This study was supported by a grant from the Dutch Kidney Foundation (KJPB 08.0003). 
Carolien Rothuizen is acknowledged for her contribution to the study. Hoang Pham is acknowledged for his assistance with the pathology work-up.

Todos os experimentos foram aprovados pelo comitê de bem-estar animal, do Centro Médico da Universidade de Leiden. 1. Preparação Animal e Anestesia <ol><li> Anestesiar o rato (1-3 meses de idade) numa câmara de indução da anestesia contínua cheio com 3-4% de isoflurano. </li><li> Raspar o lado ventral do pescoço e a parte interna da perna esquerda superior usando uma máquina de barbear eléctrica e usar um pedaço de fita adesiva para remover o cabelo. </li><li> Aplicar pomada ocular em ambos os olhos. </li><li> Posicionar o animal sobre a manta de aquecimento em que a máscara de ventilação nariz é fixo. Fixar a cabeça para a máscara do nariz usando fita adesiva. <ol><li> Deliver isoflurano a uma concentração de 1,5-2,0%, utilizando o oxigénio do ar enriquecido conjunto no seguinte fluxo: ar de 0,3 L / min, 100% de oxigénio de 0,2 L / min. Comprima a pele entre os dedos dos pés para avaliar a profundidade da anestesia e ajustar a concentração de isoflurano, se necessário. </li></ol></li><li> Fixar o animal aocobertor de aquecimento com fita adesiva em todos os membros que está ajustada para aproximadamente 37 ° C numa posição de supinação. </li><li> Injectar buprenorfina dissolvido em cloreto de sódio (NaCl) a 0,9% a uma dosagem de 0,1 mg / kg em conjunto com 0,5 ml de solução estéril de NaCl a 0,9% por via subcutânea no flanco do animal. </li><li> Aplicar clorexidina tintura de 0,5% para a área preparada. </li></ol> Incisão 2. Pele <ol><li> Posicionar o animal sob o microscópio com a sua cabeça para o cirurgião. </li><li> Adicione uma incisão longitudinal de aproximadamente 1,5 cm na linha média da região ventral do pescoço utilizando uma micro-tesoura. </li><li> Agarre as glândulas salivares com uma pinça e deslocar a glândula salivar direita cranial até que esteja parcialmente fora da ferida. </li></ol> 3. Dissecando e preparar o Vein <ol><li> Identificar e sem rodeios dissecar o ramo dorsomedial da veia jugular externa direita completamente do seu tecido perivascular, espalhando apenas umLongside e na direcção do vaso sanguíneo. </li><li> Coloque um loop (10,0 sutura) em torno da veia dissecada e aplicar um nó sem bloqueá-lo. </li></ol> 4. Retirar o músculo Sternocleidomastoid <ol><li> Utilizando uma pinça, sem rodeios dissecar o músculo esternocleidomastóideo direito do seu entorno, espalhando ao longo de sua fronteira. Coloque um par de pinças abertas sob o músculo isolado e ligar-lo proximal e distalmente com um fio de sutura 6.0. Subsequentemente, excisar o músculo utilizando um cauterizador. </li></ol> 5. Dissecando e preparar a artéria carótida comum <ol><li> Identificar e dissecção da artéria carótida comum direita usando uma pinça. Coloque um fio de sutura 6.0 em torno da artéria para auxiliar na manipulação da artéria. </li><li> Aplicar pinças vasculares como distal e proximal possível. </li><li> Adicione uma incisão longitudinal no meio da artéria de aproximadamente 1 mm usando o micro tesoura especializado. </li><li> Lavar oartéria com uma solução de heparina [100 IU / ml], até que o recipiente esteja livre de sangue. </li><li> Medir e ajustar o comprimento da incisão, se necessário, para coincidir com a largura da pinça vascular [1,1 milímetros]. </li></ol> 6. ligadura da veia <ol><li> Aplique uma pinça vascular proximal da veia já preparada e coloque um fio de sutura em torno da veia. </li><li> Aplicar tracção caudal suave para a veia utilizando um hemostático que é colocado sobre o fio de sutura e ligar a veia como distalmente quanto possível, utilizando a sutura 10.0 que foi colocado anteriormente. </li><li> Cortar a veia usando uma tesoura apenas proximal ao ligadura. </li><li> Utilizando uma pinça vascular, abra suavemente o lúmen da veia e lave-o com uma solução de heparina [100 IU / ml]. </li></ol> 7. Criação de anastomose Parte 1 <ol><li> Ligar a veia para a artéria com uma sutura interrompida (10,0) utilizando 2 nós quadrados na posição de doze horas, seguida por um fio de sutura, das seis O &#39;relógio posição. Certifique-se de conectar a veia para a artéria, sem permitir que ela gire em torno de seu próprio eixo. Rodar o animal, a fim de melhorar a exposição cirúrgica. </li><li> Posicionar um fio de sutura em torno da veia e aplicar tração lateral suave usando um hemostático. </li><li> Usando os mesmos pontos separados, completar a anastomose, colocando aproximadamente 3-4 suturas adicionais no lado ventral visível da anastomose. </li></ol> 8. Administração de Heparina <ol><li> Gire aquecimento cobertor 180 ° e concentrar o microscópio sobre a parte superior da perna esquerda. </li><li> Identificar o feixe veia / artéria / nervo femoral pela procura de uma estrutura vascular que corre longitudinalmente no lado medial da parte superior da perna e pode ser visto através da pele. Realizar uma incisão com o micro tesoura de