de 20 mmol/L.</li>
	<li>Anestesia os ratos com cetamina (100 mg/kg de peso) e xilazina (20 mg/kg de peso corporal) por injeção intraperitoneal com agulha 26-G.</li>
	<li>Coloque os ratos em posição supina sobre uma placa térmica quente (37 ° C). Verifique a profundidade da anestesia por beliscar o dedo do pé.</li>
	<li>Limpe a região abdominal com solução de iodo-povidona (desinfectante da pele). Para evitar os olhos de secagem, mantê-los húmida com pomada. Nota: Em caso de C57Bl/6 raspe a região abdominal.</li>
</ol>4. ilhéu transplante através da rota Intraportal
<ol><li>No dia 0, começa com uma laparotomia, mantendo a pele com pinças e fazendo um 2-3 cm-incisão longa com uma tesoura. Lugar uma gaze esterilizada molhada comprimir em torno das bordas da ferida para mantê-lo úmido.</li>
	<li>Coloque a compressa de gaze estéril, o situs intestino para a esquerda e fora da cavidade abdominal. Nota: Mantenha o intestino húmidas durante todo o processo com solução de NaCl 0,9% previamente aquecida ou gaze estéril embebido em solução salina normal.</li>
	<li>Mova o duodeno e localize o pâncreas. Localize a veia portal situada no local ventral, empurrando para cima o pâncreas. Usar um dedo e o polegar para expor a veia porta e, uma vez expostos, punção-lo perto do fígado.</li>
	<li>Dentro de 1 min, lentamente e com firmeza Injete os ilhéus (0,2 mL do passo 3.4) a veia porta sob a inspeção de lupa para evitar qualquer vazamento de Ilhéus. Nota: Após a injeção, verifique sempre para Ilhéus remanescentes na seringa. Tome 1 mL de mídia P/FCS em uma seringa, embutida em um prato de Petri fresco e contagem sob um microscópio.</li>
	<li>Coloque um medidor estéril no local da punção e aplicar pressão com o dedo indicador (profundidade de penetração: 0,5-1 cm) por 6 min parar o sangramento. Nota: O volume injetado não deve exceder 0,2 mL. Pressão e volume excessivo podem danificar os ilhéus devido à tensão de cisalhamento.</li>
	<li>Coloque cuidadosamente o intestino situs, peritônio, camada muscular abdominal e fáscia volta à sua posição.</li>
	<li>Usando fórceps, levantar a pele, fechar a incisão do músculo com sutura de seda e a camada superior da pele com clipes de 2-3.</li>
	<li>Limpe a superfície com desinfectante da pele, aplique um curandeiro de ferida e coloque os ratinhos em gaiolas individuais. Ferida curandeiro fornece uma camada de acrilato resistente à água e proteger de maceração e infecção secundária. Nota: Observar os ratos até eles recuperaram a consciência suficiente, fornecer tramadol (0,2 mg / rato) juntamente com líquidos. Neste protocolo, os ratos recuperaram da anestesia rapidamente e administração de tramadol reduziu a dor. Após 7 dias, deve curar a ferida.</li>
	<li>Alimente os ratos com água e comida normal. Excreção de urina e consumo de alimentos e água aumentará após a injeção de estreptozotocina.</li>
	<li>Medir os níveis de glicose de sangue todos os dias durante a primeira semana e depois uma vez por semana em ratos syngeneic. Em camundongos alogênico, medir os níveis de glicose do sangue, três vezes por semana por até 15 dias.</li>
</ol>

