This work was supported by funding from the March of Dimes FY12-133 (S.K.E.) and the National Institutes of Health R01HD037831 (S.K.E.). We would like to thank Dr. Deborah Frank for critical review of the manuscript.

NOTA: Todos os procedimentos com animais respeitadas as diretrizes para o cuidado e uso de animais estabelecidos pelo National Institutes of Health. Todos os protocolos foram aprovados pelo Comitê da Universidade de Washington em St. Louis a estudos com animais. NOTA: materiais e equipamentos específicos para este protocolo estão listados Materiais e Equipamentos tabela. 1. Criação de temporizado Feminino Mice <ol><li> Raça fêmeas adultas entre dois e seis meses de idade, em uma janela de tempo de 2 horas com os machos. H reprodução específica não são necessários, desde que estes sejam mantidos consistentes para a totalidade da experiência. </li><li> Confirmar gravidez pela presença de um tampão copulatória. Designar o dia e hora em que a ficha é visto pela primeira vez como o dia de pós-coito 0 (dpc0). Lembre-se da hora específica quando iniciar a gravação de pressão. </li><li> Realizar cirurgias em dpc8-13. A exacta dpc dependerá da estirpe de ratinho. </li><li> Para determinar se um rato está grávida antes da cirurgia, peultra-sonografia rform do abdômen com um transdutor linear de 40 MHz acoplado a um sistema de imagem antes de administrar anestesia. Isto irá permitir a visualização dos sacos gestacionais. Se um sistema de ultra-som não está disponível, utilizar o peso do rato. NOTA: Um ganho de peso de pelo menos 2 g entre dpc0 e dpc8 e distensão do abdome é um bom indicador da gravidez. </li></ol> 2. Telemetry Cirurgia Prep <ol><li> Use um autoclave para esterilizar instrumentos, gazes, cotonetes, campos cirúrgicos, e pontas de pipeta dentro de 24 horas antes da cirurgia. </li><li> Superfície cirurgia limpa com etanol 70%. </li><li> Ligue almofada de aquecimento (especializado para roedores) e talão esterilizador. </li></ol> 3. Telemetry Cirurgia <ol><li> Para anestesia, utilizar 4-5% de isoflurano com uma taxa de fluxo de oxigénio de 1-2 l / min. </li><li> Aguarde até que o rato mostra sinais de anestesia e depois injetar por via subcutânea com buprenorfina (analgésico: 0,1 ml por 20 g). </li><li> Raspar o abdómen do rato a partir do fundo das nervuras para baixo para a bexiga. </li><li> Traga o mouse para a mesa de cirurgia em almofada de aquecimento pré-aquecido, e cobrir os olhos do rato com pomada oftálmica para evitar a secagem. </li><li> Comece a técnica estéril. Calçar luvas estéreis e manter o wrapper como uma superfície estéril em que para colocar os iodo e etanol scrubs. </li><li> Retire o telêmetro a partir do pacote ou soro fisiológico e usar uma pinça estéril, para colocá-lo em gaze estéril. <ol><li> Usar a seringa (fornecido pelo fornecedor telémetro) para encher a ponta do cateter com gel. Suavemente apertar o cateter criando uma pressão negativa suficiente para extrair o gel no interior do cateter. </li></ol></li><li> Coloque uma ponta estéril sobre a pipeta. Pipetar 2 ul de cola cirúrgica e definir a pipeta sobre uma superfície estéril. </li><li> Para limpar o local cirúrgico, limpar a área com iodo e, em seguida, com etanol a 70%. </li><li> Corte um buraco na cobertura cirúrgica grande o suficientepara a área necessária para a incisão e coloque sobre o mouse. </li><li> Realizar uma pitada pé (resposta pedal) para garantir o mouse é completamente anestesiados antes de fazer uma incisão. </li><li> Faça uma pequena incisão na linha média vertical. Em seguida, faça uma incisão semelhante através do músculo parede do corpo subjacente. </li><li> Puxe cuidadosamente o útero fora da cavidade do corpo e localizar o corno uterino contendo a maioria dos filhotes que são viáveis. <ol><li> Opcional - Para além de miRNA, injetar vetor viral na camada muscular do útero antes da inserção de telêmetro. </li></ol></li><li> Faça uma pequena incisão na ponta do corno uterino com três milímetros de corte tesoura primavera ponta. </li><li> Passe o cateter, sem apertar, entre a parede uterina e sacos fetais. Insira o cateter