aproximadamente 1 cm acima directamente na veia femoral de um modo longitudinal. </li><li> Dissecar cuidadosamente o tecido perivascular da veia femoral e injetar heparina na dose oF 0,2 UI / g de peso corporal na veia femoral. </li></ol> 9. Criação de anastomose Parte 2 <ol><li> Voltar para a área do pescoço e remover o fio de sutura que foi colocada em torno da veia. </li><li> Coloque um fio de sutura (6.0), que subsequentemente passa por baixo da artéria carótida, através da veia e de novo abaixo da artéria carótida. (Figura 1D) </li><li> Remover o grampo vascular venoso. </li><li> Em seguida, a metade torcer concluída fístula artério-venosa de 180 ° ao longo do eixo da artéria carótida no sentido horário por torção simultaneamente ambos os grampos vasculares e aplicar tração medial para as extremidades do fio de sutura. </li><li> Conclua a anastomose da mesma maneira como é descrito na etapa 7.2. </li></ol> 10. Vascular braçadeira Remoção <ol><li> Remova o fio de sutura e gire o vascular grampos 180 ° no sentido horário. </li><li> Retire o grampo vascular distal seguido pelo grampo vascular proximal. </li> &lt;li&gt; Avaliar a permeabilidade da anastomose por oclusão suavemente a via de saída venosa com a pinça vasculares. No caso de uma anastomose de patente, o troço venoso pré-oclus vai expandir de uma maneira pulsátil. Mudar o local da glândula salivar direito à sua posição usando uma pinça anatómica original. </li></ol> 11. Pele Encerramento e Cuidados Pós-Operatórios <ol><li> Fechar a pele da parte superior da perna com uma sutura contínua (6,0). </li><li> Fechar a pele do pescoço com uma sutura contínua (6,0). </li><li> Remover o animal da manta de aquecimento e injectar 0,5 ml de NaCl a 0,9% por via subcutânea. </li><li> Colocar o animal em uma gaiola escurecida que é aquecido com uma lâmpada de aquecimento e deixá-lo recuperar totalmente. Quando um animal não se recuperar totalmente, se certificar de que não sofre um choque hemodinâmico devido ao sangramento na área cirúrgica. Olhe para os sinais, como um pescoço inchado e o vazamento de sangue. </li><li> Após cerca de 6 horas após a primeira injecção de buprenorphine, injectar uma dose única de uma formulação de libertação sustentada de buprenorfina por via subcutânea numa dose que é recomendado pelo fabricante, a fim de proporcionar uma analgesia adequada para uma 72 horas adicionais. </li></ol> Colheita 12. Tissue <ol><li> Anestesiar os animais com uma mistura de anestésicos-contendo midazolam (5 mg / kg), medetomidina (0,5 mg / kg) e fentanil (0,05 mg / kg) administrada intraperitonealmente. </li><li> Fixar o animal em decúbito dorsal através da inserção de agulhas através de suas patas e bochechas em um tapete de silicone não-aquecida. </li><li> Realizar uma incisão de aproximadamente 1,5-2,0 cm de altura sobre a cicatriz no pescoço com o micro-tesoura. </li><li> Dissecar o AVF, e coloque fios de sutura (6.0) em todo o proximal-, artéria distal e via de saída venosa para facilitar a identificação. </li><li> Avaliar a permeabilidade da FAV como é descrito na secção 10.3 do protocolo. </li><li> Abrir a cavidade peritoneal através da realização de uma laparotomia mediana Lcantar a micro tesoura. <ol><li> Corte completamente a caixa torácica ventral esquerda e direita usando um par de tesouras de partida caudal na linha hemiclavicular. Usando um micro tesoura, transecto o diafragma que é anexado a parte do meio da caixa torácica ventral e posteriormente deslocar esta secção cranial usando uma pinça. </li><li> Localizar a veia cava inferior e transecto-lo com um micro tesoura. </li></ol></li><li> Inserir uma agulha de calibre 23 de agulha no ventrículo esquerdo e perfundir com PBS a uma pressão de cerca de 100 mm Hg até o fluido intravasal praticamente límpida. </li><li> Sem retirar a agulha, agora perfundir com formalina a 4% durante 10 min a uma pressão de cerca de 100 mmHg. </li><li> Extirpar o AVF, ao seccionar ambas as artérias e veias com o micro tesoura e mergulhe-o em solução de formalina a 4% O / N. </li></ol> 13. Tissue Embedding e Seccionamento <ol><li> Processar o tecido de parafina de acordo com Protoco da fabricaçãoeu. O protocolo aplicado consistiu no seguinte: <ol><li> Mergulhar em etanol a 70% durante 1 h à TA. Repetir 2x. </li><li> Mergulhar em etanol a 96% durante 1 h à TA. Repetir 2x. </li><li> Mergulhar em etanol 99,5% durante 1 h à TA. Repetir uma vez. </li><li> Mergulhar em etanol 99,5% durante 1,5 h à TA. </li><li> Mergulhar em xileno a 100% durante 1 h à TA. Repetir uma vez. </li><li> Mergulhar na parafina durante 1 hora a 62 ° C. Repetir 2x. </li></ol></li><li> Incorporar a FAV em parafina para que a via de saída venosa é orientado perpendicular ao cassete incorporação. </li><li> Utilizando um micrótomo, criar 12 secções em série cada um consistindo de 30 secções com uma espessura de 5 um. Em seguida, de cada secção em série, uma secção de colocar em um único copo de montagem a partir de uma posição de um máximo de doze de forma ordenada começando com a área mais próxima da anastomose. </li></ol>