<ol>
	<li>McCall, M., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+islet+transplantation.">Update on islet transplantation.</a> Cold Spring Harbor Perspectives in Medicine. 2 (7), a007823 (2012).</li><li>Shapiro, A. M., 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=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> New England Journal of Medicine. 343 (4), 230-238 (2000).</li><li>Cantarelli, E., 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=Bone+marrow+as+an+alternative+site+for+islet+transplantation.">Bone marrow as an alternative site for islet transplantation.</a> Blood. 114 (20), 4566-4574 (2009).</li><li>Echeverri, G. J., 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=Endoscopic+gastric+submucosal+transplantation+of+islets+(ENDO-STI):+technique+and+initial+results+in+diabetic+pigs.">Endoscopic gastric submucosal transplantation of islets (ENDO-STI): technique and initial results in diabetic pigs.</a> American Journal of Transplantation. 9 (11), 2485-2496 (2009).</li><li>Korsgren, O., 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=Optimising+islet+engraftment+is+critical+for+successful+clinical+islet+transplantation.">Optimising islet engraftment is critical for successful clinical islet transplantation.</a> Diabetologia. 51 (2), 227-232 (2008).</li><li>Merani, S., Toso, C., Emamaullee, J., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> British Journal of Surgery. 95 (12), 1449-1461 (2008).</li><li>Lingwal, N., 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=Inhibition+of+gelatinase+B+(matrix+metalloprotease-9)+activity+reduces+cellular+inflammation+and+restores+function+of+transplanted+pancreatic+islets.">Inhibition of gelatinase B (matrix metalloprotease-9) activity reduces cellular inflammation and restores function of transplanted pancreatic islets.</a> Diabetes. 61 (8), 2045-2053 (2012).</li><li>Chen, C., 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=Improved+intraportal+islet+transplantation+outcome+by+systemic+IKK-beta+inhibition:+NF-kappaB+activity+in+pancreatic+islets+depends+on+oxygen+availability.">Improved intraportal islet transplantation outcome by systemic IKK-beta inhibition: NF-kappaB activity in pancreatic islets depends on oxygen availability.</a> American Journal of Transplantation. 11 (2), 215-224 (2011).</li><li>Wang, Y., Wang, T., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+aspects+are+critical+for+carrying+out+intraportal+islet+transplants+successfully+in+a+diabetes+mouse+model.">Three aspects are critical for carrying out intraportal islet transplants successfully in a diabetes mouse model.</a> Experimental and Clinical Transplantation. 10 (3), 302-304 (2012).</li><li>Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination.">A method for mouse pancreatic islet isolation and intracellular cAMP determination.</a> Journal of Visualized Experiments. (88), e50374 (2014).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> Journal of Visualized Experiments. (50), (2011).</li><li>Rother, K. I., Harlan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+facing+islet+transplantation+for+the+treatment+of+type+1+diabetes+mellitus.">Challenges facing islet transplantation for the treatment of type 1 diabetes mellitus.</a> Journal of Clinical Investigation. 114 (7), 877-883 (2004).</li><li>Su, Z., 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=Small+islets+are+essential+for+successful+intraportal+transplantation+in+a+diabetes+mouse+model.">Small islets are essential for successful intraportal transplantation in a diabetes mouse model.</a> Scandinavian Journal of Immunology. 72 (6), 504-510 (2010).</li><li>Rekittke, N. E., Ang, M., Rawat, D., Khatri, R., Linn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+Therapy+of+Type+1+Diabetes+Mellitus:+From+Pancreatic+Islet+Transplantation+to+Mesenchymal+Stem+Cells.">Regenerative Therapy of Type 1 Diabetes Mellitus: From Pancreatic Islet Transplantation to Mesenchymal Stem Cells.</a> Stem Cells International. 2016, 22 (2016).</li></ol>

Todos os autores declaram que não têm nenhum conflito de interesses.

Neste estudo, a rota intraportal de transplante de ilhotas é explorada. Este protocolo é altamente eficaz no alívio de estresse diabética e oferece uma oportunidade em profundidade para explorar estudos de transplante de ilhotas. Este protocolo confirma que cerca de 350 murino ilhotas são capazes de reverter o estado hiperglicêmico. Além disso, nenhum dos ratos morreu durante o procedimento, e o controle glicêmico foi alcançado em todos os destinatários. Níveis de glicose no corpo peso e sangue foram gravados ...

