Os autores gostariam de agradecer a Adriane Mosley e Libuse Jerabek (Stanford) para obter assistência com a técnica cirúrgica.

Todos os estudos em animais foram realizados de acordo com as diretrizes de cuidados com os animais e uso comitê da UCLA e os Institutos Nacionais de Saúde Guia para o Cuidado e Uso de Animais de Laboratório. A duração do procedimento descrito abaixo é cerca de 45-60 minutos do começo ao fim. 1. Preparação de campo cirúrgico <ol><li> Realize o procedimento em uma sala de cirurgia animal limpo. </li><li> Equipamento: isoflurano vaporizador, Gaymer T Bomba com almofada de aquecimento. </li><li> Ferramentas estéreis: duas pinças, tesouras curvas finas, porta-agulha. </li><li> Luvas estéreis deve ser usado durante todo o procedimento. </li></ol> 2. Preparação de Animais <ol><li> Colocar dois ratos machos ou fêmeas, do mesmo fundo genético, de peso e de tamanho semelhante na mesma gaiola e para monitorizar, pelo menos, duas semanas, para assegurar convivência harmoniosa. Ratinhos fêmeas são preferidos devido ao seu comportamento menos agressivo. </li><li> Anestesiar animais porutilizando um vaporizador de isoflurano. Ratinhos lugar em uma indução Secção Posi-Seal ligado ao vaporizador de isoflurano (4-5% v / v). Uma vez que a anestesia é induzida, transferir o animal para a área de raspar a pele e manter a anestesia ao longo do processo por meio de um cone de nariz ligada ao isoflurano (1,5-2% v / v). Aplicar pomada oftálmica com um Q-ponta para evitar os olhos secos. </li><li> Colocar o animal em decúbito dorsal. Absolutamente raspar o lado esquerdo do rato colocado no lado esquerdo e do lado direito do rato colocado no lado direito a partir de cerca de 1 cm acima do cotovelo de 1 cm abaixo do joelho. </li><li> Assepticamente preparar as áreas raspadas através de uma profunda limpeza (2-3x) com panos encharcados de Betadine seguidos de algodão embebido em álcool. Colocar os ratos numa almofada aquecida coberto por uma compressa estéril. </li><li> Para analgesia, administrar carprofeno e buprenorfina por via intraperitoneal ou por via subcutânea na dose de 10 mg / kg e 0,1 mg / kg, respectivamente. </li><li> Coloque os animais do seu lado, de costas, com umdjacent áreas raspadas viradas para cima. Para evitar a contaminação da zona cirúrgica, cobrir os ratos com um campo estéril expondo somente a área de operação. Criar uma pequena abertura cortina de ficar estéril ao realizar a cirurgia. Fizemos janela a cortina grande para ter uma visão melhor durante a filmagem. </li></ol> 3. Parabiose <ol><li> Usando uma tesoura afiada, faça incisões cutâneas longitudinais nas laterais raspadas de cada partida animais a 0,5 cm acima do cotovelo todo o caminho até 0,5 cm abaixo da articulação do joelho (Figura 1). Após a incisão, retirar suavemente a pele da fáscia subcutânea, mantendo a pele com um par de fórceps curvos e separar a fáscia com um segundo par para criar 0,5 centímetro de pele livre. Realize esta separação ao longo de toda a incisão. </li><li> Comece a juntar, anexando o olécrano esquerda um animal para o olécrano direito do outro. Ambos olecranons e articulações do joelho são claramente distinguíveis seguinte ªe incisão na pele. Para facilitar a adesão, dobrar o cotovelo do primeiro mouse e passar a agulha da não-absorvível 3-0 sutura sob o olécrano. Do mesmo modo, dobrar o cotovelo do segundo rato e passar o mesmo por baixo da sutura. Fixe firmemente juntas por um nó cirúrgico casal. </li><li> Ligar as articulações do joelho, seguindo o mesmo procedimento. </li><li> Após a fixação das articulações, ligar a pele dos dois animais com um absorvível 5-0 Vicryl contínuo iniciando ventral do cotovelo para o joelho. Para evitar a ruptura da pele e de separação realizar uma sutura de encerramento hermético da pele, na área em torno dos cotovelos e joelhos. Uma vez que a fixação da pele ventral foi concluída, execute um nó cirúrgico casal. Coloque os ratos na posição prona e continuar a sutura dorsal terminando com um nó cirúrgico casal. Verificar