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A FAV é considerado o calcanhar de Aquiles em tratamento de hemodiálise. Infelizmente, a FAV sofre ainda de um número elevado de falha 8-10. Apesar de extensa pesquisa sobre os mecanismos subjacentes, a fisiopatologia exata permanece desconhecida. Numerosos modelos murinos para o fracasso FAV já foram descritos na literatura 2,3,11,12. No entanto, nenhum desses modelos incorporam uma extremidade venosa a configuração anastomose arterial que é mais utilizado em cada situação clínica. Isto ...

A conduta de acesso vascular funcional é de vital importância para os pacientes com insuficiência renal que dependem de hemodiálise crônica para se manter vivo. A construção de uma fístula arteriovenosa (FAV) é atualmente a escolha preferida para acesso vascular. No entanto, as complicações relacionadas AVF constituem uma das principais causas de morbidade para pacientes em hemodiálise crónica. Apesar das extensas esforços científicos, nenhuma das novas abordagens para reduzir as complicações relacionadas ao acesso FAV resultou em melhoria substancial da AVF durabilidade. Parte deste progresso decepcionante diz respeito a compreensão incompleta da fisiopatologia subjacente da falha de acesso de hemodiálise. Para desvendar a fisiopatologia da insuficiência de acesso AV, modelos animais que imitam de perto patologia humana são de extrema importância. A este respeito, não só as espécies animais, mas também o local da anastomose, a terapia anti-coagulante necessária e a duração do acompanhamento após surtoRY deve ser tomado em consideração um. Enquanto animais de grande porte são os mais adequados para estudos de intervenção destinadas a desenvolver novas estratégias terapêuticas, modelos murinos têm o maior potencial para ter mais conhecimento dos mecanismos moleculares subjacentes falha de acesso AV, devido à disponibilidade de ratinhos transgénicos. Além disso, um grande número de ratinhos podem ser utilizados para este fim a custos mais baixos em comparação com o uso de animais maiores. O primeiro modelo murino de falha FAV foi descrita em 2004 por kwei e et ai. 2 Neste modelo, FAVs foram construídos utilizando a artéria carótida e a veia jugular de uma maneira extremo-a-extremidade utilizando um cateter intravascular. Este modelo pode ser útil para estudar a adaptação venosa no início FAVs embora a configuração de ponta a ponta e a presença de um cateter intravascular limitar a validade deste modelo para FAVs humanos. Um modelo FAV melhorada foi introduzido por Castier e 3 et ai. Na qual a extremidade dea artéria carótida é ligada para o lado da veia jugular. No entanto, em pacientes em hemodiálise FAVs são geralmente construídos por anatomizing o fim de uma veia para o lado de uma artéria. A configuração exacta da FAV é uma característica essencial de um modelo de acesso AV, uma vez que determina o perfil hemodinâmico no interior da conduta 4. Este último é um importante contribuinte para a disfunção endotelial e subsequente desenvolvimento de hiperplasia da íntima (IH) 5. Um modelo murino romance foi recentemente desenvolvido com uma configuração anatómica idêntica como é utilizado em seres humanos 6. Neste modelo, são FAV criado em ratinhos C57BL / 6 por anastomose da extremidade de um ramo da veia jugular externa para o lado da artéria carótida comum com suturas interrompidas. No presente artigo, vamos nos concentrar sobre o procedimento microcirúrgico deste modelo, a fim de facilitar a utilização generalizada deste modelo murino, com o objetivo de desvendar a fisiopatologia complexada falha de acesso de hemodiálise.