Transplante de ilhotas é uma abordagem promissora para tratar o tipo 1 diabetes mellitus1. A primeira tentativa de transferir um fragmento do pâncreas de ovelhas em um paciente diabético foi realizada por Watson Williams em 1893. No entanto, um grande avanço em transplante clínico ilhéu foi alcançado com o protocolo de Edmonton, e daí em diante, uma série de programas nacionais foram desenvolvidos2. No passado, vários sites, tais como a medula óssea, bolsa omental, região intramuscular, superfície da mucosa gástrica, baço e cápsula renal, foram explorados em modelos pré-clínicos, mas o sistema venoso portal é considerado como um do local adequado e eficaz para programas clínicos3,4,5,6.
Administração exógena de insulina pode ser substituída por infusão ilhéu na veia portal. Isto é considerado um local preferido, porque o suprimento de oxigênio é comparável ao que no pâncreas nativo devido a localização a jusante da confluência do portal artéria e veia. Além disso, há uma grande área de superfície, para que a estrutura tridimensional dos ilhéus pode ser preservada, e vascularização poderia ser facilitado5. Em um rato diabético destinatário, 2.000 equivalentes humanos ou suínos ilhéu ou 350 rato ilhotas são suficientes para reverter o estado hiperglicêmico7,8. Euglicêmico níveis foram relatados por 15 dias no modelo destinatário de rato de ilhotas xenogénicas e no modelo do rato para alogênico e por mais de 120 dias no syngeneic-rato para transplante.
Fatores relevantes para a eficácia do transplante de ilhotas através da veia porta são anestesia adequada, método de punção e hemostasia9. Anestesia pode ser induzida por inalação (5% de isoflurano) ou injeção (cetamina, xilazina ou pentobarbital), e as substâncias são muitas vezes combinadas. Para controle da profundidade e tempo de anestesia, deve ser dada atenção ao estado do mouse, como a cor da membrana mucosa, tampa do olho, reflexo corneal, frequência respiratória e temperatura corporal. É importante assegurar que o animal não está se esforçando e que sobreviva a operação. A temperatura pode ser mantida por aquecimento de diferentes dispositivos, como almofadas de aquecimento, lâmpadas de luz vermelha, etc. A quente chapa térmica tem sido usada para manter uma temperatura de 25-30 ° C entre o rato e a mesa de operação, o que impede a ocorrência de hipotermia. As doses dos anestésicos são importantes, porque todos eles são metabolizados pelo fígado, e a função hepática pode ser transitoriamente desordenada por infusão de suspensão o ilhéu. O ponto de punção ideal para a veia porta é a posição entre a primeira e a segunda veia tributário.
O número de Ilhéus e o volume de injeção se destina também influencia o resultado do transplante, como um volume excessivo pode aumentar a tensão de cisalhamento. Um volume de 0,2 mL é considerado adequado para o transplante de ilhotas na veia portal. A ferida puncionar (agulha de calibre 26) induz sangramento da veia portal, que precisa ser interrompido de forma oportuna e eficaz. Gaze estéril ou um dedo pode ser aplicado com pressão mínima no local da punção por cerca de 6 min parar o sangramento. Tomados juntos, portal ilhéu injeção é eficaz e oferece o Regulamento sobre níveis de glicose em modelos do rato diabético quimicamente induzida.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="20">
			<td>C57Bl/6 mice&#160;</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td>BALB/c mice</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td>NMRI nude mice</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine</td>
			<td>Bela-pharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylazine</td>
			<td>CEVA TIERGESUNDHEIT GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe, 23G and 26G needle</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dish</td>
			<td>Falcon</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Carl Roth</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Gauze</td>
			<td>Fuhrmann</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shaking Water bath</td>
			<td>Kotterman</td>
			<td>1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Braun</td>
			<td>1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose Meter&#160;</td>
			<td>One Touch</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Warm Thermal Plate</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Braunol (povidone-iodide solution)</td>
			<td>B.Braun Melsungen AG</td>
			<td>3864154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liposic Edo - Sprinkled Ointment (eye ointment)</td>
			<td colspan="2">Dr. G Mann Chem.-Pharm.&#160; GmbH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator</td>
			<td>Fa. Roth</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Flow Bench</td>
			<td>Herdus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ficoll</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-0 Silk Suture Vicryl</td>
			<td>Braun</td>
			<td></td>
			<td>(7.5 *1.75mm)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Michel Clamps (clips)</td>
			<td>Aesculap&#160;</td>
			<td>BN507R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P/FCS Solution</td>
			<td>Gibco</td>
			<td>21180-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCM-199 (10x)</td>
			<td>Gibco</td>
			<td>21180-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1 M)</td>
			<td>Biochrom</td>
			<td>L 1613</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamycin</td>
			<td>Ratiopharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ciprofloxacin</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#39;s Solution</td>
			<td>Gibco</td>
			<td>14060-040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Roche</td>
			<td>11213865001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptozotocin</td>
			<td>Calbiochem</td>
			<td>572204</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPSITE-spray (wound healer)</td>
			<td>Smith and nephew</td>
			<td>66004978</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Reugular food</td>
			<td>Altromin</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Head light LED 16042 (magnifying glass)</td>
			<td>Eschenbach</td>
			<td>16042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat anti-mouse PECAM-1(CD31) monoclonal primary antibody</td>
			<td>Millipore, Chemicon</td>
			<td>CBL1337</td>
			<td>Dilution (1:100)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-rat secondary antibody</td>
			<td>Dianova</td>
			<td>712095153</td>
			<td>Dilution (1:400)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyclonal guinea pig anti-insulin primay antibody</td>
			<td>Dako</td>
			<td>A0564</td>
			<td>Dilution (1:500)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-guinea pig secondary antibody</td>
			<td>Dianova</td>
			<td>706-259-148</td>
			<td>Dilution (1:400)</td>
		</tr>
	</tbody>
</table>