a continuidade do fio de sutura e confirmar a falta de aberturas. </li><li> Administrar 0,5 ml de NaCl a 0,9% por via subcutânea a cada rato para evitar desidrataçãodration. </li></ol> 4. Recuperação pós-operatória <ol><li> Manter os animais em pad aquecido até a recuperação. </li><li> Após a recuperação, fornecer analgésicos carprofen e buprenorfina. Repetir intraperitoneal ou por via subcutânea em 24 e 12 horas, respectivamente, durante 48 horas nas mesmas doses descritas acima (passo 2.5). Monitorar os animais para os sinais de dor e angústia, como tremores, letargia, mastigação de cauda, ​​costas arqueadas, a falta de higiene, etc diariamente durante duas semanas. </li><li> Profilaticamente, tratar ratos com sulfametoxazol / trimetoprima suspensão oral em sua garrafa de água 2 mg sulfa / ml 0,4 mg / ml aparar por 10 dias para evitar infecções bacterianas. </li><li> Casa cada par parabiótica em uma gaiola limpa de material de cama monolítico (por exemplo, papel toalha ou almofada absorvente estéril) para evitar a aspiração de material de cama. Retornar os animais para uma gaiola cheia de cama quando eles são capazes de manter decúbito esternal com a cabeça erguida. Para minimizar a tensãode pegar comida enquanto ajusta a existência parabiótica, coloque os grãos de ração umedecida no chão da gaiola. Fornecer material de nidificação. Em 1-2 semanas camundongos parabiótico têm a capacidade de deambular normalmente em pares cirurgicamente frente-e membros pélvicos. </li><li> Quimerismo sanguíneo ocorre 10-14 dias após a cirurgia. </li></ol> 5. Confirmação e procedimento inverso <ol><li> Depois de duas semanas extrair o sangue das veias da cauda de cada rato para análise por citometria de fluxo para confirmar quimerismo sanguíneo (Figura 3). </li><li> Processamento de sangue para análise de citometria de fluxo: diluir 2-3 gotas de sangue de 500 ul de EDTA 10 mM. Adicionar 500 ul de 2% de dextrano (diluído em PBS), homogeneizar e incubar a 37 ° C durante 30 min. Transferir o sobrenadante num novo tubo, centrifugação a 1.200 rpm durante 5 minutos e voltar a suspender as células peletizadas em PBS (ou outro tampão) para a análise de citometria de fluxo. </li><li> Dependendo delineamento experimental, o par parabiótico podem ser separados umt pontos de tempo posteriores. Em nossa experiência, temos mantido pares parabiosed para até 9 meses, sem quaisquer complicações. </li><li> Realizar procedimento inverso sobre uma superfície cirúrgico estéril. Anestesiar os ratos colocando-os em uma câmara de indução ligada a um vaporizador de isoflurano (4-5%) e manter a anestesia com 1,5-2% de isoflurano, conforme descrito acima (passo 2.2). </li><li> Remover os pêlos da área em torno da sutura inicial e assepticamente preparar as áreas raspadas, como descrito acima (passos 2.3, 2.4). </li><li> Para analgesia, administrar carprofeno e buprenorfina por via intraperitoneal ou por via subcutânea na dose de 10 mg / kg e 0,1 mg / kg, respectivamente. </li><li> Cobrir a área cirúrgica com um campo estéril, tal como descrito acima (passo 2.6). </li><li> Utilizando uma tesoura afiada, separar os ratos por meio de uma incisão longitudinal ao longo da sutura lateral e separar cuidadosamente a fáscia recém formado entre os dois ratinhos com um par de pinças curvas. Para separar as articulações, cortar os nós de suturaconectá-los. Apare a pele ao longo da incisão para alcançar as bordas lisas e recolocar a pele com um contínuo 5-0 revestido sutura Vicryl absorvível. </li><li> Para prevenir a desidratação, administrar 0,5 ml de NaCl a 0,9% por via subcutânea, a cada murganho. Manter os animais em pad aquecido até a recuperação. </li><li> Após a recuperação, repetir administração de analgésicos, carprofeno (10 mg / kg) a cada 24 horas e buprenorfina (0,1 mg / kg) a cada 12 horas por 48 horas. </li><li> Para evitar infecções bacterianas tratar ratos com sulfametoxazol / trimetoprima suspensão oral (2 mg sulfa / ml 0,4 mg aparar / ml) em sua garrafa de água durante 10 dias. </li></ol>