passado vários sacos para reduzir a sua remoção acidental. Certifique-se que os sacos fetais não são perturbados pela inserção. </li><li> Pipetar a cola cirúrgica para o sítio de inserção do telémetro para aderir o cateter para a uchifre terine e impedir o cateter de deslizar para fora da trompa uterina. Espere alguns segundos para a cola para tornar-se rígida, e em seguida, coloque cuidadosamente o útero de volta para dentro da cavidade do corpo. </li><li> Inserir o telémetro na cavidade do corpo no lado oposto do sítio da inserção do cateter. </li><li> Suturar a cavidade do corpo fechado com 6,0 suturas absorvíveis. Em primeiro lugar, fazer um ponto em bolsa e, em seguida, 3-4 pontos individuais. </li><li> Suturar a pele, utilizando a mesma técnica, mas com 6-0 não absorvíveis. </li><li> Dibucaína Swab (analgésico tópico) pomada sobre o local da incisão. </li><li> Injectar subcutaneamente 0,3-0,4 ml de solução salina estéril para ajudar a hidratar o animal. </li><li> Monitorar o animal após a cirurgia até que esteja completamente acordado. Não coloque o mouse de volta em sua gaiola até que ele é capaz de sustentar livremente sua cabeça acima da tabela como certos tipos de roupa de cama pode sufocar um rato pesadamente anestesiado. Para um rato que foi anestesiado para 30-45 min, deve-se esperar the mouse para ser totalmente acordado por 1 hora e 15 min. </li><li> Se a realização de cirurgias adicionais, limpe instrumentos com etanol 70% e talão esterilizar antes da reutilização. </li></ol> 4. recuperação pós-operatória <ol><li> Monitorar diariamente o progresso pós-cirurgia em um log. Catálogo ganho de peso e perda e prestar atenção para letargia, sangramento, ou fechamento incompleto da incisão cirúrgica. <ol><li> Alimente o mouse ração rato amolecida (~ a consistência de purê de maçã) ou recuperação dieta especializada. </li><li> Examine as suturas e se certificar que não são puxados para fora, mas não tente lidar com o mouse excessivamente. </li></ol></li></ol> 5. Telemetry Recording <ol><li> No dia do interesse (pelo menos um de dois a três dias após o mouse se recuperou da cirurgia), coloque um ímã perto do mouse para ligar o telêmetro. Coloque a gaiola do mouse sobre o receptor. A luz no receptor deve ligar se o telêmetro é ativado. </li><li> Calibrar novo Teleme<html> ters com calibrações específicas fabricante fornecido. Especificações de calibração Telemeter reutilizados serão mantidos em software. </li><li> Dentro do software, começar a gravar com o botão direito em &quot;animal&quot; e clique em &quot;iniciar a amostragem, contínuo&quot;, que permite a amostragem em taxas de até 500 Hz. Verifique se o software está definido para salvar e rastrear. NOTA: Quando o ícone do mouse amostragem fica verde, as pressões estão sendo gravadas. Perturbar o mouse durante o parto pode interromper ou alterar o trabalho, por isso observar com cautela. </li><li> Após o parto ou o período de interesse, parar a gravação com o botão direito em &quot;animal&quot; e selecionando &quot;parar as gravações, tudo&quot;. Use um ímã para desligar telêmetro para salvar a vida da bateria, como a bateria telemeter normalmente duram apenas 1,5 meses de gravação contínua. </li><li> Eutanásia o mouse por CO 2 overdose e recuperar o telêmetro. Os filhotes são sacrificados por decapitação. </li></ol>tle &quot;&gt; 6. Telemeter Esterilização <ol><li> Inserir o telémetro numa solução de 1% de detergente enzimática durante a noite em água desionizada à temperatura ambiente. </li><li> Lavar o telêmetro em água deionizada e remover todo o tecido e cola, tomando cuidado para não apertar o cateter. </li><li> Incubar o telémetro em glutaraldeído a 2% durante 24 horas à temperatura ambiente para esterilizar. </li><li> Lavar o telêmetro com solução salina estéril e armazená-lo em solução salina estéril até a próxima implantação ou a ser enviado de volta ao fabricante para ser reformado. Telémetros deve ser renovado se o tecido está localizado no interior do cateter, após a esterilização. Normalmente telémetros pode ser usado 4-5 vezes antes de precisar ser remodelado. </li></ol>