intraportal transplantation of pancreatic islets in mouse model

Transplante de ilhotas pancreáticas para reduzir a hiperglicemia é altamente bem sucedido em roedores com diabetes quimicamente induzida. O local mais comum de transplante em transplante experimental de ilhotas é a cápsula renal. No entanto, quanto menos se sabe sobre a interação das ilhotas pancreáticas com componentes do sangue, também faz sentido utilizar a abordagem da veia porta em transplante experimental de ilhotas.
Este protocolo demonstra uma técnica de transplante de ilhotas intraportal em camundongos NMRI. Estreptozotocina (180 mg/kg) é injetada intraperitonealmente para induzir hiperglicemia em ratos do destinatários. Eles são considerados como diabético a um nível de glicose do sangue não-jejum maiores que 20 mmol/L. Um dia antes da transplantação, ilhotas pancreáticas do mouse são isoladas do pâncreas de doador por digestão de colagenase; um mínimo de 350 ilhéus são utilizados por beneficiário diabético. Dependendo do rendimento de isolamento ilhéu, dois ou mais ratos de doador são utilizados por beneficiário. Depois da cultura durante a noite a 37 ° C, ilhotas são administradas para o destinatário fígado através da veia portal. Após a cirurgia, os ratos são protegidos em casas de Makrolon vermelhas e observados até está acordado. Esse protocolo mantém controle glicêmico para 120 dias em camundongos syngeneic e 15 dias em ratos alogênico.

Estudou-se a competência de ilhotas transplantadas em ratos diabéticos induzidos quimicamente. Os ilhéus foram transplantados através do sistema venoso portal no modelo de ratos diabéticos. A relação de doador-beneficiário foi 2:1. No modelo de rato syngeneic, dois ratos de doadores foram utilizados para obter 350 Ilhéus. Os ilhéus foram então transplantados em ratos diabéticos. No dia 1 observou-se uma redução no nível de glicose no sangue após transplante, sugerindo um p...

Watch this Scientific Journal Video about Intraportal Transplantation of Pancreatic Islets in Mouse Model at JoVE.com

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Transplante de ilhotas pancreáticas é uma maneira de conseguir normoglycemia no diabetes tipo 1. Este artigo centra-se na técnica de transplante através da rota intraportal para manter os níveis normais de glicose em ratos diabéticos.

Intraportal transplante de ilhotas pancreáticas no modelo de Mouse

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

intraportal-transplantation-of-pancreatic-islets-in-mouse-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.4K visualizações.  Clinical Research Lab. Transplante de ilhotas pancreáticas é uma maneira de conseguir normoglycemia no diabetes tipo 1. Este artigo centra-se na técnica de transplante através da rota intraportal para manter os níveis normais de glicose em ratos diabéticos.

Vídeo: Intraportal Transplantation of Pancreatic Islets in Mouse Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Um modelo do rato de No Utero Transplante

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Medicina

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

Modelo do rato de endometriose cirurgicamente induzida pela Auto-transplante de tecido uterino

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Protocolos de coloração de ilhotas pancreáticas humanas

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Procedimentos cirúrgicos para um modelo de rato de Transplante de Fígado parcial ortotópico com reconstrução arterial hepática

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocolo melhorado para microdissecção a laser de ilhotas pancreáticas humanas a partir de espécimes cirúrgicos

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

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.

O transplante de válvula pulmonar Usando um modelo do rato de Heterotópica Transplante de Coração

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

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; Db</sup&gt; Mouse Modelo de Diabetes Tipo 2: Isolamento de ilhotas pancreáticas e células vivas 2-Photon Imagens de ilhotas intactas

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

Ortotópico Hind Limb Transplantation no mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Usando a técnica de manga em um modelo do rato da transplantação da aorta - um vídeo instrutivo

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Intraportal transplante de ilhotas pancreáticas no modelo de Mouse

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