<ol>
	<li>Bert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exp&#233;riences+et+consid&#233;rations+sur+la+greffe+animale.">Exp&#233;riences et consid&#233;rations sur la greffe animale.</a> Journal de l'Anatomie et de la Physiologie. 1, 69-87 .</li><li>Bunster, E., Meyer, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+of+parabiosis.">Improved methods of parabiosis.</a> Anat. Rec. 57, 339-380 (1933).</li><li>Waskow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+parabiotic+mice+for+the+study+of+DC+and+DC+precursor+circulation.">Generation of parabiotic mice for the study of DC and DC precursor circulation.</a> Methods Mol. Biol. 595, 413-428 (2010).</li><li>Finerty, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parabiosis+in+physiological+studies.">Parabiosis in physiological studies.</a> Physiol. Rev. 32, 277-302 (1952).</li><li>Aicher, A., Heeschen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonbone+marrow-derived+endothelial+progenitor+cells:+what+is+their+exact+location.">Nonbone marrow-derived endothelial progenitor cells: what is their exact location.</a> Circ. Res. 101, e102 (2007).</li><li>Abe, S., Boyer, 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=Cells+derived+from+the+circulation+contribute+to+the+repair+of+lung+injury.">Cells derived from the circulation contribute to the repair of lung injury.</a> Am. J. Respir. Crit. Care Med. 170, 1158-1163 (2004).</li><li>Donskoy, E., Goldschneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymocytopoiesis+is+maintained+by+blood-borne+precursors+throughout+postnatal+life.+A+study+in+parabiotic+mice.">Thymocytopoiesis is maintained by blood-borne precursors throughout postnatal life. A study in parabiotic mice.</a> J. Immunol. 148, 1604-1612 (1992).</li><li>Powell, A. E., Anderson, E. 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=Fusion+between+Intestinal+epithelial+cells+and+macrophages+in+a+cancer+context+results+in+nuclear+reprogramming.">Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.</a> Cancer Res. 71, 1497-1505 (2011).</li><li>Duyverman, A. M., Kohno, 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=A+transient+parabiosis+skin+transplantation+model+in+mice.">A transient parabiosis skin transplantation model in mice.</a> Nat. Protoc. 7, 763-770 (2012).</li><li>Ajami, B., Bennett, J. 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=Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life.">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nat. Neurosci. 10, 1538-1543 (2007).</li><li>Weissman, I. L., Jerabek, 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=Tolerance+and+the+H-Y+antigen:+Requirement+for+male+T+cells,+but+not+B+cells,+to+induce+tolerance+in+neonatal+female+mice.">Tolerance and the H-Y antigen: Requirement for male T cells, but not B cells, to induce tolerance in neonatal female mice.</a> Transplantation. 37, 3-6 (1984).</li></ol>

Os autores não têm interesses financeiros concorrentes.

O método parabiose discutido aqui apresenta dificuldades e resultados técnicos mínimos em baixas taxas de mortalidade. A fixação do joelho e cotovelo é uma melhoria significativa da Bunster e técnica Meyer. No entanto, o procedimento invasivo permanece, assim, a manutenção de condições estéreis durante toda a cirurgia é imperativo. Para prevenir novas infecções do sítio cirúrgico, é importante que os animais parabiosed receber uma combinação de antibióticos e ser monitorizados regularmente. Para ass...