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:247px;">Dissecting microsocpe</td>
			<td style="width:219px;">Leica</td>
			<td style="width:117px;">M80</td>
			<td style="width:115px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Forceps</td>
			<td>Medicon</td>
			<td>07.61.25</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vascular forceps</td>
			<td>S&#38;T</td>
			<td>JFL-3D.2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vascular forceps</td>
			<td>S&#38;T</td>
			<td>D-5a.2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Forceps</td>
			<td>Roboz</td>
			<td>SS/45</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro scissor 5 mm blade</td>
			<td>Fine science tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro scissor 2 mm blade</td>
			<td>Fine science tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scissor</td>
			<td>Medicon</td>
			<td>05.12.21</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Clip applier 1</td>
			<td>S&#38;T</td>
			<td>CAF-4</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vascular clamp 1</td>
			<td>S&#38;T</td>
			<td>B-1V</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Clip applier 2</td>
			<td>BBraun</td>
			<td>FE572K</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vascular clamp 2</td>
			<td>BBraun</td>
			<td>FE740K</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hemostatic forceps</td>
			<td>BBraun</td>
			<td>BH110</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">10.0 sutures</td>
			<td>BBraun</td>
			<td>G1117041</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">6.0 sutures</td>
			<td>BBraun</td>
			<td>768464</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cauterizer</td>
			<td>Fine science tools</td>
			<td>18010-00</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Needle holder</td>
			<td>Medicon</td>
			<td>11.82.18</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ocular ointment</td>
			<td>Pharmachemie</td>
			<td>41821101</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Chlorhexidine tincture 0,5%</td>
			<td>Leiden University Medical Center</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heparin</td>
			<td>Leo Pharma</td>
			<td>012866-08</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Buprenorphin</td>
			<td>RB Pharmaceuticals&#160;</td>
			<td>283732</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Isoflurane</td>
			<td>Pharmachemie</td>
			<td>45,112,110</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anesthesia mask</td>
			<td>Maastricht university</td>
			<td>custom made</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Midazolam</td>
			<td>Actavis</td>
			<td>AAAC6877</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dexmedetomidine</td>
			<td>Orion</td>
			<td>141-267</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fentanyl</td>
			<td>Bipharma</td>
			<td>15923002</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Continuous anaesthetic induction chamber</td>
			<td>Vet-tech solutions</td>
			<td>AN010R</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel murine model of arteriovenous fistula failure: the surgical procedure in detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Após a criação da anastomose (Figura 1), a permeabilidade deve ser avaliada por pouco oclusão da via de saída venosa com uma pinça vascular. Quando a anastomose é patente, o tracto proximal vascular para a oclusão deve expandir claramente de uma maneira pulsátil. Além disso, a permeabilidade é confirmada através da utilização de fluoroscopia perto de infravermelhos (NIRF) que funciona efectivamente como uma angiografia (Figura 2). Uma falha no procedimento cirúrgico pode ...