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	<li>Slattery, M. M., Morrison, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preterm+delivery.">Preterm delivery.</a> Lancet. 360, 1489-1497 (2002).</li><li>Goldenberg, R. L., Culhane, J. F., Iams, J. D., Romero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+causes+of+preterm+birth.">Epidemiology and causes of preterm birth.</a> Lancet. 371, 75-84 (2008).</li><li>Keelan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inhibition+of+inflammatory+pathways+for+the+prevention+of+preterm+birth.">Pharmacological inhibition of inflammatory pathways for the prevention of preterm birth.</a> Journal Of Reproductive Immunology. 88, 176-184 (2011).</li><li>Voltolini, 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=Understanding+spontaneous+preterm+birth:+from+underlying+mechanisms+to+predictive+and+preventive+interventions.">Understanding spontaneous preterm birth: from underlying mechanisms to predictive and preventive interventions.</a> Reproductive Sciences. 20, 1274-1292 (2013).</li><li>Ratajczak, C. K., Muglia, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+parturition+biology+from+genetically+altered+mice.">Insights into parturition biology from genetically altered mice.</a> Pediatric research. 64, 581-589 (2008).</li><li>Rajakumar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+HIF-1+alpha,+HIF-2+alpha,+membrane+and+soluble+VEGF+receptor-1+proteins+are+not+increased+in+normotensive+pregnancies+complicated+by+late-onset+intrauterine+growth+restriction.">Placental HIF-1 alpha, HIF-2 alpha, membrane and soluble VEGF receptor-1 proteins are not increased in normotensive pregnancies complicated by late-onset intrauterine growth restriction.</a> American Journal Of Physiology. Regulatory, Integrative And Comparative Physiology. 293, (2007).</li><li>Whitesall, S. E., Hoff, J. B., Vollmer, A. P., D'Alecy, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+simultaneous+measurement+of+mouse+systolic+arterial+blood+pressure+by+radiotelemetry+and+tail-cuff+methods.+American+journal+of+physiology.">Comparison of simultaneous measurement of mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods. American journal of physiology.</a> Heart And Circulatory Physiology. 286, H2408-H2415 (2004).</li><li>Mackay, L. B., Shi, L. B., Maul, H. .., Maner, W. L., Garfield, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+bilateral+pelvic+neurectomy+on+cervical+ripening+in+pregnant+rats.">The effect of bilateral pelvic neurectomy on cervical ripening in pregnant rats.</a> Journal Of Perinatal Medicine. 37, 263-269 (2009).</li><li>Pierce, S. L., Kutschke, W., Cabeza, R., England, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+intrauterine+pressure+by+telemetry:+a+new+approach+for+studying+parturition+in+mouse+models.">In vivo measurement of intrauterine pressure by telemetry: a new approach for studying parturition in mouse models.</a> Physiol Genomics. 42, 310-316 (2010).</li><li>Imamura, T., Luedke, C. E., Vogt, S. K., Muglia, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+modulates+the+onset+of+murine+parturition+by+competing+ovarian+and+uterine+effects.">Oxytocin modulates the onset of murine parturition by competing ovarian and uterine effects.</a> American Journal Of Physiology. Regulatory, Integrative And Comparative Physiology. 279, R1061-R1067 (2000).</li><li>McCloskey, 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=The+inwardly+rectifying+K++channel+KIR7.1+controls+uterine+excitability+throughout+pregnancy.">The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy.</a> EMBO Mol Med. 6 (9), 1161-1174 (2014).</li></ol>