Parabiose, juntando-se a cirurgia de dois organismos, foi descrita pela primeira vez em 1864 por Paul Bert, como forma de desenvolver um modelo para o estudo de sistemas circulatórios compartilhados e consistiu na junção da pele e paredes musculares de dois ratos 1. Parabiose promove a formação de microvasos no local de inflamação 3 e tem tido várias aplicações em estudos fisiológicos, tais como a comunicação hormonal entre a glândula pituitária e gónadas, bem como o papel da hipertensão renal em 4. Foi ainda utilizado para investigar o recrutamento ea integração de células progenitoras na neovascularização 5, migração de células-tronco hematopoéticas 6 e tráfico de linfócitos 7, bem como o papel ea cinética de circular células inflamatórias ou resultam em metástase do tumor 8,9, e doenças neurodegenerativas 10. Uma vantagem significativa de mentiras Parabioseem que os animais parceria compartilham antígenos circulantes comuns, permitindo a migração de células e neovascularização, sem desencadear uma reação imunológica. Importante, Weissman et al. Demonstraram que parabiose entre ratinhos macho e fêmea não conduz à formação de anticorpos anti HY 11. No protocolo original descrito por Paul Bert os dois animais foram unidos por meio de ligação da pele e do músculo paredes 1. Este método, contudo, provocou pressão significativa para os animais e resultou na elevada taxa de mortalidade devido à infecção da ferida. Desde então, a técnica parabiose foi revisto por vários grupos, sendo os mais predominante o protocolo proposto por Bunster e Meyer em 1933 2. Seu método incluído união das articulações escápula, cavidades do corpo e da pele, permitindo um melhor apoio e menos dor para os animais. Ao mesmo tempo, o novo método resultou em cuidados pós-operatórios mínimos e signifisignificativamente diminuído as taxas de mortalidade. O protocolo aqui descrito é uma modificação da técnica de Bunster e Meyer, que é menos invasivo e permite juntar mais firme. Nomeadamente, os ratinhos são ligados através do cotovelo e do joelho, assim como a pele. Esta união impede a extensão da pele e, por conseguinte, provoca menos dor e as complicações. Aqui nós descrevemos a união de um tipo selvagem (WT) rato adulto com uma GFP constitutivo expressar mouse. Nós mostramos que duas semanas após a cirurgia que podemos alcançar 50% de quimerismo sangue que demonstra a eficácia deste procedimento cirúrgico para criar um sistema circulatório compartilhado.

<table border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>Isoflurane-Phoenix</td>
			<td>Clipper</td>
			<td>NDC 57319-559-06</td>
		</tr>
		<tr>
			<td>Posi-Seal Induction Chamber</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0530-SM</td>
		</tr>
		<tr>
			<td>Portable Anesthesia System</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0007</td>
		</tr>
		<tr>
			<td>Gaymer T Pump</td>
			<td>Gaymar Industries, Inc</td>
			<td>TP650</td>
		</tr>
		<tr>
			<td>Warming Blanket (Heating pad)</td>
			<td>Kent Scientific Corp</td>
			<td>TP-22G</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Roboz</td>
			<td>RS-5101</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>FST 14063-09</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>FST</td>
			<td>FST 12501-13</td>
		</tr>
		<tr>
			<td>Electrical shaver</td>
			<td>Oster</td>
			<td>Golden A5</td>
		</tr>
	</tbody>
</table>

parabiosis in mice: a detailed protocol

Parabiose é uma união cirúrgica de dois organismos, permitindo a partilha da circulação sanguínea. Colocar a pele de dois animais promove a formação de microvasos no local da inflamação. Parceiros parabiótico compartilhar seus antígenos circulantes e, portanto, estão livres de reação imune adversa. Descrita pela primeira vez por Paul Bert, em 1864 1, a cirurgia parabiose foi refinado por Bunster e Meyer em 1933 para melhorar a sobrevivência de animais 2. No protocolo de corrente, dois ratinhos são unidas cirurgicamente na sequência de uma alteração da técnica de Bunster e Meyer. Os animais são conectadas através das articulações do cotovelo e joelho seguido por uma ligação da pele permitindo um apoio firme que impede pressão sobre a pele suturada. Relata-se em detalhe a parabiótico união de um GFP onipresente expressar mouse para um tipo selvagem (WT) mouse. Duas semanas após o procedimento, o par é separada e as células positivas para GFP pode ser detectada por meio de análise de citometria de fluxo no sangue circulanteíon do camundongo WT. O quimerismo sanguíneo permite examinar a contribuição das células circulantes de um animal no outro.