Watch this Scientific Journal Video about A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail at JoVE.com

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

Um Modelo murino Novel de Fístula Arteriovenosa Falha: O procedimento cirúrgico em Detalhe

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

a-novel-murine-model-arteriovenous-fistula-failure-surgical-procedure

Research

JoVE Journal

Medicine

12.9K visualizações.  Leiden University Medical Center. Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

Vídeo: A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Uma nova abordagem cirúrgica para administração intratraqueal de agentes bioativos em um modelo de camundongo Fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Medicina

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Modelo Murino de Cicatrização

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Aspectos técnicos da artocava Fístula Rato

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Um procedimento cirúrgico para ressecção do mouse Rib: Um Modelo de Grande Escala Repair longo osso

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Drug Delivery intraluminal para o Rato Fístula Arteriovenosa endotélio

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

Um modelo do rato de ortotópico transplante hepático, usando uma técnica de anastomose magnético romance para reconstrução de veia Cava Suprahepatic

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Murino colostomia Distal, um novo modelo de colite de diversão em camundongos C57BL/6

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ligation of the inferior vena cava (IVC) in the outflow of the fistula, mimicking central venous stenosis. Technical aspects of this model are introduced. The aorta and IVC are exposed, following an abdominal incision. The infra-renal aorta and IVC are dissected for proximal clamping, and the distal aorta is exposed for puncture. The IVC at the midpoint between the left renal vein and the aortic bifurcation is carefully dissected to place an 8-0 suture beneath the IVC. After clamping the aorta and IVC, an AVF is created by puncturing the infra-renal aorta through both walls into the IVC with a 25 G needle, followed by ligating a 22 G intra-venous (IV) catheter and IVC together. The catheter is then removed, creating a reproducible venous stenosis without occlusion. The aorta and IVC are unclamped after confirming primary hemostasis. This novel model of central vein stenosis is easy to perform, reproducible, and will facilitate studies on AVF failure.

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

Modelo murino de estenose venosa central usando fístula Aortocaval com estenose de saída

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ...

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. An aneurysm forms as a result of progressive weakening and eventual dilation of the aorta, which can rupture or tear once it reaches a critical diameter. Aneurysms of the descending aorta in the chest, called descending thoracic aortic aneurysms (dTAA), make up a large proportion of aneurysm cases in the United States. Uncontained dTAA rupture is almost universally lethal, and elective repair has a high rate of morbidity and mortality. The purpose of our model is to study dTAA specifically, to elucidate the pathophysiology of dTAA and to search for molecular targets to halt the growth or reduce the size of dTAA. By having a murine model to study thoracic pathology precisely, targeted therapies can be developed to specifically test dTAA. The method is based on the placement of porcine pancreatic elastase (PPE) directly on the outer murine aortic wall after surgical exposure. This creates a destructive and inflammatory reaction, which weakens the aortic wall and allows for aneurysm formation over weeks to months. Though murine models possess limitations, our dTAA model produces robust aneurysms of predictable size. Furthermore, this model can be used to test genetic and pharmaceutical targets which may arrest dTAA growth or prevent rupture. In human patients, interventions such as these could help avoid aneurysm rupture, and difficult surgical intervention.

We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall.

Modelo cirúrgico murino de elastase tópica induzida por aneurisma de aorta torácica descendente

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. ...

Creating Radio-cephalic Arteriovenous Fistula in the Forearm with a Modified No-Touch Technique

Autologous arteriovenous fistula (AVF) is the primary and best option to obtain vascular access for hemodialysis treatment; other options are arteriovenous graft (AVG) and central venous catheterization (CVC). The implementation of radio-cephalic autologous arteriovenous fistula (RC-AVF) in the forearm was preferred among patients with superior vascular conditions. However, there is a high rate of early fistula failure. The chosen surgical method is understood to have an effect on the maturation of the fistula. New surgical procedures such as radial artery deviation and reimplantation (RADAR) have been significantly improved for juxta-anastomotic stenosis. Nevertheless, new problems such as stenosis of arteries and narrowing of surgical indication were also found. In this report, we presented a modified no-touch technique (MNTT) to create an RC-AVF, in which the venous and arterial wall avoid devascularization and the radial artery does not sever.

We present a modified no-touch technique (MNTT) to create a radio-cephalic arteriovenous fistula (RC-AVF) in which the venous and arterial wall avoid devascularization and the radial artery does not sever.