The authors have nothing to disclose.

As investigações sobre a contratilidade do miométrio têm contado com ex vivo medições da tensão muscular. Esta metodologia pode ser útil para testar em estágio inicial de drogas uterotônicas recentemente desenvolvidos que não podem ser administrados aos animais vivos. No entanto, abordagens in vivo são necessários para compreender longitudinalmente a progressão do trabalho de parto. As medições in vivo servir a vários propósitos. Primeiro, eles permitem a captura de uma image...

O nascimento prematuro é a principal causa de morbidade e mortalidade nos países desenvolvidos perinatal; é responsável por 50% de morbidade perinatal e 75% de 1,2 a mortalidade perinatal. Trabalho de parto prematuro é multifacetada e pode ser idiopática. Apesar de muita pesquisa surgiu nas vias moleculares e elétricas transformando o miométrio, de um tecido de repouso em um contrátil um, a fisiopatologia exata do trabalho de parto prematuro permanece indefinida. Endocrine, inflamatório, e regulação de genes têm sido relacionados ao trabalho de parto prematuro 3,4. No entanto, as preocupações éticas limitar a capacidade de realizar pesquisas sobre os mecanismos de trabalho de parto prematuro em seres humanos. Dadas estas limitações, muitos investigadores voltaram-se para os ratos como um sistema modelo com que no estudo da fisiologia de parto. Os ratos têm gestações curta duração de aproximadamente três semanas e pode ser facilmente manipulado geneticamente. Além disso, vários modelos genéticos do rato temfoi desenvolvido para determinar as vias de sinalização que são essenciais para o trabalho 5. Apesar das diferenças fundamentais entre o mouse eo parto humano, ratos e humanos compartilham muitos dos mesmos mecanismos que são essenciais para o trabalho, incluindo a inflamação intra-uterina e infecção 6. Assim, os ratos servir como uma ferramenta valiosa para examinar a atividade uterina. Até à data, o padrão-ouro para medir a contratilidade uterina em camundongos foi ex vivo gravações de tensão isométrica; no entanto, isto é limitado a um ponto de tempo gestacional por experiência e requer a remoção do útero do seu ambiente fisiológico. Outro factor importante é o elevado número de animais necessários para essas investigações. Por último, esta metodologia não permite estudos longitudinais que examinam a indução do trabalho de parto pré-termo e fisiopatologia. Os recentes avanços feitos em dispositivos de telemetria radiométricos usados ​​para estudar a pressão arterial muda 7 em ratos e intpressão rauterine em ratos 8 pediu a questão para saber se a mesma tecnologia pode ser utilizada para estudar alterações na pressão intra-uterina em ratinhos durante a gravidez. Depois de solução de problemas inicial, foi desenvolvido um método com sucesso para medir a indução do parto e progressão em um mouse. Esta abordagem in vivo pode medir a transição do estado de baixa pressão do útero quiescente para o estado de alta pressão indicativa de contrações fortes. Este método também foi capaz de detectar diferenças de pressão significativas entre ratinhos transgénicos com parto comprometida e ratinhos do tipo selvagem, demonstrando o impacto fisiológico da expressão dos genes 9. Este protocolo fornece em tempo real, no método in vivo para estudar a pressão rato uterino durante a gravidez.

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			<td style="width:105px;">&#160;Data Sciences International</td>
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intrauterine telemetry to measure mouse contractile pressure in vivo

Um complexo de integração de sinais moleculares e eléctricos é necessário para transformar um útero quiescente para um órgão contráctil no final da gravidez. Apesar da descoberta de reguladores-chave da contractilidade uterina, este processo ainda não está completamente entendido. Os ratinhos transgénicos proporcionar um modelo ideal para estudar o parto. Anteriormente, o único método para estudar a contractilidade uterina no rato foi ex vivo gravações de tensão isométrica, que são sub-óptima por várias razões. O útero tem de ser removido do seu ambiente fisiológico, um campo de investigação tempo limitado é possível, e os ratinhos deve ser sacrificado. O desenvolvimento recente de telemetria radiométrica permitiu longitudinais, as medições em tempo real da pressão intra-uterina in vivo em ratinhos. Aqui, a implantação de um telêmetro intra-uterina para medir as mudanças de pressão no útero de camundongos a partir de meados de gravidez até o parto é descrito. Ao comparar as diferenças de imprensamentos entre o tipo selvagem e ratinhos transgénicos, o impacto fisiológico de um gene de interesse pode ser elucidado. Esta técnica deve acelerar o desenvolvimento de terapias usadas para tratar distúrbios do miométrio durante a gravidez, incluindo o trabalho de parto prematuro.

Em tempo real das pressões intra-uterinos vivo pode ser gravado utilizando um sistema de aquisição de telemetria. Amostragem Pressão emparelhado com gravação simultânea de vídeo do mouse foi usado para capturar o momento exato da entrega de cada filhote e correlacionar o tempo de entrega para a pressão intra-uterina da mãe. Todos os pontos de dados podem ser representados graficamente, em seguida, durante toda a gravação para gerar uma pressão versus tempo trama (Figura 1A).</stron...

Watch this Scientific Journal Video about Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo at JoVE.com

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Telemetria Intrauterine medir Rato contrátil Pressão<em&gt; In Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

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Medicine

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

9.6K visualizações.  Washington University in St. Louis. This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy. 

Vídeo: Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Ventilação a pressão controlada para induzir lesão pulmonar aguda em ratos

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Medicina

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Ecocardiografia fetal e ultra-som Doppler pulsátil em um modelo de coelho de restrição do crescimento intra-uterino

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI para Rastreamento celular

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Precisão em Medicina Dentária, uma maneira nova de medir justeza e precisão

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

Medição da curva pressão-volume no mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

Response malha trabecular para elevação de pressão na Vida Olho Humano

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

O cateterismo cardíaco em camundongos para medir o volume Pressão do relacionamento: Investigando o efeito Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

Desenvolvimento de um algoritmo para realizar um estudo abrangente de Disreflexia Autonômica em animais com alta Spinal Cord Injury Usando um dispositivo de telemetria

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

Longo prazo medição de pressão arterial em ratos movimentando-se livremente utilizar telemetria

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...