O resultado esperado de parabiose de dois organismos é a contribuição igual de sistema circulatório do animal para cada uma circulação de sangue comum (Figura 2). Pode-se facilmente verificar o equilíbrio bem sucedido do sangue dos ratinhos WT positivo e GFP parabiosed por análise de citometria de fluxo. Aqui, o sangue venoso foi obtida a partir das caudas de ambos parabionts às 2 semanas após a cirurgia e foi fraccionado por células sanguíneas periféricas (para excluir eritrócitos). A fra...

Watch this Scientific Journal Video about Parabiosis in Mice: A Detailed Protocol at JoVE.com

Parabiosis in Mice: A Detailed Protocol

Parabiótico junção dos dois organismos conduz ao desenvolvimento de um sistema circulatório compartilhado. Neste protocolo, descrevemos os passos cirúrgicos para formar uma conexão parabiótico entre um rato do tipo selvagem e um rato expressando GFP constitutiva.

Parabiose em Ratos: Um protocolo detalhado

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Research

JoVE Journal

Medicine

73.7K visualizações.  University of California, Los Angeles.  Parabiótico junção dos dois organismos conduz ao desenvolvimento de um sistema circulatório compartilhado. Neste protocolo, descrevemos os passos cirúrgicos para formar uma conexão parabiótico entre um rato do tipo selvagem e um rato expressando GFP constitutiva. 

Vídeo: Parabiosis in Mice: A Detailed Protocol

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

O Trier Protocolo de teste de estresse social para induzir Estresse Psicológico

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

Medicina

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Protocolo para Longa Duração Hipertermia corpo inteiro em Ratos

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

Protocolo de Coleta para Pâncreas Humano

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusões artéria carótida para farmacocinéticas e farmacodinâmicas Análise de taxanos em Ratos

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Um Defeito modelo simples Critical porte Femoral em Ratos

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Heterotópico Renal autotransplante em um modelo suíno: Um Protocolo Passo-a-Passo

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

A Passo a Passo Protocolo de Cirurgia sub-retiniana em Coelhos

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

Criação de abdominais aderências em Ratos

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Um protocolo detalhado para Monitoramento transpiração Usando um Novel, Pequeno, dispositivo sem fios

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

A Detailed Protocol for Physiological Parameters Acquisition and Analysis in Neurosurgical Critical Patients

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ICP waveform, amplitude of pulse (AMP), the correlation of ICP amplitude and ICP mean (RAP), pressure reactivity index (PRx), ICP and arterial blood pressure (ABP) wave amplitude correlation (IAAC), and so on, can reflect intracranial status, predict prognosis, and can also be used as guidance of proper treatment. However, most of the clinicians focus only on the mean ICP value while ignoring these parameters because of the limitations of the current devices. We have recently developed a multimodality monitoring system to address these drawbacks. This portable, user-friendly system will use a data collecting and storing device to continuously acquire patients&#39; physiological parameters first, i.e., ABP, ICP, and oxygen saturation, and then analyze these physiological parameters. We hope that the multimodality monitoring system will be accepted as a key measure to monitor physiological parameters, to analyze the current clinical status, and to predict the prognosis of the neurosurgical critical patients.

Recently, we have developed a portable multimodality monitoring system for the monitoring of various physiological parameters in neurosurgical critical patients. Detailed protocols on how to use this multimodality monitoring system are presented here.

Um protocolo detalhado para parâmetros fisiológicos aquisição e análise em pacientes críticos neurocirúrgicos

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ...